UNIT 1A.1
One of the most important emerging technologies used by microbiologists and other
life scientists and laboratory workers that handle pathogenic and infectious agents is
the technology that manifests in what is collectively referred to as biosafety. Biosafety
measures designed to ensure the safety of laboratory workers include the use of various
primary and secondary barriers, many of which are due to the advent of new technologies
in the fields of material science and engineering. Persons carrying out the protocols in this
manual may encounter potentially hazardous materials such as pathogenic and infectious
biological agents, as well as toxic chemicals and carcinogenic, mutagenic, or teratogenic
reagents (see UNIT 1A.3). In the case of biological agents, it has long been recognized
that laboratory workers can acquire infections from the agents they manipulate thus
making the very nature of their work an occupational hazard. Bacterial agents cause the
most commonly acquired laboratory infections but pathogenic agents belonging to all
categories of microorganisms can cause infections.
New biosafety technologies and associated evolving guidelines have emerged to significantly improve ways to safely handle microbiological material. In addition, a better
understanding of the risks associated with various manipulations of many agents transmissible by different routes has facilitated our ability to apply appropriate biosafety
practices to specific laboratory arenas. As this knowledge base grows and new biosafety
technologies emerge, evolving safety guidelines will continue to benefit laboratory
workers. A combination of engineering controls, management policies, work practices and procedures, as well as medical interventions, collectively defines these safety
guidelines.
Several biosafety levels, described in this unit, have been developed for microbiological
and biomedical laboratories to provide increasing levels of personnel and environmental
protection. UNIT 1A.2 will provide information related to biosafety practices associated
with potential agents of biocrime and biowarfare. UNIT 1A.3 will provide guidelines for the
safe use of hazardous chemicals.
It is important to note that most governments regulate the use of biohazardous materials.
Therefore, it is essential that they be used in strict accordance with local and national
regulations (see APPENDIX 1B). Cautionary notes are included in many instances throughout
the manual, and some specific guidelines are provided below (and in references therein).
However, we emphasize that users must proceed with the prudence and precautions
associated with good laboratory practice, under the supervision of personnel responsible
for implementing laboratory safety programs at their institutions. Guidelines for the safe
use of radioisotopes are presented in APPENDIX 1C.
Precautions described in this unit should be applied to the routine handling of viable
pathogenic microorganisms, as well as all human-derived materials, because they may
harbor dangerous pathogens such as human immunodeficiency virus (HIV), hepatitis B
virus (HBV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and a host of bacterial pathogens. In addition to the guidelines provided herein, experimenters can find a
wealth of information about handling infectious agents in the appropriate government
publications (see Literature Cited and APPENDIX 1B).
Emerging
Technologies
Contributed by Richard Coico and George Lunn
Current Protocols in Microbiology (2005) 1A.1.1-1A.1.8
C 2005 by John Wiley & Sons, Inc.
Copyright
1A.1.1
Biosafety
1A.1.2
Current Protocols in Microbiology
for less dangerous waste, red ones for suspected HIV-containing material). All of
these bags are marked with the universal biohazard symbol.
2. At time of disposal, the bags are loosely closed (not completely sealed) with
temperature-sensitive autoclave tape (also widely available from supply houses),
placed in an autoclavable basin, and sterilized at 121 C. When the tape indicates
that sterilization temperature has been achieved, it is then possible to dispose of the
waste by ordinary means.
3. At many institutions, contaminated hypodermic needles, scalpels, broken glass, and
other sharp objects must be disposed of separately. These must be placed in appropriate
sharps containers (e.g., Baxter), which may be autoclaved when full.
BIOSAFETY LEVELS
For each biosafety level there are specific supervisory qualifications as assurance that
laboratory workers are provided appropriate role models and knowledgeable mentors.
Various types of specialized equipment are used to provide primary barriers between
the microorganism and the laboratory worker. These range from disposable gloves and
other personnel protective equipment to complex biosafety cabinets or other containment
devices.
The laboratory director is specifically and primarily responsible for the safe operation
of the laboratory. His/her knowledge and judgment are critical in assessing risks and
appropriately applying these recommendations. The recommended biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special
characteristics of the agents used, the training and experience of personnel, and the nature or function of the laboratory may further influence the director in applying these
recommendations.
The U.S. Centers for Disease Control and Prevention (CDC; see Internet Resources)
defines four levels of biosafety, which are outlined below. Selection of an appropriate
biosafety level for work with a particular agent or animal study (see Animal Facilities) depends upon a number of factors. Some of the most important are the virulence,
pathogenicity, biological stability, route of spread, and communicability of the agent;
the nature or function of the laboratory; the procedures and manipulations involving the
agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic
measures.
Table 1A.1.1 provides a summary of recommended biosafety levels for infectious agents.
For regulations and guidelines applicable outside of the U.S., please refer to APPENDIX 1B
and Internet Resources.
NOTE: The following information has been adapted from Biosafety in Microbiological and Biomedical Laboratories, 4th Ed. (BMBL, 4th Ed.; GPO S/N 017-04000547-4), which is published jointly by the U.S. Centers for Disease Control and
Prevention (CDC) and National Institutes of Health (NIH), and is available online at
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm (see Internet Resources for other
online versions). Readers are strongly urged to review this publication prior to initiating
any experiment outlined in this manual.
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1A.1.3
Current Protocols in Microbiology
Table 1A.1.1 CDC Summary of Recommended Biosafety Levels for Infectious Agentsa, b
Biosafety
level
Agent characteristics
BSL-1
Not known to
Standard microbiological None required
consistently cause disease practices
in healthy adults
BSL-2
BSL-3
Indigenous or exotic
agents with potential for
aerosol transmission;
disease may have serious
or lethal consequences
Dangerous/exotic agents
which pose high risk of
life-threatening disease,
aerosol-transmitted lab
infections; or related
agents with unknown risk
of transmission
BSL-4
Practices
Decontamination of all
waste
Decontamination of lab
clothing before
laundering
Baseline serum
Safety equipment
(primary barriers)c
Facilities (secondary
barriers)
Open bench-top sink
Class I or II BSCs or
other physical
containment devices used
for all open
manipulations of agents
Physical separation
from access corridors
Self-closing,
Protective lab clothing
double-door access
and gloves
Exhausted air not
Respiratory protection as
recirculated
needed
Negative airflow into
laboratory
a Adapted from Biosafety in Microbiological and Biomedical Laboratories, 4th Ed. (GPO S/N 017-040-00547-4), available online at
required practices, equipment, and/or facilities described for the BSL in question.
c See http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm for more information concerning biological safety cabinets (BSCs).
Biosafety
educational training and teaching laboratories, and for other laboratories in which work
is done with defined and characterized strains of viable microorganisms not known
to consistently cause disease in healthy adult humans. Bacillus subtilis, Naegleria
gruberi, infectious canine hepatitis virus, Escherichia coli K-12 (see BMBL for
restrictions), and exempt organisms under the NIH Recombinant DNA Guidelines
(http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html) are representative of microorganisms meeting these criteria. Many agents not ordinarily associated with disease
processes in humans are, however, opportunistic pathogens and may cause infection in
1A.1.4
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Technologies
1A.1.5
Current Protocols in Microbiology
The primary hazards to personnel working with Biosafety Level 4 agents are respiratory
exposure to infectious aerosols, mucous membrane or broken skin exposure to infectious
1A.1.6
Current Protocols in Microbiology
ANIMAL FACILITIES
The CDC defines four biosafety levels for activities involving infectious disease work
with experimental animals. These combinations of practices, safety equipment, and
facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing
levels of protection to personnel and the environment.
In this manual, when these conditions are necessary, a note is provided in the unit or
protocol introduction with the following format, where x is the appropriate ABSL.
CAUTION: Protocols using live animals must first be reviewed and approved by
an Institutional Animal Care and Use Committee (IACUC) or must conform to
governmental regulations regarding the care and use of laboratory animals. This
experiment requires Animal Biosafety Level x (ABSL-x) conditions. Follow all
appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1 and
other pertinent resources (APPENDIX 1B) for more information.
For more information, refer to the Section IV of the BMBL, 4th Ed., available online at
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s4.htm.
CLINICAL LABORATORIES
Clinical laboratories, especially those in health care facilities, receive clinical specimens
with requests for a variety of diagnostic and clinical support services. Typically, the
infectious nature of clinical material is unknown, and specimens are often submitted
with a broad request for microbiological examination for multiple agents (e.g., sputa
submitted for "routine," acid-fast, and fungal cultures). It is the responsibility of the
laboratory director to establish standard procedures in the laboratory, which realistically
addresses the issue of the infective hazard of clinical specimens.
Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial
processing of clinical specimens and serological identification of isolates can be done
safely at Biosafety Level 2 (see above), the recommended level for work with bloodborne
pathogens such as hepatitis B virus and HIV. The containment elements described in
BSL-2 are consistent with the OSHA standard, Occupational Exposure to Bloodborne
Pathogens (Richmond, 1994) from the Occupational Safety and Health Administration
(OSHA; see Internet Resources). This requires the use of specific precautions with all
clinical specimens of blood or other potentially infectious material (Universal or Standard
Precautions; MMWR, 1988). Additionally, other recommendations specific for clinical
laboratories may be obtained from the U.S. National Committee for Clinical Laboratory
Standards (NCCLS, 1977).
Biosafety Level 2 recommendations and OSHA requirements focus on the prevention
of percutaneous and mucous membrane exposures to clinical material. Primary barriers
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Current Protocols in Microbiology
Literature Cited
Morbidity and Mortality Weekly Report (MMWR),
Centers for Disease Control 1988. Update: Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus and other bloodborne pathogens in
healthcare settings. MMWR 37:377-388.
National Committee for Clinical Laboratory Standards (NCCLS) 1997. Protection of Laboratory
Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. Approved Guideline, Dec. 1977,
NCCLS Doc. M29-A. NCCLS, Wayne, Penn.
Occupational Safety and Health Administration
(OSHA), U.S. Department of Labor 1991. Occupational exposure to bloodborne pathogens,
final rule. In Federal Register Volume 56,
pp. 64175-64182. U.S. Government Printing Office, Washington D.C.
Richmond, J.Y. 1994. HIV Biosafety: Guidelines
and Regulations. In AIDS Testing, Edition 2 (G.
Schochetman and J. R. George, eds.) pp. 346360. Springer-Verlag, New York.
Key References
U.S. Department of Health and Human Services,
Public Health Service, Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH) 1999. Biosafety in
Microbiological and Biomedical Laboratories
(BMBL), 4th Ed. 1999. (J.Y. Richmond and
R.W. McKenney, eds.) U.S. Government Printing Office, Washington D.C. (GPO S/N 017040-00547-4).
Fuscaldo, A.A., Erlick, B.J., and Hindman, B.
(eds.) 1980. Laboratory Safety, Theory and
Practice. Academic Press, San Diego.
Internet Resources
http://www.OSHA.gov
OSHA web site.
http://tis.eh.doe.gov/docs/osh tr/ch5.html
DOE OSH technical reference chapter on personal
protective equipment.
http://www.cdc.gov/od/ohs/biosfty/bmbl4/
bmbl4toc.htm
Online version of the Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 4th Ed. See
Key References for print version information.
http://bmbl.od.nih.gov
BMBL version with longer than average web pages
and minimal graphics for ease of printing.
http://www.cdc.gov/od/ohs/pdffiles/
4th%20BMBL.pdf
PDF version of the BMBL. See above for other
versions.
http://www.cdc.gov/od/ohs/biosfty/bmbl4/
bmbl4s6.htm
Provides information on recommended biosafety
levels for infectious agents and infected animals.
http://www.absa.org/resguides.html
The American Biological Safety Association
(ABSA) Biosafety Guidelines website. This page
provides links to international biosafety guidelines
and websites.
http://www.ebsa.be
The European Biosafety Association homepage. In
addition to being a source of information in and of
itself, it is also host to the International Biosafety
Working Group (IBWG), a compendium of international regulations and guidelines with descriptions and URLs. (Access by clicking International
Biosafety from the menu bar on the top of the
screen.)
http://www.cdc.gov
The Centers for Disease Control and Prevention
website.
George Lunn
Baltimore, Maryland
Biosafety
1A.1.8
Current Protocols in Microbiology
UNIT 1A.2
RISK IDENTIFICATION
To most effectively determine appropriate
biosecurity measures, an inventory of assets
is warranted. In other words, laboratory directors should ask themselves and their research staff, What needs protection from theft
or removal? The scenarios presented in this
unit are most specifically directed towards protection of potentially dangerous microorganisms and toxins, but research activities also
involve many other assets such as research
equipment (e.g., analytical balances, computer
equipment), proprietary materials, chemicals,
radioisotopes, research animals, experimental
data, and more. Stolen research materials, at
the very least, are likely to represent a loss of
time and intellectual capital.
RISK ASSESSMENT
While a biosafety risk assessment is based
on identifying the potential for the microbe
to cause infection and/or illness during routine laboratory use, a biosecurity risk assessment involves evaluating the same organism
for its potential for intended removal and malicious use, and the consequences of such use.
In addition, an assessment of laboratory activities (e.g., multiple transfers) or an enhanced
threat environment (e.g., suspicious activities)
are factors in completing a biosecurity risk assessment.
Many dangerous pathogens may present
limited biosecurity risk because they are difficult to grow or to disseminate in the form
of an active bioweapon. Likewise, other less
virulent, easily contained microbes may be of
a lower safety concern but may represent a
desirable tool for bioterrorism.
A risk assessment for malicious use of biological agents includes consideration of factors
that influence both the potential for an organism to be used in a threatening manner (threat
potential) and the consequences that might result from the intentional use of the agent to
inflict harm (Fig. 1A.2.1). Factors influencing
the risk for malicious use have been described
by Gaudioso et al. (2005).
Determining the consequences of the release of the agent can include the following:
Emerging
Technologies
1A.2.1
Supplement 3
Figure 1A.2.1 Graphic guideline for determining the risk for malicious use of a biological agent.
Based on a graphic developed by Sandia Laboratories and used with their kind permission.
Biosafety
Practices
Associated with
Potential Agents
of Biocrime and
Biowarfare
RISK MANAGEMENT
Both the World Health Organization
(WHO, 2004) and the U.S. Centers for Disease Control and Prevention (CDC/NIH, 1999;
MMWR, 2002) have recently included biosecurity discussions as part of commonly used
biosafety guidelines. As described by these
discussions and elaborated by Sandia National Laboratories (2005), the key components to managing biosecurity risks are
(1) physical security measures, (2) personnel security/reliability, (3) materials control
and accountability, (4) transfer security, and
(5) information security. All of these components are overlapping and interactive in nature
(Fig. 1A.2.2). Of particular importance to individuals in microbiology laboratories are physical security, personnel reliability, and materials control and accountability.
Physical Security
Physical security is intended to deter and
detect unauthorized access to research areas
containing materials of concern. Physical security may be as simple as keeping doors
closed at all times and locking the doors when
1A.2.2
Supplement 3
Personnel Reliability
Physical security may deter an outsider, but
will present no barrier to an insider with full
access. Personnel reliability is critical in limiting concerns for insider theft. Clearly not
all positions within an institution require the
same level of scrutinythe determination of
Emerging
Technologies
1A.2.3
Current Protocols in Microbiology
Supplement 3
(3) retrieval of security badges, keys, electronic equipment, etc.; and (4) deactivation of
computers and electronic access accounts.
Very few, if any, research operations function without requiring access for some sort of
visitor. There are several factors to consider
with regard to visitors. Determine the different categories of visitorssome visitors are
repair or sales persons who provide critical
expertise and products for a laboratory, some
are observers, and some are working visitors
with various lengths of stay. Also, host and
escorting procedures and assignments must be
documented and followed.
Materials
It is impossible to describe microorganisms
in an absolute number because amplification
and inactivation are so easily accomplished. A
general accounting of the number of containers is more reasonable in this case. A basic
inventory is required for the organisms held
in repositories, working stocks, master stocks,
clinical specimens, genetic material, as well as
contaminated materials, such as waste.
Accountability
Control
Controls are developed to assure that materials stay, or are transported, as intended
Biosafety
Practices
Associated with
Potential Agents
of Biocrime and
Biowarfare
While it is important to inventory and provide control measures for materials of concern, it is imperative that the responsibility for
Figure 1A.2.3 An example of a simple log sheet that may be used to record access to materials
of concern in the laboratory.
1A.2.4
Supplement 3
acknowledge that to be truly effective, any decision on how to manage dual-use research
must be made and implemented internationally.
DUAL-USE RESEARCH
LITERATURE CITED
Gaudioso, J., Salerno, R., and Barnett, N. 2005.
Principles of Laboratory Biosecurity presented
at the American Biological Safety Association
(ABSA) Summer Seminar Series. Presentation
posted at http://www.biosecurity.sandia.gov.
Morbidity and Mortality Weekly Report (MMWR),
Centers for Disease Control and Prevention,
2002. Laboratory Security and Emergency Response Guidance for Laboratories Working with
Select Agents. MMWR 51 No. RR-19.
National Research Council. 2004. Biotechnology
Research in an Age of Terrorism. National
Academies Press, Washington, D.C.
Sandia National Laboratories. 2005. Laboratory
Biosecurity Implementation Guidelines. Sandia
Report SAND2005-248P, Albuquerque, N.
Mex.
U.S. Department of Health and Human Services,
Public Health Service. Centers for Disease
Control and Prevention (CDC) and National
Institutes of Health (NIH). 1999. Biosafety in
Microbiological and Biomedical Laboratories
(BMBL), 4th Ed (J.Y. Richmond and R.W.
McKenney, eds.) U.S. Government Printing
Office, Washington, D.C.
INTERNET RESOURCES
http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl04/index.html
World Health Organization Laboratory Biosafety
Manual, 3rd edition, 2004
http://www.biosecurity.sandia.gov
Resources collected and published by the Biosecurity Team at Sandia National Laboratories.
http://www.biosecurityboard.gov
Website of the U.S. National Science Advisory
Board on Biosecurity.
Emerging
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1A.2.5
Current Protocols in Microbiology
Supplement 3
UNIT 1A.3
Persons carrying out the protocols in this manual may encounter toxic chemicals, including carcinogenic, mutagenic, or teratogenic reagents (see Table 1A.3.1). Most governments regulate the use of these materials; it is essential that they be used in strict
accordance with local and national regulations. Cautionary notes and some specific
guidelines are included in many instances throughout the book; however, users must proceed with the prudence and precautions associated with good laboratory practice, under
the supervision of those responsible for implementing laboratory safety programs at their
institutions. Guidelines for the safe use of radioisotopes are presented in APPENDIX 1C.
It is not possible in the space available to list all the precautions required for handling
hazardous chemicals. Many texts have been written about laboratory safety (see Literature
Cited and Key References). Obviously, all national and local laws should be obeyed, as
well as all institutional regulations. Controlled substances are regulated by the Drug
Enforcement Administration (http://www.usdoj.gov/dea). By law, Material Safety Data
Sheets (MSDSs) must be readily available. All laboratories should have a Chemical
Hygiene Plan (29 CFR Part 1910.1450); institutional safety officers should be consulted
as to its implementation. Help is (or should be) available from your institutional Safety
Office: use it.
Chemicals must be stored properly for safety. Certain chemicals cannot be easily or safely
mixed with and should not be stored near certain other chemicals, because their reaction
is violently exothermic or yields a toxic product. Some examples of incompatibility are
listed in Table 1A.3.2. When in doubt, always consult a current MSDS for information
on reactivity, handling, and storage. Chemicals should be separated into general hazard
classes and stored appropriately. For example, flammable chemicals such as alcohols,
ketones, aliphatic and aromatic hydrocarbons, and other materials labeled flammable
should be stored in approved flammable storage cabinets, with those also requiring
refrigeration being kept in explosion-proof refrigerators. Strong oxidizers must be segregated. Strong acids (e.g., sulfuric, hydrochloric, nitric, perchloric, and hydrofluoric)
should be stored in a separate cabinet well removed from strong bases and from flammable
organics. An exception is glacial acetic acid, which is both corrosive and flammable, and
which must be stored with the flammables.
Facilities should be appropriate for working with hazardous chemicals. In particular,
hazardous chemicals should be handled only in chemical fume hoods, not in laminar
flow cabinets. The functioning of the fume hoods should be checked periodically. Laboratories should also be equipped with safety showers and eye-wash facilities. Again, this
equipment should be tested periodically to ensure that it functions correctly. Other safety
equipment may be required depending on the nature of the materials being handled. In
addition, researchers should be trained in the proper procedures for handling hazardous
chemicals as well as other laboratory operationse.g., handling of compressed gases,
use of cryogenic liquids, operation of high-voltage power supplies, and operation of
lasers of all types.
Before starting work, know the physical and chemical hazards of the reagents used.
Wear appropriate protective clothing and have a plan for dealing with spills or
accidents; coming up with a good plan on the spur of the moment is very difficult. For
example, have the appropriate decontaminating or neutralizing agents prepared and close
at hand. Small spills can probably be cleaned by the researcher. In the case of larger spills,
Emerging
Technologies
Contributed by George Lunn and Gretchen Lawler
Current Protocols in Microbiology (2005) 1A.3.1-1A.3.36
C 2005 by John Wiley & Sons, Inc.
Copyright
1A.3.1
Chemical
Hazards
Remarks
Enzyme inhibitor
Carcinogenic
Mutagenic, enzyme inhibitor
Safe Use of
Hazardous
Chemicals
1A.3.2
Current Protocols in Microbiology
Chemical
Hazards
Teratogenic, toxic
Mutagenic
Carcinogenic
Toxic
Diethylamine (DEA)
Diethylpyrocarbonate (DEPC)
Diethyl sulfate
Diisopropyl fluorophosphate (DFP)
Dimethyl sulfate (DMS)
Dimethyl sulfoxide (DMSO)
Diphenylamine (DPA)
2,5-Diphenyloxazole (PPO)
Dithiothreitol (DTT)
Eosin B
Erythrosin B
Ether
Carcinogenic, mutagenic
Flammable liquid, toxic
Formamide
Formic acid
Mutagenic, toxic
Carcinogenic, toxic
Carcinogenic, toxic
Teratogenic, toxic
Toxic
Carcinogenic, flammable liquid,
teratogenic, toxic
Teratogenic, toxic
Corrosive, toxic
Glutaraldehyde
Guanidinium thiocyanate
Hoechst 33258 dye
Hydrochloric acid, concentrated
Remarks
continued
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1A.3.3
Current Protocols in Microbiology
Chemical
Hazards
Remarks
Hydroxylamine
Propane sultone
Propidium iodide (PI)
Teratogenic, toxic
Mutagenic
Safe Use of
Hazardous
Chemicals
1A.3.4
Current Protocols in Microbiology
Chemical
Hazards
Pyridine
Rhodamine and derivatives
Rose Bengal
Safranine O
Sodium azide
Sodium nitrite
Sulfuric acid, concentrated
Carcinogenic
Corrosive, oxidizer, teratogenic,
toxic
SYTO dyes
Tetramethylammonium chloride (TMAC)
N,N,N ,N -Tetramethyl-ethylenediamine
(TEMED)
Texas Red (sulforhodamine 101, acid
chloride)
Toluene
Toluidine blue O
N-p-Tosyl-L-lysine chloromethyl ketone
(TLCK)
N-p-Tosyl-L-phenylalanine chloromethyl
ketone (TPCK)
Trichloroacetic acid (TCA)
Toxic
Toxic
Corrosive, flammable liquid, toxic
Corrosive, toxic
Remarks
Toxic
Flammable liquid, teratogenic, toxic
Mutagenic, toxic
See Basic Protocol 2
Toxic, enzyme inhibitor
See Basic Protocol 11
Toxic, mutagenic, enzyme inhibitor
a CAUTION: These chemicals should be handled only in a chemical fume hood by knowledgeable workers equipped with eye protection, lab coat, and
gloves. The laboratory should be equipped with a safety shower and eye wash. Additional protective equipment may be required.
b For extensive information on the hazards of these and other chemicals as well as cautionary details, see Bretherick (1990), Lunn and Sansone (1994a),
Bretherick et al. (1999), Lewis (1999), Furr (2000), and ONeil (2001).
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1A.3.5
Current Protocols in Microbiology
Chemical
Incompatible with
Acetic acid
Acetone
Acetylene
Alkali metals, alkaline earth
metals
Ammonia (anhydrous)
Ammonium nitrate
Aniline
Arsenical materials
Azides
Bromine
Calcium oxide
Carbon (activated)
Carbon tetrachloride
Chlorates
Chlorine
Chlorine dioxide
Copper
Cumene hydroperoxide
Cyanides
Flammable liquids
Safe Use of
Hazardous
Chemicals
continued
1A.3.6
Current Protocols in Microbiology
Chemical
Incompatible with
Fluorine
Hydrocarbons (liquid or gas)
Hydrocyanic acid
Hydrofluoric acid
Hydrogen peroxide
Hydrogen sulfide
Hydroperoxide
Hypochlorites
Iodine
Mercury
Nitric acid
Nitrites
Nitroparaffins
Oxalic acid
Oxygen
Perchloric acid
Peroxides, organic
Phosphorus (white)
Potassium chlorate
Potassium perchlorate
Potassium permanganate
Selenides and tellurides
Silver
Sodium
Sodium nitrate
Sodium peroxide
Sulfides
Sulfuric acid
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1A.3.7
Current Protocols in Microbiology
the area should be evacuated and help should be sought from those experienced in and
equipped for dealing with spillse.g., the institutional Safety Office.
Protective equipment should include, at a minimum, eye protection, a lab coat, and
gloves. In certain circumstances, other items of protective equipment may be necessary
(e.g., a face shield). Different types of gloves exhibit different resistance properties (Table
1A.3.3). No gloves resist all chemicals, and no gloves resist any chemicals indefinitely.
Disposable gloves labeled exam or examination are primarily for protection from
biological materials (e.g., viruses, bacteria, feces, blood). They are not designed for and
usually have not been tested for resistance to chemicals. Disposable gloves generally
offer extremely marginal protection from chemical hazards in most cases and should
be removed immediately upon contamination before the chemical can pass through. If
possible, design handling procedures to eliminate or reduce potential for contamination.
Never assume that disposable gloves will offer the same protection or even have the
same properties as nondisposables. Select gloves carefully and always look for some
evidence that they will protect against the materials being used. Inspect all gloves before
every use for possible holes, tears, or weak areas. Never reuse disposable gloves. Clean
reusable gloves after each use and dry carefully inside and out. Observe all commonsense precautionse.g., do not pipet by mouth, keep unauthorized persons away from
hazardous chemicals, do not eat or drink in the laboratory, wear proper clothing in the
laboratory (sandals, open-toed shoes, and shorts are not appropriate).
Order hazardous chemicals only in quantities that are likely to be used in a reasonable
time. Buying large quantities at a lower unit cost is no bargain if someone (perhaps you)
has to pay to dispose of surplus quantities. Substitute alcohol-filled thermometers for
mercury-filled thermometers, which are a hazardous chemical spill waiting to happen.
Although any number of chemicals commonly used in laboratories are toxic if used
improperly, the toxic properties of a number of reagents require special mention. Chemicals that exhibit carcinogenic, corrosive, flammable, lachrymatory, mutagenic, oxidizing,
teratogenic, toxic, or other hazardous properties are listed in Table 1A.3.1. Chemicals
listed as carcinogenic range from those accepted by expert review groups as causing
cancer in humans to those for which only minimal evidence of carcinogenicity exists.
No effort has been made to differentiate the carcinogenic potential of the compounds in
Table 1A.3.1. Oxidizers may react violently with oxidizable material (e.g., hydrocarbons,
wood, and cellulose). Before using any of these chemicals, thoroughly investigate all its
characteristics. Material Safety Data Sheets are readily available; they list some hazards
but vary widely in quality. A number of texts describing hazardous properties are listed
at the end of this unit (see Literature Cited). In particular, Saxs Dangerous Properties
of Industrial Materials, 11th ed. (Lewis, 2004), and the Handbook of Reactive Chemical
Hazards, 6th ed. (Bretherick et al., 1999), give comprehensive listings of known hazardous properties; however, these texts list only the known properties. Many chemicals,
especially fluorochromes, have been tested only partially or not at all. Prudence dictates
that, unless there is good reason for believing otherwise, all chemicals should be regarded
as volatile, highly toxic, flammable human carcinogens and should be handled with great
care.
Safe Use of
Hazardous
Chemicals
1A.3.8
Current Protocols in Microbiology
Chemical
Acetaldehyde
Acetic acid
Acetone
Ammonium hydroxide
Amyl acetate
Neoprene gloves
Nitrile gloves
VG
VG
VG
VG
VG
VG
VG
VG
VG
VG
VG
VG
Aniline
Benzaldehyde
Benzene
Butyl acetate
Butyl alcohol
VG
VG
VG
VG
Carbon disulfide
Carbon tetrachloride
Chlorobenzene
Chloroform
Chloronaphthalene
Cyclohexanol
VG
Dibutyl phthalate
Diisobutyl ketone
Dimethylformamide
Dioctyl phthalate
VG
VG
VG
VG
VG
Ethyl acetate
Ethyl alcohol
VG
VG
VG
VG
Ethyl ether
VG
VG
Ethylene dichloride
Ethylene glycol
VG
VG
VG
VG
Formaldehyde
VG
VG
VG
VG
Formic acid
VG
VG
VG
VG
Glycerin
VG
VG
VG
VG
Hexane
Hydrochloric acid
VG
VG
Ketones
VG
VG
VG
VG
VG
VG
Linseed oil
VG
VG
Furfural
continued
Emerging
Technologies
1A.3.9
Current Protocols in Microbiology
Chemical
Methyl alcohol
Neoprene gloves
Nitrile gloves
VG
VG
VG
VG
Methylamine
Methyl bromide
VG
Methyl isobutylketone
VG
Methyl methacrylate
VG
Monoethanolamine
VG
VG
VG
Morpholine
VG
VG
VG
Naphthalene
Naphthas, aliphatic
VG
VG
Naphthas, aromatic
Nitropropane (95.5%)
Oleic acid
VG
VG
Oxalic acid
VG
VG
VG
VG
Palmitic acid
VG
VG
VG
VG
VG
Phenol
VG
Phosphoric acid
VG
VG
VG
Potassium hydroxide
VG
VG
VG
VG
i-Propyl alcohol
VG
VG
VG
VG
n-Propyl alcohol
VG
VG
VG
VG
Sodium hydroxide
VG
VG
VG
VG
Styrene (100%)
Sulfuric acid
Tetrahydrofuran
Toluene diisocyanate
Triethanolamine
VG
VG
Tung oil
VG
VG
Turpentine
VG
Nitric acid
Perchloroethylene
Propyl acetate
Toluene
Trichloroethylene
Xylene
a Performance varies with glove thickness and duration of contact. An asterisk indicates limited use. Abbreviations: VG,
Safe Use of
Hazardous
Chemicals
b Adapted from the July 8, 1998, version of the DOE OSH Technical Reference Chapter 5 (APPENDIX C at
http://eh.doe.gov/docs/osh tr/ch5c.html). For more information, also see Forsberg and Keith (1999) and Forsberg and
Mansdort (2003).
1A.3.10
Current Protocols in Microbiology
Protocol
Method
Basic Protocol 1
Alternate Protocol 1
Alternate Protocol 2
Support Protocol 1
Basic Protocol 2
Alternate Protocol 3
Support Protocol 2
Basic Protocol 3
Basic Protocol 4
Support Protocol 3
Basic Protocol 5
Support Protocol 4
Basic Protocol 6
Alternate Protocol 4
Alternate Protocol 5
Alternate Protocol 6
Support Protocol 5
Basic Protocol 7
Basic Protocol 8
Disposal of Iodine
Basic Protocol 9
Alternate Protocol 7
Support Protocol 6
Basic Protocol 10
Support Protocol 7
Support Protocol 8
Basic Protocol 11
Support Protocol 9
Basic Protocol 12
Alternate Protocol 8
Alternate Protocol 9
Support Protocol 10
Emerging
Technologies
1A.3.11
DISPOSAL METHODS
A number of procedures for the disposal of hazardous chemicals are available; protocols for the disposal and decontamination of some hazardous chemicals commonly
encountered in microbiology laboratories are listed in Table 1A.3.4. These procedures
are necessarily brief; for full details consult the original references or a collection of
these procedures (see Lunn and Sansone, 1994a).
CAUTION: These disposal methods should be carried out only in a chemical fume hood
by workers equipped with eye protection, a lab coat, and gloves. Additional protective
equipment may be necessary.
BASIC
PROTOCOL 1
Materials
Benzidine or diaminobenzidine tetrahydrochloride dihydrate
0.1 M HCl (for benzidine)
0.2 M potassium permanganate: prepare immediately before use
2 M sulfuric acid
Sodium metabisulfite
10 M potassium hydroxide (KOH)
Additional reagents and equipment for testing for the presence of aromatic amines
(see Support Protocol 1)
1. For each 9 mg benzidine, add 10 ml of 0.1 M HCl or for each 9 mg diaminobenzidine
tetrahydrochloride dihydrate, add 10 ml water. Stir the solution until the aromatic
amine has completely dissolved.
2. For each 10 ml of solution, add 5 ml freshly prepared 0.2 M potassium permanganate
and 5 ml of 2 M sulfuric acid. Allow the mixture to stand for 10 hr.
3. Add sodium metabisulfite until the solution is decolorized.
4. Add 10 M KOH to make the solution strongly basic, pH >12.
CAUTION: This reaction is exothermic.
5. Dilute with 5 vol water and pass through filter paper to remove manganese compounds.
Safe Use of
Hazardous
Chemicals
6. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzidine; see Support Protocol 1).
7. Neutralize the filtrate with acid and discard.
1A.3.12
Current Protocols in Microbiology
ALTERNATE
PROTOCOL 1
6. Dilute with 5 vol water and filter through filter paper to remove manganese compounds.
7. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzidine; see Support Protocol 1).
8. Neutralize the filtrate with acid and discard it.
9. Verify complete decontamination by wiping the surface with a paper towel moistened
with water and squeezing the liquid out of the towel. Test the liquid for the presence
of benzidine or diaminobenzidine (see Support Protocol 1). Repeat steps 1 to 9 as
necessary.
ALTERNATE
PROTOCOL 2
The enzyme horseradish peroxidase catalyzes the oxidation of the amine to a radical
which diffuses into solution and polymerizes. The polymers are insoluble and fall out of
solution.
Emerging
Technologies
1A.3.13
Current Protocols in Microbiology
Additional reagents and equipment for testing for the presence of aromatic amines
(see Support Protocol 1)
1. Adjust the pH of the aqueous benzidine or diaminobenzidine solution to 5 to 7 with
1 N HCl or NaOH as required, and dilute so the concentration of aromatic amines is
100 mg/liter.
2. For each liter of solution, add 3 ml of 3% hydrogen peroxide and 300 U horseradish
peroxidase. Let the mixture stand 3 hr.
3. Remove the precipitate by filtering the solution through a porous glass filter or
by centrifuging 5 min at room temperature in a benchtop centrifuge to pellet the
precipitate.
The precipitate is mutagenic and should be treated as hazardous waste.
4. Immerse the porous glass filter in 1:1 (v/v) 0.2 M potassium permanganate/2 M
sulfuric acid. Clean the filter in a conventional fashion and discard potassium permanganate/sulfuric acid solution as described for benzidine and diaminobenzidine
(see Basic Protocol 1).
5. For each liter of filtrate, add 100 ml of 5% ascorbic acid.
6. Test the filtrate for the presence of aromatic amines (see Support Protocol 1).
7. Discard the decontaminated filtrate.
SUPPORT
PROTOCOL 1
Materials
Decontaminated aromatic amine solution
10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer (1.5
mM K2 HPO4 /1.5 mM KH2 PO4 ) (benzidine) or 75:25 (v/v) methanol/1.5 mM
potassium phosphate buffer (diaminobenzidine)
250-mm 4.6-mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or
equivalent
Additional reagents and equipment for reversed-phase liquid chromatography
(Snyder et al., 1997)
Analyze the decontaminated aromatic amine solution by reversed-phase HPLC using a
250-mm 4.6-mm-i.d. Microsorb C-8 column or equivalent (Snyder et al., 1997). To
detect benzidine, elute with 10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium
phosphate buffer at a flow rate of 1.5 ml/min and UV detection at 285 nm. To detect
diaminobenzidine, elute with 75:25 (v/v) methanol/1.5 mM potassium phosphate buffer
at a flow rate of 1 ml/min and UV detection at 300 nm.
BASIC
PROTOCOL 2
Safe Use of
Hazardous
Chemicals
1A.3.14
Current Protocols in Microbiology
Acridine orange
18 hr
20
10 min
500
18 hr
Azure A
10 min
80
Azure B
10 min
80
Brilliant blue R
2 hr
80
Congo red
2 hr
40
2 hr
80
2 hr
40
Crystal violet
30 min
200
Eosin B
30 min
40
Erythrosin B
18 hr
10
Ethidium bromide
4 hr
20
Janus green B
30 min
80
Methylene blue
30 min
80
Neutral red
10 min
500
Nigrosin
2 hr
80
Orcein
2 hr
200
Propidium iodide
2 hr
20
Rose Bengal
3 hr
20
Safranine O
1 hr
20
30 min
80
2 hr
40
Compound
Alizarin red S
Toluidine blue O
Trypan blue
limit of detection (see Support Protocol 2 and Table 1A.3.5). In each case, decontamination should be >99%. This protocol describes a method for batch decontamination in
which the resin is stirred in the solution to be decontaminated and removed by filtration
at the end of the reaction time. Large volumes of biological stain can be decontaminated
using a column (see Alternate Protocol 3). For full details refer to the original literature
(Lunn and Sansone, 1991b) or a compilation (Lunn and Sansone, 1994a).
Materials
Amberlite XAD-16 resin (Supelco)
100 g/ml biological stain in water
Additional reagents and equipment for testing for the presence of biological stain
(see Support Protocol 2)
For batch decontamination of 20 ml stain
1a. Add 1 g Amberlite XAD-16 to 20 ml of 100 g/ml biological stain in water.
For aqueous solutions having stain concentrations other than 100 g/ml, use proportionately greater or lesser amounts of resin to achieve complete decontamination.
For solutions of erythrosin B, use 2 g Amberlite XAD-16 for 20 ml stain.
2a. Stir the mixture for at least the time indicated in Table 1A.3.5.
Emerging
Technologies
1A.3.15
Current Protocols in Microbiology
3. Pour the resin slurry into the column through a funnel. As the resin settles, tap the
column with a rubber stopper fitted over a pencil to encourage even packing. Attach
a QG 20 lab pump.
1A.3.16
Current Protocols in Microbiology
Compound
1 ppm
5 ppm
Acridine orange
465
>990
>990
Alizarin red S
120
150
240
Azure A
615
810
>975
Azure B
630
882
>1209
706
>1396
>1396
Crystal violet
1020
>1630
>1630
Ethidium bromide
260
312
416
Janus green B
170
650
>870
Methylene blue
420
645
1050
Neutral red
>2480
>2480
>2480
Safranine O
365
438
584
Toluidine blue O
353
494
606
4. Pump the 25-g/ml biological stain solution through the column at 2 ml/min.
Alternatively, gravity flow coupled with periodic adjustment of the flow-regulating valve
can be used.
5. Check the effluent from the column for the presence of biological stain (see Support
Protocol 2). Stop the pump when stain is detected.
Table 1A.3.6 lists breakthrough volumes at different detection levels for a number of
biological stains.
SUPPORT
PROTOCOL 2
Materials
Filtrate or eluate from biological stain decontamination (see Basic Protocol 2 or
Alternate Protocol 3)
DNA solution: 20 g/ml calf thymus DNA in TBE electrophoresis buffer, pH 8.1
(APPENDIX 2A)
1 M KOH solution
pH 5 buffer (see recipe)
Test the filtrate or eluate from the biological stain decontamination procedure using the
appropriate method as indicated in Table 1A.3.7.
Traces of acid or base on the resin may induce color changes in the stain. Accordingly, with cresyl
violet acetate or neutral red, mix aliquots of the filtrate with 1 vol pH 5 buffer before analyzing.
With alizarin red S and orcein, mix aliquots of the filtrate with 1 vol of 1 M KOH solution before
analyzing.
Emerging
Technologies
1A.3.17
Current Protocols in Microbiology
Compound
Reagentb
Procedure
Wavelength(s)
(nm)
Limit of detection
(ppm)
Acridine orange
DNA
solution
ex 492, em 528
0.0032
615
0.9
556
0.46
Azure A
633
0.15
Azure B
648
0.13
Brilliant blue R
585
1.0
Congo red
497
0.25
Coomassie brilliant
blue G
610
1.7
ex 588, em 618
0.021
Crystal violet
588
0.1
Eosin B
514
0.21
Erythrosin B
ex 488, em 556
0.025
ex 540, em 590
0.05
Janus green B
660
0.6
Methylene blue
661
0.13
540
0.6
570
0.8
Ethidium bromide
Neutral red
1 M KOH
pH 5 buffer
DNA
solution
pH 5 buffer
Nigrosin
Orcein
1 M KOH
579
1.15
Propidium iodide
DNA
solution
ex 350, em 600
0.1
Rose Bengal
ex 520, em 576
0.04
Safranine O
ex 460, em 582
0.03
Toluidine blue O
626
0.2
Trypan blue
607
0.22
Increase the fluorescence of solutions of acridine orange, ethidium bromide, and propidium iodide
by mixing an aliquot of the filtrate with an equal volume of DNA solution. Let the solution stand
15 min before measuring the fluorescence.
BASIC
PROTOCOL 3
Safe Use of
Hazardous
Chemicals
2. Remove any insoluble material by filtration and discard it with the solid or liquid
hazardous waste.
3. Neutralize the aqueous layer with base and discard it.
1A.3.18
Current Protocols in Microbiology
BASIC
PROTOCOL 4
Materials
Cyanide (e.g., NaCN) or cyanogen bromide (CNBr)
1 M NaOH
5.25% (v/v) sodium hypochlorite (NaOCl; i.e., standard household bleach)
Additional reagents and equipment for testing for the presence of cyanide (see
Support Protocol 3)
1. Dissolve cyanide in water to give a concentration 25 mg/ml or dissolve CNBr in
water to give a concentration 60 mg/ml.
If necessary, dilute aqueous solutions so the concentration of NaCN or CNBr does not
exceed the limit.
2. Mix 1 vol NaCN or CNBr solution with 1 vol 1 M NaOH and 2 vol fresh 5.25%
NaOCl. Stir the mixture 3 hr.
IMPORTANT NOTE: With age, bleach may become ineffective; use of fresh bleach is
strongly recommended.
3. Test the reaction mixture for the presence of cyanide (see Support Protocol 3).
4. Neutralize the reaction mixture and discard it.
SUPPORT
PROTOCOL 3
Materials
Cyanide or cyanogen bromide decontamination reaction mixture (see Basic
Protocol 4)
Phosphate buffer (see recipe)
10 mg/ml sodium ascorbate in water: prepare fresh daily
100 mg/liter NaCN in water: prepare fresh weekly
10 mg/ml chloramine-T in water: prepare fresh daily
Cyanide detection reagent (see recipe)
Sorvall GLC-1 centrifuge or equivalent
1. If necessary to remove suspended solids, centrifuge two 1-ml aliquots of the cyanide
or cyanogen bromide decontamination reaction mixture 5 min in a benchtop centrifuge, room temperature. Add each supernatant to 4 ml phosphate buffer in separate
tubes.
2. If an orange or yellow color appears, add 10 mg/ml freshly prepared sodium ascorbate
dropwise until the mixture is colorless, but do not add more than 2 ml.
3. Add 200 l of 100 mg/liter NaCN to one reaction mixture (control solution).
4. Add 1 ml freshly prepared 10 mg/ml chloramine-T to each tube. Shake the tubes and
let them stand 1 to 2 min.
5. Add 1 ml cyanide detection reagent, shake, and let stand 5 min.
Emerging
Technologies
1A.3.19
Current Protocols in Microbiology
A blue color indicates the presence of cyanide. If destruction has been complete and the
analytical procedure has been carried out correctly, the treated reaction mixture should
be colorless and the control solution, which contains NaCN, should be blue.
6. If necessary, remove suspended solids by centrifuging tubes 5 min at room temperature. Measure the absorbance at 605 nm with appropriate standards and blanks.
BASIC
PROTOCOL 5
Materials
Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl
methanesulfonate, diepoxybutane, or 1,3-propane sultone
5 M NaOH
Acid
Additional reagents and equipment for testing for the presence of dimethyl sulfate,
diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate,
diepoxybutane, or 1,3-propane sultone (see Support Protocol 4)
For bulk quantities of dimethyl sulfate
1a. Add 100 ml dimethyl sulfate to 1 liter of 5 M NaOH. Stir the reaction mixture.
2a. Fifteen minutes after all the dimethyl sulfate has gone into solution, neutralize the
reaction mixture with acid.
Safe Use of
Hazardous
Chemicals
3. Test the reaction mixture for the presence of the original compound (see Support
Protocol 4).
4. Discard the decontaminated reaction mix.
1A.3.20
Current Protocols in Microbiology
SUPPORT
PROTOCOL 4
Materials
Reaction mixture containing dimethyl sulfate, diethyl sulfate, methyl
methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propane
sultone
98:2 (v/v) 2-methoxyethanol/acetic acid
5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol
Piperidine
2-Methoxyethanol
1. Dilute an aliquot of the reaction mixture with 4 vol water.
2. Add 100 l diluted reaction mixture to 1 ml of 98:2 (v/v) 2-methoxyethanol/acetic
acid. Swirl to mix.
3. Add 1 ml of 5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol. Heat 10 min
at 100 C, then cool 5 min in ice.
4. Add 0.5 ml piperidine and 2 ml of 2-methoxyethanol.
5. Measure the absorbance of the violet reaction mixture at 560 nm against an appropriate blank.
The absorbance of a decontaminated solution should be 0.000.
BASIC
PROTOCOL 6
Materials
Ethidium bromide or propidium iodidecontaining solution in water, buffer, or 1
g/ml cesium chloride
5% (v/v) hypophosphorous acid: prepare fresh daily by diluting commercial 50%
reagent 1/10
0.5 M sodium nitrite: prepare fresh daily
Sodium bicarbonate
Additional reagents and equipment for testing for the presence of ethidium
bromide or propidium iodide (see Support Protocol 5)
Emerging
Technologies
1A.3.21
Current Protocols in Microbiology
Materials
Equipment contaminated with ethidium bromide
Decontamination solution (see recipe)
Sodium bicarbonate
Additional reagents and equipment for testing for the presence of ethidium
bromide (see Support Protocol 5)
1. Wash the equipment contaminated with ethidium bromide once with a paper towel
soaked in decontamination solution.
The pH of decontamination solution is 1.8. If this is too corrosive for the surface being
decontaminated, wash with a paper towel soaked in water instead.
2. Wash the surface five times with paper towels soaked in water using a fresh towel
each time.
3. Soak all the towels 1 hr in decontamination solution.
4. Neutralize the decontamination solution by adding sodium bicarbonate until the
evolution of gas ceases.
5. Test the decontamination solution for the presence of ethidium bromide (see Support
Protocol 5).
6. Discard the decontamination solution and the paper towels as nonhazardous liquid
and solid wastes.
ALTERNATE
PROTOCOL 5
Safe Use of
Hazardous
Chemicals
Ethidium bromide
Isopropanol saturated with cesium chloride
Decontamination solution (see recipe)
Sodium bicarbonate
Additional reagents and equipment for testing for the presence of ethidium
bromide (see Support Protocol 5)
1A.3.22
Current Protocols in Microbiology
1. If necessary, dilute the ethidium bromide in isopropanol saturated with cesium chloride so the concentration of ethidium bromide is 1 mg/ml.
2. For each volume of ethidium bromide solution, add 4 vol decontamination solution.
Stir the reaction mixture 20 hr.
3. Neutralize the reaction mixture by adding sodium bicarbonate until the evolution of
gas ceases.
4. Test the reaction mixture for the presence of ethidium bromide (see Support
Protocol 5).
5. Discard the decontaminated solution.
ALTERNATE
PROTOCOL 6
Materials
Ethidium bromide
Isoamyl alcohol or 1-butanol
Decontamination solution (see recipe)
Activated charcoal
Sodium bicarbonate
Separatory funnel
Additional reagents and equipment for testing for the presence of ethidium
bromide (see Support Protocol 5)
1. If necessary, dilute the ethidium bromide in isoamyl alcohol or 1-butanol so the
concentration is 1 mg/ml final.
2. For each volume of ethidium bromide solution, add 4 vol decontamination solution.
Stir the two-phase reaction mixture rapidly for 72 hr.
3. For each 100 ml of reaction mixture, add 2 g activated charcoal. Stir another 30 min.
4. Filter the reaction mixture.
5. Neutralize the filtrate by adding sodium bicarbonate until the evolution of gas ceases.
Separate the layers using a separatory funnel.
More alcohol may tend to separate from the aqueous layer on standing.
6. Test the alcohol and aqueous layers for the presence of ethidium bromide.
7. Discard the alcohol and aqueous layers appropriately. Discard the activated charcoal
as solid waste.
The aqueous layer contains 4.6% 1-butanol or 2.3% isoamyl alcohol.
SUPPORT
PROTOCOL 5
This protocol is used to verify that solutions no longer contain ethidium bromide or
propidium iodide. The limits of detection are 0.05 parts per million (ppm) for ethidium
bromide and 0.1 ppm for propidium iodide.
Materials
Reaction mixture containing ethidium bromide or propidium iodide
TBE buffer, pH 8.1 (APPENDIX 2A)
20 g/ml calf thymus DNA in TBE buffer, pH 8.1
Emerging
Technologies
1A.3.23
Current Protocols in Microbiology
1. Mix 100 l reaction mixture containing ethidium bromide or propidium iodide with
900 l TBE buffer, pH 8.1.
2. Add 1 ml of 20 g/ml calf thymus DNA in TBE, pH 8.1. Prepare a blank solution
(100 l water + 900 l TBE + 1 ml of 20 g/ml calf thymus DNA) and control
solutions containing known quantities of ethidium bromide or propidium iodide. Let
the mixtures stand 15 min.
3. To detect ethidium bromide, measure the fluorescence with an excitation wavelength
of 540 nm and an emission wavelength of 590 nm. To detect propidium iodide,
measure the fluorescence with an excitation wavelength of 350 nm and an emission
wavelength of 600 nm.
If a spectrophotofluorometer is not available, fluorescence of ethidium bromide can be
qualitatively determined using a hand-held UV lamp on the long-wavelength setting (Lunn
and Sansone, 1991c).
BASIC
PROTOCOL 7
Materials
30% hydrogen peroxide
10% (w/v) sodium metabisulfite
10% (w/v) potassium iodide
1 M HCl
1% (w/v) starch indicator solution
1. Add 5 ml of 30% hydrogen peroxide to 100 ml of 10% sodium metabisulfite. Stir
the mixture at room temperature until the temperature starts to drop, indicating that
the reaction is over.
2. Test for the presence of hydrogen peroxide by adding a few drops of the reaction
mixture to an equal volume of 10% potassium iodide. Add a few drops of 1 M HCl
to acidify the reaction mixture, then add a drop of 1% starch indicator solution.
A deep blue color indicates the presence of excess oxidant. If necessary, add more 10%
sodium metabisulfite until the starch test is negative.
DISPOSAL OF IODINE
Iodine is reduced with sodium metabisulfite to iodide (Lunn and Sansone, 1994b).
Materials
Iodine
10% (w/v) sodium metabisulfite
1 M HCl
1% (w/v) starch indicator solution
1. Add 5 g iodine to 100 ml of 10% sodium metabisulfite. Stir the mixture until the
iodine has completely dissolved.
2. Acidify a few drops of the reaction mixture by adding a few drops of 1 M HCl. Add
1 drop of 1% starch indicator solution.
Safe Use of
Hazardous
Chemicals
1A.3.24
A deep blue color indicates the presence of iodine. If reduction is not complete, add more
sodium metabisulfite solution.
BASIC
PROTOCOL 9
Materials
Solution containing 1600 ppm mercuric acetate or 1350 ppm mercuric chloride
Dowex 50X8-100 ion-exchange (mercuric acotate) or Amberlite IRA-400(Cl)
ion-exchange (mercuric chloride) resin
Additional reagents and equipment to test for the presence of mercury (see Support
Protocol 6)
1a. For mercuric acetate: For each 200 ml of solution containing 1600 ppm mercuric
acetate, add 1 g Dowex 50X8-100 ion-exchange resin. Stir the mixture 1 hr, then
filter through filter paper.
1b. For mercuric chloride: For each 200 ml of solution containing 1350 ppm mercuric
chloride, add 1 g Amberlite IRA-400(Cl) ion-exchange resin. Stir the mixture 6 hr,
then filter through filter paper.
The speed and efficiency of decontamination will depend on factors such as the size and
shape of the flask and the rate of stirring.
2. Test the filtrate for the presence of mercury (see Support Protocol 6).
3. Discard the decontaminated filtrate and the mercury-containing resin appropriately.
ALTERNATE
PROTOCOL 7
Materials
Iron powder, 60 mesh
Waste water containing 2.5 ppm mercury
6-mm-i.d. column
1. Pack a 6-mm-i.d. column with 1 g of 60-mesh iron powder.
Use a fresh column for each treatment.
2. Pass 2 liters of water containing 2.5 ppm of mercury through the column at a
flow rate of 250 ml/hr.
Emerging
Technologies
1A.3.25
Current Protocols in Microbiology
Solutions containing a higher concentration of mercury may also be treated, but this will
result in a higher final concentration of mercury (e.g., treating a 100-ppm solution in this
fashion yielded 33 ppb final).
Some iron ends up in solution and can be removed by adjusting the pH to 8. The resulting
precipitated Fe(OH)3 can then be removed by filtration.
3. Aerate the resulting effluent to remove traces of metallic mercury and continue
aeration 30 min after the last of the effluent has emerged from the column. Vent
the metallic mercury removed from the solution by aeration into the fume hood or
capture it in a mercury trap.
The effluent contains <5 ppb mercury. The presence of iodide or polypeptone may necessitate several treatments to reduce the mercury to an acceptable level.
SUPPORT
PROTOCOL 6
BASIC
PROTOCOL 10
Materials
Sodium azide or solution containing sodium azide
Ceric ammonium nitrate
10% (w/v) potassium iodide
1 M HCl
1% (w/v) starch indicator solution
Sodium nitrite
4 M sulfuric acid
Additional reagents and equipment to test for the presence of sodium azide (see
Support Protocol 7) or nitrite (see Support Protocol 8)
Decontamination using ceric ammonium nitrate
1a. For each gram of sodium azide, add 9 g ceric ammonium nitrate to 30 ml water, and
stir until it has dissolved.
Safe Use of
Hazardous
Chemicals
2a. Dissolve each gram of sodium azide in 5 ml water. Add this solution to the ceric
ammonium nitrate solution at the rate of 1 ml each min. Stir 1 hr.
If the reaction is carried out on a larger scale, an ice bath may be required for cooling.
1A.3.26
Current Protocols in Microbiology
3a. Check that the reaction is still oxidizing by adding a few drops of the reaction mixture
to an equal volume of 10% potassium iodide. Acidify the mixture with 1 drop of 1 M
HCl and add 1 drop of 1% starch indicator solution.
The deep blue color of the starch-iodine complex indicates that excess oxidant is present.
If excess oxidant is not present, add more ceric ammonium nitrate.
4a. Test for the presence of sodium azide (see Support Protocol 8).
5a. Discard the decontaminated reaction mixture.
3b. Check that there is excess nitrous acid in the reaction. Add a few drops of the reaction
mixture to an equal volume of 10% potassium iodide. Acidify the mixture with 1
drop 1 M HCl. Add 1 drop starch indicator solution.
The deep blue color of the starch-iodine complex indicates that excess nitrous acid is
present. If excess nitrous acid is not present, add more sodium nitrite.
4b. If excess nitrous acid is present, test for the presence of sodium azide (see Support
Protocol 7).
5b. Discard the decontaminated reaction mixture.
SUPPORT
PROTOCOL 7
Materials
Reaction mixture from sodium azide treated with ceric ammonium nitrate or
sodium nitrite
1 M KOH
Acetonitrile
Sodium azide indicator solution (see recipe)
0.2 M HCl
Emerging
Technologies
1A.3.27
Current Protocols in Microbiology
2b. Add 1 drop sodium azide indicator solution, then basify the mixture by adding 1 M
KOH until it turns purple (typically, 3 to 10 ml are required).
3b. Add 2 ml acetonitrile. Add 0.2 M HCl dropwise until the mixture turns yellow, then
add 1 drop more.
If >1 ml sulfamic acid is used, add 4 ml acetonitrile.
1A.3.28
Current Protocols in Microbiology
SUPPORT
PROTOCOL 8
Materials
-Naphthylamine
15% (v/v) aqueous acetic acid
Sulfanilic acid solution (see recipe)
Reaction mixture treated to remove excess nitrite (see Support Protocol 7, step 1b)
1. Prepare the modified Griess reagent by boiling 0.1 g -naphthylamine in 20 ml water
until it dissolves. While the solution is still hot, pour it into 150 ml of 15% aqueous
acetic acid. Add 150 ml sulfanilic acid solution.
CAUTION: -Naphthylamine is a carcinogen.
This reagent should be stored at room temperature in a brown bottle.
BASIC
PROTOCOL 11
Materials
Solutions of APMSF, AEBSF, PMSF, DFP, TLCK, or TPCK in buffer, DMSO,
isopropanol, or water
1 M NaOH
Glacial acetic acid
Additional reagents and equipment for testing for the presence of the enzyme
inhibitors (see Support Protocol 9)
1. If necessary, dilute the solutions with the same solvent so that the concentrations
given in Table 1A.3.8 are not exceeded.
Bulk quantities of AEBSF, PMSF, and TPCK may be dissolved in isopropanol and bulk
quantities of APMSF and TLCK may be dissolved in water at the concentrations shown
in Table 1A.3.8. Bulk quantities of DFP (a liquid) may be mixed directly with 1 M NaOH,
taking care to make sure that all the DFP has mixed thoroughly, in the ratio shown in
Table 1A.3.8 (e.g., 40 l DFP with 1 ml of 1 M NaOH).
2. Add 1 M NaOH so that the ratio of solution to 1 M NaOH is that listed in Table
1A.3.8.
3. Shake to ensure complete mixing, check that the solution is strongly basic (pH 12),
and allow to stand for the time given in Table 1A.3.8.
Emerging
Technologies
1A.3.29
Current Protocols in Microbiology
Compound
Concentration
Solvent
Ratioa
Time
AEBSF
1 mM
Buffer (pH
5.0-8.0)
1:0.1
1 hr
AEBSF
20 mM
DMSO
1:10
24 hr
AEBSF
20 mM
Isopropanol
1:10
24 hr
APMSF
2.5 mM
Buffer (pH
5.0-8.0)
1:0.1
1 hr
APMSF
25 mM
DMSO
1:5
24 hr
APMSF
25 mM
50:50
isopropanol:pH 3
buffer
1:5
24 hr
APMSF
100 mM
Water
1:5
24 hr
DFP
10 mM
Buffer (pH
6.4-7.2)
1:0.2
18 hr
DFP
200 mM
DMF
1:2
18 hr
DFP
pure
1:25
1 hr
DFP
10 mM
Water
1:0.2
18 hr
PMSF
10 mM
Buffer (pH
5.0-8.0)
1:0.1
1 hr
PMSF
100 mM
DMSO
1:5
24 hr
PMSF
100 mM
Isopropanol
1:5
24 hr
TLCK
1 mM
Buffer (pH
5.0-8.0)
1:0.1
18 hr
TLCK
5 mM
DMSO
1:5
18 hr
TLCK
5 mM
Water
1:0.1
18 hr
TPCK
1 mM
Buffer (pH
5.0-8.0)
1:0.1
18 hr
TPCK
1 mM
DMSO
1:0.1
18 hr
TPCK
1 mM
Isopropanol
1:0.1
18 hr
a Ratio refers to the proportion of solution to sodium hydroxide, i.e., solution (compound + solvent):1
M NaOH.
4. Neutralize the reaction mixture with glacial acetic acid and test for the presence of
residual enzyme inhibitor (see Support Protocol 9).
5. Discard the decontaminated reaction mixture.
SUPPORT
PROTOCOL 9
Safe Use of
Hazardous
Chemicals
1A.3.30
Current Protocols in Microbiology
Compound
Mobile phase
UV detector
wavelength
Retention
time
Limit of
detection
AEBSF
225 nm
9.5 min
0.1 g/ml
APMSF
232 nm
7.7 min
0.5 g/ml
PMSF
220 nm
8 min
0.9 g/ml
TLCK
228 nm
9.5 min
0.37 g/ml
TPCK
228 nm
10.5 min
2 g/ml
Materials
Decontaminated enzyme inhibitor solutions
Acetonitrile, HPLC grade
Water, HPLC grade
0.1% (v/v) trifluoroacetic acid in water
10 mM phosphate buffer, pH 7
Compound of interest
250-mm 4.6 mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or
equivalent
Additional reagents and equipment for reversed-phase liquid chromatography
(Snyder et al., 1997)
Analyze the decontaminated enzyme inhibitor solutions by reversed-phase HPLC using
a 250-mm 4.6-mm-i.d. Microsorb C-8 reversed-phase column, or equivalent, using
the conditions shown in Table 1A.3.9. In each case, the injection volume was 20 l, the
separation occurred at ambient temperature, and the flow rate was 1 ml/min. Check the
analytical procedures by spiking an aliquot of the acidified reaction mixture with a small
quantity of a dilute solution of the compound of interest.
BASIC
PROTOCOL 12
Note that the following procedures may only be used to degrade bulk quantities of
MNNG, solutions in methanol (or ethanol), contaminated glassware, or spills. Also the
methods are not appropriate for other nitrosamides (e.g., N-methyl-N-nitrosourea) or
the chemically similar nitrosamines (e.g., dimethylnitrosamine). For a full discussion of
procedures for the degradation of these compounds see Lunn and Sansone, 1994a.
Materials
N-Methyl-N -nitro-N-nitrosoguanidine (MNNG) as a solid or in solution in
methanol or ethanol
6 M hydrochloric acid (APPENDIX 2A)
Sulfamic acid
Neutralizing solution (e.g., aqueous NaOH)
Additional reagents and equipment for detecting the presence of MNNG (see
Support Protocol 10)
Emerging
Technologies
1A.3.31
Current Protocols in Microbiology
1. Take up bulk quantities of MNNG in methanol so that the concentration does not
exceed 30 g/L. If necessary, dilute methanol or ethanol solutions with the same
solvent so that the concentration of MNNG does not exceed 30 g/liter.
2. Slowly add an equal volume of 6 M HCl with stirring.
3. For each 1 liter of the resulting solution, add 35 g sulfamic acid. Stir 24 hr.
4. Neutralize the reaction mixture, e.g., with sodium hydroxide solution.
5. Test the reaction mixture for the presence of MNNG (see Support Protocol 10).
6. Discard the decontaminated reaction mixture.
ALTERNATE
PROTOCOL 8
ALTERNATE
PROTOCOL 9
DECONTAMINATION OF SPILLS OF
N-METHYL-N -NITRO-N-NITROSOGUANIDINE (MNNG)
Materials
Methanol
70 g/liter sulfamic acid in 2 M hydrochloric acid
Neutralizing solution (e.g., aqueous NaOH)
Additional reagents and equipment for detecting the presence of MNNG (see
Support Protocol 10)
1. Soak the area in methanol until the nitrosamide appears to be dissolved, then add an
approximately equal volume of 70 g/liter sulfamic acid in 2 M HCl.
2. After 6 hr, remove the reaction mixture and clean the area in a conventional fashion.
3. Check for completeness of destruction by using a wipe soaked in methanol and
analyzing it for the presence of the compound (see Support Protocol 10).
4. Neutralize the reaction mixture, e.g., with sodium hydroxide solution.
5. Test the reaction mixture for the presence of MNNG (see Support Protocol 10).
Safe Use of
Hazardous
Chemicals
1A.3.32
Current Protocols in Microbiology
SUPPORT
PROTOCOL 10
Reversed-phase HPLC (Snyder et al., 1997) is used to test for the presence of MNNG.
Materials
Neutralized reaction mixture
12:88 methanol/3.5 mM (NH4 )H2 PO4
250-mm 4.6-mm-i.d. Microsorb C-8 reversed-phase column (Varian) or
equivalent
Additional reagents and equipment for reversed-phase liquid chromatography
(Snyder et al., 1997)
Analyze the neutralized reaction mixture(s) by reversed-phase HPLC using a UV detector
operating at 254 nm and a 250-mm 4.6-mm-i.d. Microsorb C-8 column (Lunn et al.,
1988). Elute with a mobile phase of 12:88 methanol/3.5 mM (NH4 )H2 PO4 flowing at 1
ml/min at ambient temperature. Check the analytical procedure by spiking the neutralized
reaction mixture with an aliquot of a dilute solution of MNNG.
Decontamination solution
Dissolve 4.2 g sodium nitrite (0.2 M final) and 20 ml hypophosphorous acid (3.3%
w/v final) in 300 ml water. Prepare fresh.
Horseradish peroxidase
Dissolve hydrogen-peroxide oxidoreductase (EC 1.11.1.7 [Type II]; specific activity
150 to 200 purpurogallin U/mg, Sigma) in 1 g/liter sodium acetate to give 30 U/ml.
Prepare fresh daily.
For small-scale reactions, a more dilute solution can be used to avoid working with inconveniently small volumes.
pH 5 buffer
2.04 g potassium hydrogen phthalate (0.05 M final)
38 ml 0.1 M potassium hydroxide (15 mM)
H2 O to 200 ml
Store at room temperature
Phosphate buffer
13.6 g monobasic potassium phosphate (KH2 PO4 ; 0.1 M final)
0.28 g dibasic sodium phosphate (Na2 HPO4 ; 2 mM final)
3.0 g potassium bromide (KBr; 25 mM final)
1 liter H2 O
Store at room temperature
Potassium bromide is necessary to make the assay for cyanide work correctly.
Emerging
Technologies
1A.3.33
Current Protocols in Microbiology
Lunn, G. and Sansone, E.B. 1994b. Safe disposal of highly reactive chemicals. J. Chem. Educ. 71:972-976.
Lunn, G. and Sansone, E.B. 1994c. Degradation and disposal of some enzyme inhibitors. Scientific note.
Appl. Biochem. Biotechnol. 48:57-59.
1A.3.34
Current Protocols in Microbiology
Lunn, G. and Sansone, E.B., 1994d. Safe disposal of diisopropyl fluorophosphate (DFP). Appl. Biochem.
Biotechnol. 49:165-171.
Lunn, G., Sansone, E.B., Andrews, A.W., and Keefer, L.K. 1988. Decontamination and disposal of nitrosoureas and related N-nitroso compounds. Cancer Res. 48:522-526.
Lunn, G., Klausmeyer, P.K., and Sansone, E.B. 1994. Removal of biological stains from aqueous solution
using a flow-through decontamination procedure. Biotech. Histochem. 69:45-54.
Manufacturing Chemists Association. 1973. Laboratory Waste Disposal Manual. p. 136. Manufacturing
Chemists Association, Washington, D.C.
Mason, K.G. 1967. Hydrogen azide. In Mellors Comprehensive Treatise on Inorganic and Theoretical
Chemistry, Vol. VIII (Suppl. II) pp. 1-15. John Wiley & Sons, New York.
National Research Council. 1983. Prudent Practices for Disposal of Chemicals from Laboratories, p. 88.
National Academy Press, Washington, D.C.
ONeil, M.J. (ed.) 2001. The Merck Index, 13th ed. Merck & Co., Whitehouse Station, N.J.
Patnode, W. and Wilcock, D.F. 1946. Methylpolysiloxanes. J. Am. Chem. Soc. 68:358-363.
Shirakashi, T., Nakayama, K., Kakii, K., and Kuriyama, M. 1986. Removal of mercury from laboratory
waste water with iron powder. Chem. Abstr. 105:213690y.
Snyder, L.R., Kirkland, J.J., and Glajch, J.L. 1997. Practical HPLC Method Development, 2nd ed. John
Wiley & Sons, New York.
KEY REFERENCES
The following are good general references for laboratory safety.
Alaimo, R.J. (ed.) 2001. Handbook of Chemical Health and Safety. American Chemical Society, Washington,
D.C.
American Chemical Society, Committee on Chemical Safety. 1995. Safety in Academic Chemistry Laboratories 6th ed. American Chemical Society, Washington, D.C.
Castegnaro, M. and Sansone, E.B. 1986. Chemical Carcinogens. Springer-Verlag, New York.
DiBerardinis, L.J., First, M.W., Gatwood, G.T., and Seth, A.K. 2001. Guidelines for Laboratory Design,
Health and Safety Considerations, 3rd ed. John Wiley & Sons, New York.
Fleming, D.D., Richardson, J.H., Tulis, J.J., and Vesley, D. 1995. Laboratory Safety, Principles and Practices,
2nd ed. American Society for Microbiology, Washington, D.C.
Freeman, N.T. and Whitehead, J. 1982. Introduction to Safety in the Chemical Laboratory. Academic Press,
San Diego.
Fuscaldo, A.A., Erlick, B.J., and Hindman, B. (eds.) 1980. Laboratory Safety, Theory and Practice. Academic
Press, San Diego.
Lees, R. and Smith, A.F. (eds.) 1984. Design, Construction, and Refurbishment of Laboratories. Ellis
Horwood, Chichester United Kingdom.
Montesano, R., Bartsch, H., Boyland, E., Della Porta, G., Fishbein, L., Griesemer, R.A., Swan, A.B., and
Tomatis, L. (eds.) 1979. Handling Chemical Carcinogens in the Laboratory, Problems of Safety. IARC
Scientific Publications No. 33. International Agency for Research on Cancer, Lyon, France.
National Research Council. 1995. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals.
National Academy Press, Washington, D.C.
Pal, S.B. (ed.) 1991. Handbook of Laboratory Health and Safety Measures, 2nd ed. Kluwer Academic
Publishers, Hingham, Mass.
Picot, A. and Grenouillet, P. 1994. Safety in the Chemistry and Biochemistry Laboratory. John Wiley &
Sons, New York.
Rosenlund, S.J. 1987. The Chemical Laboratory: Its Design and Operation: A Practical Guide for Planners
of Industrial, Medical, or Educational Facilities. Noyes Publishers, Park Ridge, N.J.
Stricoff, R.S. and Walters, D.B. 1995. Handbook of Laboratory Health and Safety, 2nd ed. John Wiley &
Sons, New York.
Young, J.A. (ed.) 1991. Improving Safety in the Chemical Laboratory: A Practical Guide, 2nd ed. John
Wiley & Sons, New York.
Emerging
Technologies
1A.3.35
Current Protocols in Microbiology
INTERNET RESOURCES
http://www.ilpi.com/msds/index.html
Where to find MSDSs on the internet. Contains links to general sites, government and nonprofit sites,
chemical manufacturers and suppliers, pesticides, and miscellaneous sites.
http://www.OSHA.gov
The OSHA website. Standards can be accessed by clicking on the link in the right sidebar labeled Standards.
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=10106
Text of OSHA Standard 29 CFR 1910.1450: Occupational Exposure to Hazardous Chemicals in Laboratories.
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=9992
Table Z-1 of OSHA Standard 29 CFR 1910.10000, which provides a list of permissible exposure limits
(PELs) for air contaminants.
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=9993
Table Z-2 of OSHA Standard 29 CFR 1910.1000, which provides a list of PELs for toxic and hazardous
substances.
http://hazard.com/msds/index.php
Main site for Vermont SIRI. One of the best general sites to start a search. Browse manufacturers alphabetically (for sheets not in the SIRI collection) or do a keyword search in the SIRI MSDS database. Lots of
additional safety links and information.
http://siri.uvm.edu/msds
Alternate site for Vermont SIRI.
http://eh.doe.gov/docs/osh tr/ch5.html
DOE OSH technical reference chapter on personal protective equipment.
Safe Use of
Hazardous
Chemicals
1A.3.36
Current Protocols in Microbiology
UNIT 1A.4
BACKGROUND INFORMATION
The Decay Process
As anyone who has taken a basic chemistry course will remember, each element is
characterized by its atomic number (Z), defined as the number of protons in the atoms
nucleus. Isotopes of a given element exist because some atoms of each element, while by
definition having the same number of protons,
have a different number of neutrons and therefore a different nuclear weight (A), equivalent
to the atomic mass number which reflects the
total number of nucleons (protons plus neutrons). It should be noted that the number of
electrons outside the nucleus remains the same
for all isotopes of a given element, and so all
isotopes of a given element are equivalent with
respect to their chemical reactivity.
Radioactive decay occurs when energy is
released from the nucleus of an atom of a particular isotope. This often results in the conversion of an atom of one isotope to an isotope
of a different element, a process termed transmutation, as the number of protons in the atom
changes after decay. The energy released from
naturally occurring radioisotopes can be particulate ( and particles), or nonparticulate
( rays).
particles
An particle is essentially the nucleus of
a helium atom, or two protons plus two neutrons. It is a relatively large, heavy particle that
moves slowly and usually only across short
distances before it encounters some other atom
with which it interacts. These particles are released from isotopes with large nuclei (atomic
number >82; e.g., thorium, uranium); such
isotopes are not commonly used in biological
research except for specific applications such
as electron microscopy and X-ray diffraction
studies.
particles
In contrast to particles, particles are
light, high-speed, charged particles. Negatively charged particles are essentially electrons of nuclear origin that are released when
a neutron is converted to a proton. Release of
a particle thus changes the atomic number
and elemental status of the isotope.
Emerging
Technologies
1A.4.1
radiation
radiation has both particle and wave properties; its wavelength falls within the range of
X-ray wavelengths. Presently, radiation is
defined as that originating from an atomic nucleus, and X-ray radiation as that originating
from the electron cloud surrounding the nucleus. Unlike -particle release, the release
of radiation by itself produces an isotopic
change rather than an elemental one; however,
the resultant nuclei are unstable and often decay further, releasing particles.
Isotopic decay
Safe Use of
Radioisotopes
creating ions. Thus, these three types of emissions are called ionizing radiation. The formation of such ions may result in the perturbation
of biological processes: therein lies the danger
associated with radioactivity!
1A.4.2
Current Protocols in Microbiology
vials or tubes containing the radioactive material. Speed should be encouraged in all
manipulations, though not to the point of
recklessness! Have everything needed for the
experiment ready at hand before radioactivity
is introduced into the work area.
Emerging
Technologies
1A.4.3
Current Protocols in Microbiology
amounts of bremsstrahlung radiation is the reason that low-density materials are used as the
primary layer of shielding for radiation from
32
P. Water, glass, and plastic are suitable lowdensity materials (as opposed to lead). Obviously water is unsuitable as a shielding layer
for work on the bench, although it does a reasonable job when samples are incubating in a
water bath. Shields made from a thickness of
glass sufficient to stop these particles would be
extremely heavy and cumbersome (as well as
dangerous if dropped). Fortunately, plastic or
acrylic materialsvariously called Plexiglas,
Perspex, or Luciteare available for shielding against radiation from 32 P. Shields, as
well as storage boxes, constructed of various
thicknesses of Plexiglas are necessary equipment in laboratories where 32 P is used. When
mCi amounts of 32 P are used at one time, it
is necessary to also block the bremsstrahlung
radiation by adding a layer of high-density material (such as 4 to 6 mm of lead) to the outside
of the Plexiglas shield (covering the side farthest from the radioactive source).
rays released during the decay of 125 I
have much higher penetrance than the particles from 32 P decay; this radiation must be
stopped by very-high-density material, such as
lead. Lead foil of varying thicknesses (0.5 to 6
mm) can be purchased in rolls and then cut and
molded to cover any container, or taped to a
Plexiglas shield (used in this instance for support). Obviously this latter arrangement has the
disadvantage that it is impossible to see what
one is doing through the shield. For routine
shielding of manipulations involving 125 I, it is
useful to purchase a lead-impregnated Plexiglas shield that is transparent, albeit inevitably
very heavy (as well as relatively expensive).
Although it seems logical that the use
of more radioactivity necessitates the use of
thicker layers of shielding, it is also true that
no shielding material is capable of completely
stopping all radiation. When deciding how
thick is thick enough, consult the half-value
layer measurement for each type of shielding
material. This number gives the thickness of
a given material necessary to stop half the radiation from a source. In general, 1 to 2 cm
of Plexiglas and/or 0.02 mm of lead are sufficient to shield the amounts of radioactivity
used in experiments performed in the authors
laboratory.
GENERAL PRECAUTIONS
Safe Use of
Radioisotopes
1A.4.4
Current Protocols in Microbiology
isotope. This is done not only so that appropriate shielding can be placed around waste
containers, but so that some waste can be allowed to decay prior to disposal in the normal (nonradioactive) trash. With a decreasing
number of radioactive waste disposal facilities
able or willing to accept radioactive waste for
burial (and a concomitant increase in dumping
charges from those that still do), this practice
of on-site decay can save an institution thousands of dollars a year in disposal charges.
6. Label your label! It is only common
courtesy (as well as common sense) to alert
coworkers to the existence of anything and everything radioactive that is left where they may
come in contact with it! A simple piece of tape
affixed to the sample boxwith the investigators name, the amount and type of isotope,
and the dateshould suffice. Yellow hazard
tape printed with the international symbol for
radioactivity is commercially available in a variety of widths.
7. Monitor radioactivity early and often.
Portable radiation detection monitors are essential equipment for every laboratory using
radioactivity. No matter how much or how little radioactivity is being used, the investigator
should keep a hand-held monitor nearbyand
it should be on! Turn it on before touching any
radioactivity to avoid contaminating the monitors switch. Always check the batteries! Use a
monitor with the appropriate detection capacity ( for 35 S and 32 P; for 125 I) before, during,
and after all procedures. The more frequently
fingers and relevant equipment are monitored,
the more quickly a spill or glove contamination
will be detected. Such timely detection will
keep both the potential mess and cleanup time
to a minimum. While it is tempting to cover the
monitors detection tube with Parafilm to protect it from contamination, remember that this
will preclude the detection of the weaker
of 35 S! Because low-energy emitters such
as 3 H cannot be detected at all using such
monitors, wipe tests of the bench and equipment used, followed by scintillation counting
of the wiped samples, are necessary to ensure
that contamination of the work area did not
occur.
SPECIFIC PRECAUTIONS
The following sections describe precautions to be taken with individual isotopes in
specific forms. Although the sections dealing
with 35 S- or 32 P-labeling of proteins in intact cells are presented in terms of mammalian
cells, most of the instructions are also pertinent
(with minimal and obvious modifications) to
Working with 35 S
As discussed above, the radiation generated during 35 S decay is not strong enough
to make barrier forms of shielding necessary. The risk associated with 35 S comes primarily through its ingestion and subsequent
concentration in various target organs, particularly the testes. As reported several years ago
(Meisenhelder and Hunter, 1988), 35 S-labeled
methionine and cysteine, which are routinely
used to label proteins in intact cells and by
in vitro translation, break down chemically to
generate a volatile radioactive component. The
breakdown occurs independently of cellular
metabolism. Thus, the radioactive component
is generated to the same extent in stock vials as
in cell culture dishes. The process seems to be
promoted by freezing and thawing 35 S-labeled
materials. The exact identity of this component is not known, although it is probably SO2
or CH3 SH. What is known is that it dissolves
readily in water and is absorbed by activated
charcoal or copper.
The amount of this volatile radioactive
component released, despite stabilizers added
by the manufacturers, is about 1/8000 of the
total radioactivity present. The amount of this
radioactivity that a scientist is likely to inhale
while using these compounds is presumably
even smaller. Nevertheless, such a component
can potentially contaminate a wide area because of its volatility, and also tends to concentrate in target organs. Thus, it is advisable
to thaw vials of 35 S-labeled amino acids in a
controlled area such as a hood equipped with a
charcoal filter. This charcoal filter will become
quite contaminated and should be changed
every few months. If such an area is not available, the stock vial should be thawed using a
needle attached to a charcoal-packed syringe
to vent and trap the volatile compound.
Anyone who has ever added 35 S-labeled
amino acids to dishes of cells for even short periods knows that the incubators used for such
labelings quickly become highly contaminated
with 35 S. Such contamination is not limited
to the dish itself, nor to the shelf on which
the dish was placed. Rather, the radioactive
components solubility in water allows it to
circulate throughout the moist atmosphere of
the incubator and contaminate all of the inside surfaces. For this reason, in laboratories
where such metabolic labelings are routine, it
is highly convenient to designate one incubator
to be used solely for working with 35 S-labeled
Emerging
Technologies
1A.4.5
Current Protocols in Microbiology
Working with 32 P
Microcurie (Ci) amounts of 32 P
Safe Use of
Radioisotopes
contamination could easily occur though careless pipetting and the resultant creation of an
aerosol of radioactive microdroplets, because
the concentration of a typical stock solution of
labeled nucleotide may be 10 Ci/l.
For proper protection during this sort of
experiment, besides the usual personal attire
(glasses, gloves, coat, and ring and lapel safety
badges) it is necessary to use some form of
Plexiglas screen between the body and the
samples (see Fig. 1A.4.1A). Check the level
of radiation coming through the outside of the
shield with a portable monitor to be sure the
thickness of the Plexiglas is adequate. Consult your Radiation Safety Officer if the monitor reads more than 4000 cpm. Hands can be
shielded from some exposure by placing the
sample tubes in a solid Plexiglas rack, which
is also useful for transporting samples from the
bench to a centrifuge or water bath (see Fig.
1A.4.1B).
Experiments of these types often include an
incubation step performed at a specific temperature, usually in a water bath. Although the
water surrounding the tubes or hybridization
bags will effectively stop radiation, shielding should be added over the top of the tubes
(where there is no water)e.g., a simple flat
piece of Plexiglas. If the frequency of usage
justifies the expense, an entire lid for the water
bath can be constructed from Plexiglas. When
hybridization reactions are performed in bags,
care should be taken to monitor (and shield)
the apparatus used to heat-seal the bags.
The waste generated during the experiments should also be shielded. It is convenient
to have a temporary waste container right on
the bench. Discard pipet tips and other solid
waste into a beaker lined with a plastic bag and
placed behind the shield. This bag can then be
emptied into the appropriate shielded laboratory waste container when the experiment is
done. Liquid waste can be pipetted into a disposable tube set in a stable rack behind the
shield (see Fig. 1A.4.1C).
When radiolabeled probes or proteins must
be gel-purified, it may be necessary to shield
the gel apparatus during electrophoresis if the
samples are particularly hot. Be advised that
the electrophoresis buffer is likely to become
very radioactive if the unincorporated label is
allowed to run off the bottom of the gel; check
with your Radiation Safety Officer for instructions on how to dispose of such buffer. It is also
prudent to check the gel plates with a portable
detection monitor after the electrophoresis is
completed, because they sometimes become
contaminated as well.
1A.4.6
Current Protocols in Microbiology
Figure 1A.4.1 Plexiglas shielding for 32 P. (A) Two portable shields (L and T design) made of
0.5-in. (12.5 mm) Plexiglas. Either can be used to directly shield the scientist from the radioactivity
he or she is using. Turned on its side, the L-shaped shield can be used to construct two sides of
a cage around a temporary work area, providing shielding for other workers directly across or to
the sides of the person working with 32 P. (B) Tube rack for samples in microcentrifuge tubes. (C)
Tube holder for liquid waste collection.
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1A.4.7
Current Protocols in Microbiology
Figure 1A.4.2 (A) Box for cell incubation (a cell house). (B) Box for solid waste collection made
of 0.5-in. Plexiglas. (C) Sample storage rack and box made of 0.5-in. Plexiglas. (D) Box for solid
waste collection made of 0.5-in. Plexiglas. Abbreviations: ID, interior dimension.
Safe Use of
Radioisotopes
1A.4.8
Current Protocols in Microbiology
Figure 1A.4.3
Emerging
Technologies
1A.4.9
Current Protocols in Microbiology
Safe Use of
Radioisotopes
1A.4.10
Current Protocols in Microbiology
Working with 33 P
Using 33 P-labeled nucleotides to label
nucleic acid probes or proteins
Several of the major companies that manufacture radiolabeled biological molecules also
sell nucleotides labeled with 33 P (both - and
-labeled forms). 33 P offers a clear advantage
over 32 P with respect to ease of handling, because the energy of the particles emitted lies
between that of 35 S and 32 P; thus, 33 P does
not require as many layers of Plexiglas and
lead shielding as 32 P. In fact, the radiation
emitted can barely penetrate gloves and the
surface layer of skin, so the hazard associated
with exposure to even millicurie amounts of
33
P is thought to be insignificant (as reported
in the NEN Life Science product brochure).
Gel bands visualized on autoradiograms of
33
P-labeled compounds are sharper than bands
labeled with 32 P because the lower-energy
radiation does not have the scatter associated
with 32 P. The half-life of 33 P is also longer (25
days compared to 14 days for 32 P). Despite
its higher cost, these features have led many
researchers to choose 33 P-labeled nucleotides
for use in experiments such as band/gel shift
assays where discrimination of closely spaced
gel bands is important.
The best way to determine what degree of
shielding is needed when using 33 P is to monitor the source using a portable monitor and
add layers of Plexiglas as necessary, as described previously for 32 P.
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1A.4.11
Current Protocols in Microbiology
be tricky, as their anodized surfaces are sensitive to many detergents; check with the rotor
manufacturer for appropriate cleansing solutions. Many surfaces prove resistant to even
Herculean cleaning efforts; in these instances,
the best that can be done is to remove all contamination possible and then shield whatever
is left until the radioactivity decays sufficiently
for safety.
LITERATURE CITED
Board on Radiation Effects (BRER). VIII. 2005.
Health risks from exposure to low levels of
ionizing radiation: BEIR VII Phase 2. 2005.
Board on Radiation Effects Research (BRER).
The National Academies Press, Washington,
D.C.
Available online at http://www.nap.
edu/books/030909156X/html.
Bursik, S., Meisenhelder, J., and Spahn, G. 1999.
Characterization and minimization of extremity
doses during 32 P metabolic cell labeling. Health
Phys. 77:595-600.
Meisenhelder, J. and Hunter, T. 1988. Radioactive
protein-labelling techniques. Nature 335:120.
Shleien, B. (ed.) 1987. Radiation Safety Manual for
Users of Radioisotopes in Research and Academic Institutions. Nucleon Lectern Associates,
Olney, Md.
Safe Use of
Radioisotopes
1A.4.12
Current Protocols in Microbiology
UNIT 1B.1
There are a variety of systems available for examining the formation of bacterial biofilms.
In this unit, several approaches are described that are useful for studying, in particular, the
earlier stages of formation of these communities. Static biofilm systems may be preferable
to chemostatic or continuous-flow methods for a number of reasons. First, static assays
are particularly useful for examining early events in biofilm formation, in some cases
detecting biofilm formation in <60 min. For example, these assays can be used to identify
signals that modulate the transition from a planktonic to a biofilm mode of existence.
A second major advantage of these systems is the simplicity of the protocols: these
assays can be executed primarily using common laboratory equipment. Furthermore,
several of the assays outlined below have a relatively high throughput and can easily be
adapted to study a variety of biofilm formation conditions, making them excellent tools
for performing genetic screens. In addition, simple changes in the configurations of these
screens (e.g., media used, medium replacement regimens, incubation time, washing vigor)
allow examination of different aspects of these bacterial communities, such as formation
or dispersal.
The static nature of these systems does have some drawbacks. Because most of the
cultures are neither continuously supplied with fresh medium nor aerated, there may be
limitation of nutrients, and an inability to easily generate mature biofilms may be encountered. Continuous-flow and chemostat systems for the production of mature biofilms are
described in other units in this chapter.
Presented in this unit are four basic protocols for the growth and analysis of biofilms
in static systems. The microtiter plate biofilm assay (see Basic Protocol 1) is a useful
method for assessing bacterial attachment by measuring the staining of the adherent
biomass. Because it utilizes a 96-well plate format, it is suitable as a tool for screening
large numbers of bacterial strains or species. The air-liquid interface (ALI) assay (see
Basic Protocol 2) is complementary to the microtiter plate biofilm assay in that it provides
a mechanism for direct microscopic viewing of the live attached microbes. A colony-based
biofilm system is also described (see Basic Protocol 3), with this system being especially
useful for monitoring cell death in biofilms treated with antimicrobial agents. Finally, the
Kadouri system (see Basic Protocol 4) is a low-flow system that can serve as a bridge
between the static assays discussed in this unit and continuous-flow systems.
CAUTION: Follow all appropriate biosafety requirements relevant to the microorganism
under investigation. Refer to UNIT 1A.1 and other pertinent resources (see APPENDIX 1B) for
instructions on safe handling of microorganisms.
BASIC
PROTOCOL 1
Emerging
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1B.1.1
Table 1B.1.1 Typical Conditions for Developing and Performing Microtiter Plate Biofilm Assays
Organism
Reference
Agrobacterium
tumefaciens
28
Escherichia coli
25
Pseudomonas aeruginosa
25-37
95% ethanol
Pseudomonas fluorescens
25-30
95% ethanol
Staphylococcus aureus
37
Streptococcus mutans
37
100% DMSO
Vibrio cholerae
25-30
Materials
Bacterial strains of interest
Appropriate media for bacteria under study (APPENDIX 2C)
70% ethanol
0.1% (w/v) crystal violet in water
Solvent (e.g., 95% v/v ethanol; see Table 1B.1.1 for other options) for solubilizing
dye and biofilm biomass
96-well microtiter plates, not tissue culturetreated (Becton Dickinson catalog no.
353911) with lids (Becton Dickinson catalog no. 353913)
96-prong inoculating manifold, sterile (DanKar Scientific)
Small trays (e.g., large pipet tip boxes) sufficient in size to hold 96-well microtiter
plates
Optically clear flat-bottom 96-well plates, nonsterile
Plate reader or spectrophotometer
For small sample numbers (<20 strains or species):
1a. Inoculate each bacterium of interest in a 3-to-5-ml culture and grow to stationary
phase.
2a. Dilute cultures 1:100 in the desired media. Pipet 100 l of each diluted culture into
each of four wells in a fresh microtiter plate which has not been tissue culture treated.
Cover plate and incubate at optimal growth temperature for the desired amount of
time.
Lids for the microtiter dishes may be reused. Clean lids with 70% (v/v) ethanol and air-dry
prior to each experiment.
The time course for attachment varies depending on the organism and must be determined
empirically, although when using this system, many organisms commonly studied will form
a biofilm within 48 hr.
3. Set up four small trays in a series and add 1 to 2 inches of tap water to the last three.
The first tray is used to collect waste, while the others are used to wash the assay plates.
1B.1.2
Current Protocols in Microbiology
4. Remove planktonic bacteria from each microtiter dish (step 2a or 2b) by briskly
shaking the dish out over the waste tray. To wash wells, submerge plate in the first
water tray and then vigorously shake out the liquid over the waste tray. Replace water
when it becomes cloudy.
5. Add 125 l of 0.1% crystal violet solution to each well. Stain 10 min at room
temperature.
6. Shake each microtiter dish out over the waste tray to remove the crystal violet solution.
Wash dishes successively in each of the next two water trays (i.e., the two not used
in step 4), and shake out as much liquid as possible after each wash.
This step will remove any crystal violet that is not specifically staining the adherent
bacteria. The wash trays can be reused for a number of plates, but the water should be
replaced when its color becomes dark or when the efficiency of the washes is observed to
decrease.
7. Invert each microtiter dish and vigorously tap on paper towels to remove any excess
liquid. Allow plates to air-dry.
At this stage, the staining is stable and the dried plates may be stored at room temperature
for at least several weeks.
8. Add 200 l of 95% ethanol (or another appropriate solvent) to each stained well.
Allow dye to solubilize by covering plates and incubating 10 to 15 min at room
temperature.
A number of reagents may be used to solubilize biofilms. Some suggestions are listed in
Table 1B.1.1, but solvents should be tested empirically for each organism.
9. Briefly mix the contents of each well by pipetting, and then transfer 125 l of the
crystal violet/ethanol solution from each well to a separate well in an optically clear
flat-bottom 96-well plate. Measure the optical density (OD) of each of these 125-l
samples at a wavelength of 500 to 600 nm.
The authors suggest four replicate wells for each strain or species used in step 2. The absorbance values from these replicates are sufficient to determine an average and standard
deviation for each strain or species and thus provide a measure of the extent of biofilm
formation. The number of replicates can be increased as desired.
The OD wavelengths are presented as a range to allow for variations in the capabilities
of plate readers.
ALTERNATE
PROTOCOL 1
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1B.1.3
Current Protocols in Microbiology
2. Wash wells six times, each time using a multichannel pipettor to add 100 l sterile
PBS per well and then vigorously shaking out the liquid over a waste container to
remove planktonic cells.
3. Use scissors to cut each individual well from the microtiter plate. Add 100 l PBS
to each well.
4. Add each well (i.e., the actual plastic well plus its contents) to a separate 8-ml tube
containing 1.9 ml PBS (for a final liquid volume of 2 ml). Cap before and after
addition.
This step is performed so that each sample can be sonicated (step 5) in an 8-ml tube.
Microtiter wells are not suitable for use as sonication vessels, as they are too small.
5. Insert sonicator microtip and sonicate the contents of each tube for 8 sec at 40% power.
Perform viable counts (Phelan, 1996) on the resulting suspensions by plating on agar
medium to enumerate bacteria that were attached to the microtiter well surface.
To verify that the sonication procedure does not reduce cell viability, perform viable
counts on a separate culture of planktonic cells before and after sonication. If reduced
cell viability is seen, adjust the sonication regimen accordingly.
If desired, use crystal violet staining followed by microscopy to determine the efficacy of
sonication in dislodging attached cells from microtiter well walls. Perform crystal violet
assay as in Basic Protocol 1. Lack of a crystal violet ring indicates that the bacterial cells
have been removed from the wall of the well.
BASIC
PROTOCOL 2
Materials
Bacterial strains of interest
Appropriate media for bacteria under study (APPENDIX 2C)
Flat-bottom 24-well plate, sterile, with lid
Large paper clips or microcentrifuge tube rack and lab tape
Inverted microscope
1. Adjust a sterile, flat-bottom, 24-well plate so that it sits at a 30 to 50 angle from
horizontal.
Growing and
Analyzing Static
Biofilms
The method used will depend on the brand of plate and the available materials. One
approach is to affix four large paperclips to one end of the plate. The paperclips stably
elevate the end of the plate to which they are attached, while the opposite end remains low
(see Fig. 1B.1.1). Alternatively, one end of the plate may be rested on a microcentrifuge
tube rack (or anything of the desired height) and stabilized with tape to prevent slipping.
1B.1.4
Current Protocols in Microbiology
Figure 1B.1.1 (A) Schematic of angled plate for air-liquid interface (ALI) assay (back view).
(B) Side view of ALI assay setup. An angled plate (a) is supported by paper clips (b). The ALI
occurs at the center of the bottom of each well, where a meniscus forms.
Figure 1B.1.2 Phase-contrast micrographs taken at the air-liquid interfaces of biofilms formed
by the wild-type Pseudomonas aeruginosa PA14 strain and a nonflagellated mutant (flgK), both
grown as described in Basic Protocol 2.
If possible, set up the angled plate in the warm room or incubator to be used for sample
culturing, and load the wells there (step 4) to avoid potential disruption of the sample
meniscuses upon transport.
4. Carefully pipet an aliquot (300 l) of each diluted culture into a separate well in the
angled 24-well plate such that the upper edge of the aliquot just reaches the center of
the bottom of the well. Cover plate with lid and incubate at appropriate temperature
for desired period of time.
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1B.1.5
Current Protocols in Microbiology
When inoculating each well, it is important to slowly add the culture down the lower
wall to avoid wetting the entire bottom of the well. The meniscus of the liquid should pass
through the center of the well, which is often the most optically clear part. This will ensure
that the air-liquid interface is appropriately positioned for viewing.
5. Aspirate culture from the wells and gently wash twice, each time by adding 400 l
sterile medium and then aspirating. After completing these two washes, add 200 l
medium (i.e., enough to cover the bottom of the well) to each well.
Washing should remove most of the unattached bacteria from the wells. This is important,
as planktonic cells can make viewing more difficult and can continue to colonize surfaces,
potentially obscuring the air-liquid interface.
6. Lay the plate flat on the stage of an inverted microscope and visualize by phasecontrast or epifluorescent microscopy. Focus just above the center of the bottom of
the well to observe bacteria at the air-liquid interface.
Depending on the bacterial species and conditions, the bacteria may form a well-defined
aggregations at the ALI. The ALI will occur wherever the meniscus of the culture was in
contact with the plastic, but it will be most visible in the center of the well, where the well
tends to be the most optically clear. If desired, the ALI may be circled on the bottom of
the plate with a wax pencil prior to microscopic analysis to aid in its identification. Some
sample results obtained with Pseudomonas aeruginosa are shown in Figure 1B.1.2.
ALTERNATE
PROTOCOL 2
4. Insert a coverslip into each well so that it is held at a 90 angle relative to the bottom
of the well (i.e., perpendicular to the bottom of the well) so that the meniscus of
the medium is at the center of the coverslip. Cover plate and incubate at appropriate
temperature for desired period of time.
Growing and
Analyzing Static
Biofilms
5. Remove each coverslip from its well and rinse off nonadherent cells by dipping in
sterile medium.
6. Stain bacteria by submerging coverslips in 0.1% crystal violet for 10 min.
1B.1.6
Current Protocols in Microbiology
7. Rinse off excess dye by dipping each coverslip in two successive water baths, and
then allow coverslips to air-dry.
8. Visualize the bacteria at the air-liquid interface on each coverslip by microscopy.
BASIC
PROTOCOL 3
The other biofilm systems presented in this unit involve attachment of bacteria to a
surface while bathed in liquid medium. This is the only protocol where the substratum
to which the bacteria attach themselves is also the avenue for meeting nutritional needs
and removing waste. For that reason, this assay is more analogous to growing bacteria
on plates rather than in broth. In this protocol, however, the colonies can grow much
larger, because they are periodically relocated to fresh plates and thus have a fresh supply
of nutrients. An advantage of this system is that the biofilms formed are derived from
clonal growth of the original population of bacteria deposited on the filter, whereas in
liquid media, motile bacteria may be able to invade an aggregation of cells. Likewise,
in the system described below, bacteria have limited ability to detach and migrate or drift
away from the biofilm formed. The stable and spatially restricted nature of this system
makes the colony biofilm especially useful for observing the effects of antimicrobial
agents, as changes in cell number can be more easily attributed to cell death rather than
detachment.
Materials
Bacterial strain of interest
Appropriate liquid medium for bacterial strain under study (APPENDIX 2C)
Appropriate agar medium with and without antibiotic (or other) supplementation
PBS (APPENDIX 2A), sterile
Forceps, sterilized (e.g., autoclaved or flame-sterilized in 70% v/v ethanol)
Poretics 25-mm-diameter black polycarbonate membranes with pore size of
0.22 m (GE Osmonics catalog no. K02BP02500)
100-mm-diameter Petri dishes, sterile
UV light source (e.g., UVP 8-W multiple-ray laboratory lamp)
15-ml tubes, sterile, with tightly fitting lids
Vortex mixer
1. On the night before the colony biofilm assay is to be started, inoculate the bacterium
of interest in 3- to 5-ml cultures and grow overnight (i.e., to stationary phase).
Figure 1B.1.3
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Technologies
1B.1.7
Current Protocols in Microbiology
2. Using sterilized forceps, place 25-mm black polycarbonate membranes with a pore
size of 0.22 m in a sterile Petri plate. Position plate 30 cm from a UV light source
and treat with UV light in a sterile environment (e.g., a biological control hood) for
10 min per side, using the sterilized forceps to flip the plate.
Here and in all subsequent steps, the Petri plate should be kept covered whenever possible
to maintain the sterility of the membranes.
The number of membranes needed depends on the desired length of the experiment. Two
membranes are required for the initial baseline count of cells (in duplicate), and four more
are needed for each time point (for duplicate sampling of treated and untreated cells).
Additionally, a few extra membranes may be desirable as backups. For example, starting
with sixteen membranes per strain for an experiment with a 72-hr antibiotic treatment
period will allow duplicate sampling of treated and untreated cells every 24 hr, leaving
two extra backup membranes per strain.
6. After the incubation in step 5 is complete, sample biofilm growth in the following way.
a. Aseptically transfer a membrane to each of two separate 15-ml tubes containing
10 ml sterile PBS, such that each tube contains a single membrane.
The membrane can be gently folded over onto itself before it is lifted off the plate.
Doing so will allow the membrane to fit easily into a 15-ml tube.
b. Cap tubes and vortex samples for two pulses of 60 sec each (or as necessary) to
detach all bacteria.
c. Prepare dilution series of the vortexed samples, and plate each dilution on a separate
agar plate. Incubate plates overnight at the appropriate temperature and determine
the average number of colony-forming units (cfu) per membrane.
The counts obtained here will serve as the baseline for future experimentation; this
sampling time point is designated T = 0 with respect to the start of experimental
treatments.
Growing and
Analyzing Static
Biofilms
7. Transfer half of the remaining membranes (step 5) to fresh agar plates not containing
antibiotic, and transfer the other half to agar plates supplemented with antibiotic
at the appropriate concentration. Incubate all plates at appropriate temperature for
24 hr.
1B.1.8
Current Protocols in Microbiology
8. When incubation is complete, sample biofilm growth (as in step 6) in two untreated
membranes and two treated membranes. Transfer the remaining untreated membranes to fresh agar plates not containing antibiotic, and transfer the remaining treated
membranes to fresh agar plates supplemented with antibiotic at the appropriate concentration. Incubate plates at appropriate temperature for another 24 hr.
9. Repeat step 8 until all colony biofilms have been sampled.
BASIC
PROTOCOL 4
Materials
Silicone sealant
Sodium hypochlorite
70% (v/v) ethanol
Bacterial strain of interest
Appropriate liquid medium for bacterial strain under study (APPENDIX 2C)
20-G needles, sterile (Becton Dickinson catalog no. 305175)
Flat-bottom 6-well plates, sterile, with lids
1/16 1/16in. (0.16 0.16cm) straight and 90 -elbow, barbed, polypropylene
fittings (such as those included in Cole-Parmer catalog no. 6365-90)
0.8- to 1.6-mm-i.d. silicone tubing (e.g., Watson-Marlow catalog no. 913.A008.016
or 913.A016.016)
2-liter flasks
Two-hole rubber stopper (no. 8) equipped with a long glass tube (i.e., one that can
reach almost to the bottom of a 2-liter flask) and a short glass tube (for gas
exchange)
Peristaltic pump (e.g., Watson-Marlow PumpPro) equipped with Marprene
manifold tubing (inner diameter, 0.8 mm; e.g., Watson-Marlowe catalog no.
978.0165.000)
Inverted microscope (UNIT 2A.1)
Modify 6-well plate lid
1. Heat a 20-G needle in the flame of a Bunsen burner. Push the heated needle through
the lid of a 6-well plate such that it enters directly above one side of a well. Remove
the needle and repeat this process to make a hole in the lid directly above the opposite
side of the same well.
The holes should be large enough for the needle to pass through freely.
These holes will serve as ports for connecting a peristaltic pump that will supply growth
medium to and carry waste away from the well. If desired, multiple wells may be prepared
on the same plate and/or multiple plates may be run from the same pump simultaneously,
depending on the number of channels the pump has for tubing.
2. Obtain two straight 1/16 1/16in. (0.16 0.16cm) barbed polypropylene fittings
(i.e., the type used to connect two pieces of tubing). Using a sharp razor blade,
carefully cut off one of the two male ends on each fitting to give a modified fitting
with one male end and a flat base (see Fig. 1B.1.4).
Emerging
Technologies
1B.1.9
Current Protocols in Microbiology
Figure 1B.1.4
Figure 1B.1.5 Diagram of Kadouri drip-fed biofilm system. Fresh culture medium is pumped onto
a biofilm grown in the bottom of a well in a 6-well plate, while planktonic bacteria and spent medium
are removed through a needle placed on the opposite side of the well. Numbers are explained in
Basic Protocol 4, step 4.
3. Set the flat base of each modified fitting on top of the 6-well plate lid (step 1) such
that the hole in the fitting lines up with one of the holes in the lid, and then pass a
20-G needle through both the fitting and the hole in the lid to maintain the alignment.
Secure each fitting to the lid of the plate using a silicone sealant, and let the sealant
dry completely (see Fig. 1B.1.4).
Once constructed, this modified lid can be sterilized with ethanol or UV irradiation and
reused multiple times with fresh plates in subsequent experiments.
Growing and
Analyzing Static
Biofilms
1B.1.10
6. Submerge the silicone tubing from step 4, not including the Marprene manifold
tubing already installed on the pump, in a beaker containing distilled water and cover
with foil. Autoclave along with the flasks prepared in step 5.
7. Connect the plate lid to the medium reservoir line and the waste line as follows (also
see Fig. 1B.1.5).
a. Using polypropylene fittings, connect one of the intake lines on the pump to
the silicone tubing that will eventually lead to the medium reservoir. Connect the
corresponding outflow line to the end opposite the 90 elbow on one of the silicone
tubes that will eventually connect to the modified plate lid.
b. Connect another outflow line on the pump to the silicone tubing that will eventually
lead to the waste receptacle. Connect the corresponding intake line to the end
opposite the 90 elbow on the other silicone tube that will eventually connect to
the modified plate lid (Fig. 1B.1.5).
c. Attach a sterile 20-G needle to the elbow fitting on each of the two silicone tubes
that are to be connected to the modified plate lid.
8. Adjust the growth medium influx and efflux rates in the following way.
a. Place a sterile, capped microcentrifuge tube in a rack and make two small, diametrically opposed holes in the lid using a 20-G needle heated over a flame.
b. Pipet 1 ml of flow-adjustment water (step 5) into the open tube, close the lid,
and then insert the 20-G needles from step 7 through the holes in the lid.
c. Return the two-hole rubber stopper to the flask containing the flow-adjustment
water. Connect the silicone tubing that will eventually serve as the growth medium
intake line to the long glass tube on the stopper, and place the open end of the
appropriate silicone tube in the waste receptacle.
d. Start the peristaltic pump at a flow rate of 12 ml/hr (2.0 rpm on a Watson-Marlow
PumpPro). Adjust the tension on the manifold tubing such that the water level in
the microcentrifuge tube is steady or slightly decreasing.
The purpose of this step is to adjust the liquid flow such that the well will neither dry out
nor overflow during the actual experiment. Increasing the flow rate and observing for a
period of time will make subsequent recognition of steady or slightly decreasing water
levels easier.
9. Stop the pump, remove the 20-G needles from the microcentrifuge tube, and insert
them into the modified fittings on the 6-well plate lid.
10. To 500 ml sterile H2 O (step 5), add sodium hypochlorite to a final concentration of
0.05% (v/v). Remove the two-hole rubber stopper (still connected to the eventual
growth medium intake line) from the flask containing the flow-adjustment water,
squirt 70% ethanol around its sides, and transfer it to the flask containing the sodium
hypochlorite solution. Restart the pump and run the sodium hypochlorite solution
through the system for at least 3 hr at a flow rate of 12 ml/hr.
Emerging
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1B.1.11
Current Protocols in Microbiology
Rinsing with 70% ethanol solution will help ensure sterility each time the rubber stopper
is transferred to a new flask. In addition, flushing with sodium hypochlorite sterilizes the
pumps Marprene tubing, which cannot be autoclaved.
11. Turn off the pump and aseptically transfer the two-hole rubber stopper (still connected
to the eventual growth medium intake line) to the flask containing 1.5 liters of sterile
water for rinsing. Restart the pump and flush the system with this water for 3 hr or
overnight, adjusting the flow rate as necessary to equalize the inflow and ouflow of
liquid.
This rinsing step removes the sodium hypochlorite from the system and helps ensure that
the input and output flow are balanced.
20. Sterilize the system by flushing with 0.05% sodium hypochlorite as in step 10, and
then rinse by flushing with sterile water as in step 11.
1B.1.12
Current Protocols in Microbiology
21. Dismantle all tubing and coil it gently into the bottom of a 1-liter beaker. Cover with
distilled water, cover beaker with foil, and autoclave to sterilize.
After autoclaving, the tubing may be reused for future experiments; however, before setting
up a new system, be sure to examine the entire length of each piece of tubing for clogs or
holes that could cause pressure build-ups or leaks.
COMMENTARY
Background Information
Microtiter plate assay
Permutations of the microtiter dish biofilm
assay (see Basic Protocol 1) have been in use
for a number of years, due to their ease of use
and the adaptability of the general protocol to
a variety of applications. This assay is equally
well suited to the analysis of tens or hundreds
of strains or species and is particularly useful as
the basis of a genetic screen for mutants defective in surface attachment capabilities. Once
conditions have been determined in which the
wild-type strain forms a biofilm, a library of
mutants can be screened to identify those that
differ in terms of the extent or kinetics of attachment. Additionally, slight modifications to
this system would allow one to look at the influence of other variables that may affect biofilm
formation, such as nutritional provision (e.g.,
the carbon or nitrogen source used or the availability of phosphate or iron) or the presence
of antibiotics or detergents. It is important to
keep in mind that due to the indirect nature
of biofilm assessment in this system, it is desirable to pair this assay with another method
to further examine biofilm formation. For example, using direct microscopic observation of
the biofilm to confirm the crystal violet staining pattern is suggested.
Air-liquid interface assay
This technique (see Basic Protocol 2)
emerged from a simple adjustment to the typical practice of growing biofilms in multiwell
plates. Many bacteria are limited to or prefer
aerobic growth and therefore are most prone
to forming biofilms at the interface between
the medium and air. By tilting the 24-well
plate such that the air-liquid interface (ALI)
falls on an optically clear portion of the plate,
the potential for microscopic analysis is improved. This system makes an excellent companion to the microtiter dish assay (see Basic
Protocol 1), as both are useful at many of the
same time points and stages of biofilm formation. This assay also allows the visualization of
live bacteria without the need for crystal violet staining; therefore, real-time interactions of
the bacteria with the surface can be monitored
(Caiazza and OToole, 2004). The ALI assay
can be extended to later time points by replacing the growth medium periodically, but for an
accurate study of a mature biofilm, a system
in which the medium is continually replaced,
such as the Kadouri system (see Basic Protocol 4) or a flow cellbased system (UNIT 1B.2),
is recommended.
Colony biofilms
Colony biofilms (see Basic Protocol 3)
have typically been used for the purpose of
determining antibiotic resistance (Anderl
et al., 2000; Walters et al., 2003). Such systems
are well suited to studies of the viability of
cells within a biofilm in response to various
assaults, such as exposure to antimicrobial
agents or UV light.
Kadouri system
The Kadouri drip-fed biofilm system
(named for the postdoctoral researcher who developed it) is a close relative of the continuousflow biofilm systems presented in other units
in this chapter. Although the medium is constantly being exchanged in this system (see
Basic Protocol 4), the shear forces on the bacteria are minimal; therefore, this system may
apply less mechanical stress to the biofilms.
Planktonic cells in this system are not swiftly
swept away but may linger in the well for
longer periods. Hence, the system may be more
closely representative of an equilibrium state
between the attached and planktonic lifestyles.
This system is typically considered a lowflow scheme and serves as an intermediate
regimen between static assays and standard
flow cell systems; however, because the system involves continuous feeding with growth
medium, it allows observation of the entire process of biofilm formation, from reversible to
irreversible attachment and on to microcolony
formation, macrocolony formation, and detachment. One advantage this system provides
is the potential to generate and easily access
a relatively large amount of biomass that can
be scraped from the bottom of the well and
used for applications such as DNA microarrays and proteomics. This system is also useful for many of the microscopy assays typically
done by flow cellbased methods. The 6-well
plate format also has the advantage of being
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ALI
Growing and
Analyzing Static
Biofilms
inoculum has a low cell count and the incubation time is short, there may be an insufficient
number of bacteria adhered to the surface at
the time of viewing, and this may make identification of the ALI more difficult. If this is the
case, consider starting with a less dilute inoculum or increasing the incubation time. Alternatively, circling the ALI with a wax pencil may
make it easier to locate under the microscope.
Stringent washing of the wells is useful for removing any unattached bacteria; this wash step
will help prevent colonization of the formerly
dry portion of the well after the whole well
has been bathed in medium. Even under the
best washing conditions, bacteria can detach
from surfaces and colonize other portions of
the well. For this reason, it is important to work
quickly and analyze plates on a microscope immediately after they have been prepared for observation. If there are many strains or species
to analyze, it may be advisable to distribute
them over a number of plates so that only a
few need be prepared for viewing at one time.
Colony biofilms
One important aspect of this assay is the
predetermination of appropriate experimental conditions (e.g., antibiotic concentration).
The resistance of a bacterium to antimicrobial
agents in a colony biofilm may differ significantly from the resistance of the same bacterium in a different biofilm system, such as the
microtiter plate biofilm assay system described
earlier in this unit (see Basic Protocol 1). It is
recommended that the organism be tested on
various concentrations of antibiotic to find appropriate experimental conditions.
The initial inoculation of the colony biofilm
is an important step that can affect the success of the experiment. It is important to ensure
that each membrane receives an equal amount
of cells and that there is only a single point
of inoculation in the center of the membrane.
To prevent any of the culture from spraying
elsewhere on the membrane, touch the drop of
liquid at the end of the pipet tip gently to the
membrane before depressing the pipet plunger
to its final stop. A single, carefully centered
spot will ensure the greatest experimental reproducibility, as the counting is no longer accurate if the bacteria have grown off the edges of
the membrane. In addition, complete and uniform removal of the bacteria from the membrane is important for accuracy. Empirically
testing different conditions to maximize removal of bacteria from the membrane during
the vortexing step while preserving viability is
recommended.
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Kadouri system
Two common problems that are experienced in this system are overflow and drying out of the wells. Both can be addressed
by very carefully calibrating the influx and efflux rates (see Basic Protocol 4, step 8). If the
medium is evaporating too quickly from the
system, consider increasing the influx rate to
compensate. Likewise, ensuring that there is
sufficient medium in the reservoir is especially
crucial when the system will be left unattended
for long periods. Due to the nature of biofilmforming bacteria, it is also important to carefully monitor the tubing prior to each experiment. The waste tubes are especially prone to
being colonized, and the colonizing bacteria
can completely block the flow, causing pressure to build up and rupture the junctions between pieces of tubing. If blockage is found,
the piece of tubing must be trimmed to remove
the clogged portion, or else the entire section
must be replaced.
This system is designed for observation of
biofilms forming on the bottoms of the wells,
but depending on the conditions and the strain
or species used, the cells may in addition (or
instead) form a biofilm, known as a pellicle, at
the air-liquid interface. To prevent this, the environment at the bottom of the plate should be
made as hospitable as possible for the bacteria.
A common problem is the lack of oxygen at the
bottom of the well. This may be overcome by
increasing the flow rate in the system to deliver
more oxygenated medium to the cells while
more rapidly removing unattached bacteria. In
some cases, adjusting the composition of the
medium (e.g., by providing an alternative electron acceptor) will decrease the requirement
for oxygen. As with all of the systems presented in this unit, optimal conditions must be
determined empirically for each organism.
After the setup is complete, this system requires little daily attention as the cells grow.
It should be monitored periodically (twice a
day) for overflow or evaporation of the media
in the wells and for potential rupture of tubing,
and also to ensure that there is an ample supply
of medium in the reservoir. However, biofilms
may take over a week to develop to the desired
stage (as determined by microscopic observation), and this should be taken into consideration when planning experiments.
Anticipated Results
Microtiter plate assay
Using the microtiter plate assay is an effective way to monitor bacterial attachment to an
abiotic surface over time. In general, if this assay were performed over several time points,
one would expect to see a progressive increase,
possibly followed by a decrease, in attached
biomass. The eventual decrease in crystal violet staining is presumed to occur because
the lack of nutrients may stimulate the bacteria to detach from the surface (Sawyer and
Hermanowicz, 1998; Hunt et al., 2004). The
time course of biofilm growth must be determined empirically for each organism and set
of conditions used. The staining pattern also
varies from one organism to another. For example, when P. aeruginosa is grown with only
oxygen as an electron acceptor, a ring of staining forms at the air-medium interface. In contrast, when this species is grown on arginine, a
carbon source used aerobically and anaerobically by P. aeruginosa, the entire well shows
positive crystal violet staining. Examples of
typical results with this assay for P. aeruginosa
are shown in Figure 1B.1.6.
Direct enumeration of bacteria from
microtiter plate assay
When bacteria are grown in the microtiter
plate assay, one generally expects to see increased adherent biomass over a period of time
followed by a decrease as the bacteria detach
or die. The number of cfu eluted from each
well is expected to correlate with the amount
of crystal violet staining obtained at the corresponding time point.
assay
For bacterial species that form a ring around
the well in the microtiter plate assay, the airliquid interface should be heavily colonized
with attached bacteria in this system. For nonmotile or facultative anaerobic bacteria, the
boundary may not be as well defined. Some
typical results obtained with the bacterium
P. aeruginosa are displayed in Figure 1B.1.2.
ALI
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Figure 1B.1.6 Microtiter plate biofilm assay of Pseudomonas aeruginosa. (A) Crystal violetstained wells from a microtiter
dish at 0 and 10 hr postinoculation. The bacteria were grown on glucose supplemented with casamino acids. Wells were
inverted to facilitate photography. (B) Quantification of staining at various time points over 10 hr, based on absorbance
readings made at 600 nm. (C) The wells show formation of a biofilm when P. aeruginosa is grown on LB (left; requires
aerobic growth) or on arginine (right; can be utilized anaerobically).
Figure 1B.1.7 Using the Kadouri system, a biofilm of P. aeruginosa was grown in minimal medium
supplemented with arginine at room temperature and photographed by phase-contrast microscopy
at 24, 48, and 118 hr. Microcolonies can be observed as early as 24 hr, with large macrocolonies
formed by 118 hr.
Time Considerations
Growing and
Analyzing Static
Biofilms
are performed to enumerate the attached bacteria, will take longer, as more handling is involved and the plated bacteria will need to
be incubated for another 12 to 24 hr for the
colonies to grow large enough to be counted.
ALI assay
The ALI assay follows a time course similar
to that of the microtiter plate assay. The length
of the assay will again depend on the desired
time frame to be studied. Washing of plates followed by microscopic analysis takes 15 min
per well. Alternate Protocol 2 is similar to the
ALI assay in terms of its time requirements.
Colony biofilms
During the growth of colony biofilms, the
bacteria require little attention, except that
the membranes must be transferred to fresh
plates every 24 hr. Over the course of an experiment, the separation of bacteria from the
membrane and subsequent enumeration are the
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Literature Cited
Anderl, J.N., Franklin, M.J., and Stewart, P.S.
2000. Role of antibiotic penetration limitation
in Klebsiella pneumoniae biofilm resistance to
ampicillin and ciprofloxacin. Antimicrob. Agents
Chemother. 44:1818-1824.
Caiazza, N.C. and OToole, G.O. 2004. SadB is required for the transition from reversible to irreversible attachment during biofilm formation
by Pseudomonas aeruginosa PA14. J. Bacteriol.
186:4476-4485.
Christensen, G.D., Simpson, W.A., Younger, J.J.,
Baddour, L.M., Barrett, F.F., Melton, D.M., and
Beachey, E.H. 1985. Adherence of coagulasenegative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices.
J. Clin. Microbiol. 22:996-1006.
Danhorn, R., Hentzer, M., Givskov, M., Parsek,
M.R., and Fuqua, C. 2004. Phosphorus limitation enhances biofilm formation of the plant
pathogen Agrobacterium tumefaciens through
the PhoR-PhoB regulatory system. J. Bacteriol.
186:4492-4501.
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UNIT 1B.2
This unit describes methods for growing and analyzing biofilms under hydrodynamic
conditions in flow cells. The flow in these cells is essentially laminar, which means that
the biofilm can be grown under highly controlled conditions and that perturbations such
as addition of antibiotics or change of the growth medium can be done efficiently at a
defined time point. The Basic Protocol includes inoculation of the flow cells, running
of the system, microscopic inspection and imaging of flow cellgrown biofilms, image
capture and analysis, and disassembly and cleaning of the flow cells. Support Protocols
1, 2, and 3 describe, respectively, construction of a flow cell system, construction of a
bubble trap, and procedures related to fluorescent in situ hybridization (FISH) of speciesidentifying probes for a biofilm that has been grown in a flow cell.
Confocal laser scanning microscopy (CLSM) is used for monitoring and imaging of
structure development in the flow cellgrown biofilms. For a detailed description of
CLSM, see UNIT 2C.1. The COMSTAT image-analysis program is used for calculation of
objective parameters such as surface coverage, biofilm thickness, and biovolume from
the acquired biofilm images.
CAUTION: Follow all biosafety requirements relevant to the microorganism under investigation. Refer to UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for instructions
on safe handling of microorganisms.
BASIC
PROTOCOL
Materials
Inoculum, e.g., fresh overnight culture of desired microorganisms
70% and 96% (v/v) ethanol
Medium (e.g., see APPENDIX 2C)
Silicone glue (3M Super Silicone Sealant Clear)
Flow cell system (BioCentrum-DTU, Technical University of Denmark, or see
Support Protocol 1)
Syringes with needles (e.g., Terumo LU-100, 27-G (0.4 12 mm), 0.5 ml)
Clamps
Microscope (Zeiss LSM5 META; Chapter 2)
Scalpels
Computer software:
Imaris (Bitplane; http://www.bitplane.com)
COMSTAT (BioCentrum-DTU, Technical University of Denmark)
Matlab (required for COMSTAT; MathWorks; http://www.mathworks.com)
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Current Protocols in Microbiology (2005) 1B.2.1-1B.2.15
C 2005 by John Wiley & Sons, Inc.
Copyright
1B.2.1
2. Inject the inoculum carefully into the flow chamber. Wipe the tubing with 70%
ethanol after removing the needle. Reseal the tubing by applying a thin layer of
silicone glue at the needle hole.
3. After inoculation of all flow channels, clamp the effluent tubes immediately after the
flow cell and place the entire flow cell on a flat surface with the substratum downward
to allow cell adhesion to the substratum.
4. After 1 hr, remove the clamps, turn the flow cell over, and resume the medium flow.
The substratum is now on top of the flow cell.
7. Once the flow cell is on the microscope stage, manipulate the system on site, e.g., by
injecting reagents into the flow channels, stopping the flow, changing the flow rate,
or switching the medium.
Take care to avoid air bubbles at this point, since most changes must be performed
downstream of the bubble traps.
10. Remove the substratum from the flow cell using a scalpel.
If the substratum is made of glass, it will inevitably break in the process.
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13. Calculate objective parameters from the acquired images by using suitable software
such as the COMSTAT image-analysis program. Parameters include:
Biovolume
Area occupied by biomass in each layer
Thickness distribution and average thickness
Area distribution of microcolonies at substratum level
Volumes of microcolonies identified at the substratum level
Fractal dimension of each microcolony identified at substratum level
(Minkovski method)
Fractal dimension (cross-correlation function method)
Roughness coefficient
Distribution of diffusion distances and average diffusion distance
Surface area (substratum coverage) and surface to volume ratio
Maximum thickness of the biofilm.
The COMSTAT program and user instructions are available from http://www.im.dtu.dk/
comstat. The parameters obtained from COMSTAT analysis can be paired and plotted in
a diagram for ease of interpretation. By inspecting the diagrams, it is possible to compare
several samples and determine if a sample is more similar to one group of samples than to
another group, e.g., Figure 1B.2.1. For a detailed description of parameters see Heydorn
et al. (2000b).
Figure 1B.2.1 COMSTAT analysis of biolms formed by three different bacterial species. Each
point represents roughness and average thickness calculated from one biolm CLSM image. The
symbols , +, and represent each of the three species. The two-dimensional analysis of the
correlation between the roughness coefcient and average thickness of the biolms reveals that
it is possible to categorize biolms produced by the three species into three separate groups.
Analysis based on either one of the two parameters alone would not indicate a characteristic
specic to each of the three species. Combining several of the COMSTAT parameters can rene
the analysis further.
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SUPPORT
PROTOCOL 1
Materials
Silicone glue (3M Super Silicone Sealant Clear)
Medium appropriate for organisms and type of biofilm being grown (see APPENDIX
2C)
70% and 96% (v/v) ethanol
0.5% (w/v) sodium hypochlorite
H2 O, sterile
1% hydrogen peroxide
Polycarbonate sheet plastic, 6 and 35 mm thick
CNC tooling machine, or a drilling machine mounted on an upright stand and
equipped with a milling drill-tool (3 mm)
Substratum: 50 24mm glass coverslips or other appropriate material
2-ml syringe (Terumo)
Tubing:
Silicone, 3 mm outer diameter, 1 mm inner diameter
Silicone, 4 mm outer diameter, 2 mm inner diameter
Silicone, 7 mm outer diameter, 5 mm inner diameter
Marprene, 3 mm outer diameter, 1 mm inner diameter (Watson-Marlow)
Peristaltic Pump (Watson-Marlow, 205S)
Medium bottles (Schott or BIO 101)
Rubber stoppers (to fit medium bottles) with holes for glass tubes
Glass tubes, 6 mm outer diameter, 3 mm inner diameter
Air filter, autoclavable (optional)
Clear polypropylene plastic connectors and T-connectors (Cole Parmer,
E-06365-xx), 1/8 in. (3.175 mm) and 1/16 in. (1.588 mm)
Bubble traps (BioCentrum-DTU, Technical University of Denmark, or see Support
Protocol 2)
Microscope (UNIT 2A.1)
Rolling cart for flow systems and pumps (optional)
Waste container
Construct the flow cell
1. Construct the flow cell from a 6-mm thick sheet of polycarbonate according to the
working drawing shown in Figure 1B.2.2 using a CNC tooling machine, or a drilling
machine mounted on an upright stand and equipped with a milling drill-tool (3 mm).
Growing and
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1B.2.4
Current Protocols in Microbiology
Figure 1B.2.2
This leaves a central base portion (60 26 mm, 6 mm thick) with two 8 26mm
ends, each 1 mm thick. The ends facilitate the mounting of the flow cell on a standard
microscope specimen holder, like a normal microscope slide.
3. Mill three channels, 1 mm deep and 40 mm long, in the central base portion of
the plastic base. If advanced tooling equipment is available, carve connectors to the
tubing from the plastic (see Fig. 1B.2.2). Otherwise, still using the milling tool,
vertically drill 3-mm wells into the base at each end of the channels and horizontally
drill pipes (3-mm drill) from the ends of the central portion of the flow cell, until the
pipes connect to the wells.
4. Glue substratum of suitable material and size (e.g., a 50 24mm glass coverslip)
to the flow cell base using silicone glue. Apply the silicone glue as very thin threads
using a 2-ml syringe. Place the threads between each channel and along the perimeter
of the top of the base. Avoid holes in the glue threads to prevent liquid leakage from
the flow cell.
5. Place the substratum on top of the glue and press the substratum gently onto the flow
cell base. Inspect the glue through the substratum (if transparent) to ensure that the
glue is effectively sealing the substratum to the flow cell.
The handle of the syringe piston is an excellent tool for pressing the substratum onto the
flow cell base.
6. If areas with insufficient gluing at the edges are observed, reseal by applying extra
glue outside the flow cell adjacent to the potential leak. If the gluing is insufficient
between the channels, remove the substratum, clean the base with 96% ethanol, and
repeat the mounting procedure (steps 4 and 5).
7. If the flow cell has connectors, attach silicone tubing to them; otherwise, place tube
ends inside the drilled holes and attach with silicone glue.
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Figure 1B.2.3 Schematic of the ow cell system: a, medium bottle; b, peristaltic pump; c, bubble
trap; d, ow cell; e, waste container. Silicone tubing with inner diameter (i.d.) of 2 mm and outer
diameter (o.d.) of 4 mm goes from the medium bottle to the point (arrow) where the tube is fanned
out. All other tubing has an i.d. of 1 mm and o.d. of 3 mm and is made of silicone, except for the
segment that goes through the pump, which consists of Marprene tubing. The connectors used
for assembly of the system are listed under Materials in Support Protocol 1.
8. Equip the medium bottle with a rubber stopper of a suitable size with two holes.
Place one glass tube, long enough to almost reach the bottom of the medium bottle,
into one of the holes (to be used as a siphon tube for medium). Place another glass
tube, short enough to not reach into the liquid, into the other hole (to be used as an
air vent). Cover the short glass tube with metal foil or mount an air filter via a short
connecting plastic tube.
9. Connect the siphon tube to the feed silicone tubing (3 mm i.d., 1 m long). Mount
a straight connector in the end of the feed line to enable easy attachment to the
downstream part of the system after autoclaving. Cover this connector with metal
foil. Assemble and autoclave the remaining parts of the system together.
Connect all tubing either directly to the flow cells or bubble traps, or use straight or
T-shape connectors between the individual pieces of tubing.
Do not attach the medium bottle until the system is sterilized.
10. Split a common feed tube into the required number of channels in the experiment
by using T-connectors to fan out the feed supply line and reduce the tube dimension
from 3- to 1-mm i.d.
11. For each individual channel in the experiment perform the following steps:
a. Connect Marprene tubing to the feed tubing. Connect silicone tubing from the
Marprene tubing to the bubble traps.
b. Connect the bubble traps to the flow cell inlet tubing. Connect outlet tubing from the
flow cell so that it can reach a waste container.
Make sure that the inlet and outlet tubes are sufficiently long to allow the flow cell to be
moved to the microscope stage.
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1B.2.6
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13. Remove the stoppers from the top of the bubble traps and place them in 70% ethanol.
Fill the system with 0.5% sodium hypochlorite solution by pumping at a high flow
rate for 5 to 10 min.
The liquid will pour from the top of the bubble traps; there will be minimal flow downstream
from the traps.
14. Place the stoppers back onto the bubble traps and remove any air bubbles from the
system components by tapping onto a hard surface while the pump is still running.
Continue pumping the disinfectant through the system for 5 to 10 min.
After replacing the stoppers onto the bubble traps, the remainder of the system will quickly
fill with disinfectant.
15. Stop the flow and continue sterilization by allowing the sodium hypochlorite solution
to stand in the system from 2 hr to overnight.
16. Remove all traces of sodium hypochlorite by emptying the disinfectant from the
system and filling and emptying it with sterile distilled water two to three times.
Ensure all disinfectant is removed by purging the system with air between each pass
of water. Conclude by passing 1 to 2 liters sterile distilled water through the system
at lower flow rate overnight (e.g., 0.2 mm/sec, 3 ml/hr).
Emptying and purging the system with air is accomplished by running the pump with the
feed tubing taken out of the liquid.
17. Additionally, pass a solution of 1% hydrogen peroxide through the system, as this
will eliminate biomass not cleaned by the sodium hypochlorite solution.
This is mainly for systems that are not autoclaved.
18. Purge the system with air and fill with medium just prior to starting the experiment. Set the laminar flow in the channels to 0.2 mm/sec (corresponding to
3 ml/hr/channel in the flow cells described here).
SUPPORT
PROTOCOL 2
Materials
10-mm-thick polycarbonate sheet plastic (simple trap) or 35 80 45mm
polycarbonate block (advanced trap)
Drilling machine mounted on an upright stand and equipped with a 3-mm milling
drill-tool and an 8-mm pointed drill (simple trap) or CNC tooling machine
(advanced trap)
2-ml syringes with inner diameter of 8.5 mm (simple trap) or 5-ml syringes with
inner diameter of 12.5 mm (advanced trap)
9 2mm rubber gaskets (advanced trap; M-seals, 221355;
http://www.m-seals.dk/cms.ashx)
Silicone glue (3M Super Silicone Sealant Clear)
Silicone tubing, 3 mm outer diameter (simple trap)
Stoppers (e.g., EFD; 5113-B; http://www.efd-inc.com/catalogs/
EFD-Barrel-Reservoirs.pdf )
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Figure 1B.2.4
DTU, 2005).
Working drawing of the advanced bubble trap base (design copyright Biocentrum-
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SUPPORT
PROTOCOL 3
Materials
Biofilms adhered to the substratum of flow cell system (see Basic Protocol)
Paraformaldehyde fixative (see recipe)
1 phosphate buffered saline (PBS; see recipe)
20% (w/v) acrylamide monomer (200:1 acrylamide:bisacrylamide)
N,N,N ,N -tetramethylethylenediamine (TEMED)
1% (w/v) ammonium persulfate (APS) activator, freshly prepared
Fluorescently labeled oligonucleotide probes (e.g., FITC, Cy3, Cy5; e.g.,
Eurogentec)
FISH prehybridization buffer (see recipe)
FISH hybridization buffer (prehybridization buffer with an appropriate
concentration of formamide; see Stahl and Amann, 1991)
FISH washing buffer (see recipe)
Antifade agent (e.g., SlowFade, Molecular Probes)
Clamps
50-ml beaker
2-ml microcentrifuge tubes
Humidified storage container (e.g., Petri dish with wetted paper or cotton)
Microscope slides with 8- to 10-mm diameter wells for FISH (Novakemi AB)
Incubation chamber (50-ml polypropylene centrifuge tube with FISH hybridization
buffersaturated paper toweling)
Microscope slides and coverslips
Rubber gaskets, 0.7 mm thick
Additional reagents and equipment for calibrating oligonucleotide probes (e.g.,
Stahl and Amann, 1991)
Fix the biofilm
1. Stop the feed pump and clamp off the effluent tubes from the flow chambers. Cut off
the inlet tubes so that 3 to 4 cm of inlet tube is left on the flow chamber (i.e., enough
to reach the bottom of a 2-ml microcentrifuge tube).
2. Place the pump on the effluent side of the flow cell, and, after mounting the tubes to
the pump, gently remove the clamps. Using reverse flow, slowly activate the pump
so that a drop of medium appears on the end of the inlet tubes.
3. Put paraformaldehyde fixative in a 50-ml beaker and submerge the inlet tube into
the fixative. Draw the fixative into the channels using a flow rate of 0.2 mm/sec for
15 min. Stop the flow and allow the fixative to work for 1 hr.
CAUTION: Paraformaldehyde is carcinogenic. Handle in a ventilated fume hood, wearing suitable gloves (see UNIT 1A.3).
1B.2.9
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6. Reverse the flow briefly to let a small drop appear on the end of the inlet tubes.
Then insert the tubes into the activated acrylamide solution and draw it into the flow
channel at a pump speed of 0.5 mm/sec.
Note that the pump rate is increased to ensure that the chamber is filled with the acrylamide
solution quickly before it starts to solidify. In the described system, this takes exactly 1 min,
50 sec. It is recommended that the time it takes to fill the flow cell chamber be measured
using an empty channel.
7. Gently clamp off the effluent tubing and remove the pump. Allow to the acrylamide
to solidify for at least 1 hr at room temperature.
9. Clean used equipment, taking special care to remove solidified acrylamide from the
flow channels and the inlets and outlets of the flow cells. Use a dishwashing brush
for the channels and a needle for the inlets and outlets.
13. Meanwhile, add the probe(s) to 30 l-aliquots of FISH hybridization buffer for a
final concentration of 2.5 g/ml.
More than one probe can be used in each hybridization.
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14. Remove the FISH prehybridization buffer from the wells containing the slabs, add
the hybridization solution containing the probes, and incubate in the formamidesaturated incubation chamber at 37 C, in the dark for 2 hr or longer.
15. After incubation, remove the hybridization solution and wash the slabs as follows:
a. Add 50 l FISH prehybridization buffer, and incubate 30 min.
b. Remove the FISH prehybridization buffer, add 50 l FISH washing buffer, and
incubate 30 min.
c. Remove the washing buffer, add distilled water, leave for 15 to 30 sec, and remove
the water.
The incubations in the washing steps are also performed in the formamide-saturated
incubation chamber at 37 C, in the dark.
16. Mount the slabs on microscope slides with the biofilm facing up. Place a rubber
gasket to protect the embedded biofilm from being squashed by the coverslip.
17. Before sealing the sample, add a drop of antifade agent to the slab. Seal the sample
with tape and store in a dark cool place until microscopy is performed.
The mounted samples can be stored for several months at 4 C if kept moist and in the
dark.
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COMMENTARY
Background Information
Studies of the fouling of industrial tubular
systems in the early 1980s required reliable
and reproducible microscopic observations of
biofilm development and encouraged the design of the first flow cells. One of the first flow
cells used for biofilm research was the Robbins
device (McCoy et al., 1981). This apparatus is
constructed of admiralty brass and does not
allow direct microscopic inspection but rather
has replaceable plugs with sampling surfaces
allowing microscopic inspection of relatively
undisturbed biofilm. The emerging realization
in the mid-1980s of the importance of biofilms
in medical context prompted the development
of a modified Robbins device which was constructed from an acrylic block, and contained
plugs with medically relevant surfaces such
as catheter material (Nickel et al., 1985). A
need for direct microscopic observation of developing biofilms led to the development of
flow cells with glass surfaces mountable on
microscopes. One of the first flow cells of this
type consisted of two microscope slides, two
cover slips, and silicone tubing, and was assembled with silicone adhesive (Caldwell and
Lawrence, 1986). Doug Caldwells group at
the University of Saskatchewan subsequently
developed the flow cell constructed of Plexiglas and mounted with a microscope slide similar to the flow cell described in detail in this
unit (Wolfaardt et al., 1994). These flow cells
are well suited for laminar flow conditions,
whereas studies of biofilm development under
high-shear turbulent flow conditions may use
other systems such as square glass tubing flow
cells (Stoodley et al., 1999). Biofilm growth in
traditional flow cells results in relatively little
biomass, but in cases where a large amount of
biomass is needed, e.g., for proteome or transcriptome analysis, the biofilms can be grown
in silicone tubing (Sauer et al., 2002). To estimate the extent of biofilm development in the
silicone tubing, a glass capillary with a square
cross-section can be inserted into the tubing
during the setup of the system to provide a
window for microscopic investigation (Sauer
et al., 2002).
Although conventional light microscopy
can be used, confocal laser scanning microscopy (CLSM) is particularly well suited
for monitoring of three-dimensional structure
formation in flow cellgrown biofilms. As a re-
Growing and
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Biolms in Flow
Cells
1B.2.12
Current Protocols in Microbiology
Backgrowth
In the standard flow cell setup, there are
no special precautions to prevent fouling of
the tubing upstream of the flow cells. In most
cases there will be little backgrowth, but, if
the cells are highly motile, it may occur. Because biofilm in the upstream tubing will use
substrate components and release waste products, it may affect the biofilm in the flow cell
and result in less controlled conditions: therefore, it should be removed. For the same reasons, microscopic inspection and bioimaging
of flow cellgrown biofilms should be done
near the inlet. One way to reduce backgrowth
is to quickly disconnect the inlet tubing from
the flow cell periodically (e.g., once a day), cut
off a few centimeters, and reconnect. It may
also be necessary to exchange effluent tubes if
they become heavily fouled.
Arrangement of the effluent tubes
The effluent tubes must be arranged carefully, to avoid reverse siphoning from the
waste container (by placing the end of the tubes
above the surface of the waste reservoir), and
to avoid accidental entrance of air in case of
leaks (by placing the effluent tubes above the
level of the flow cells).
Flow stoppage
The biofilms formed by some microbial species are intolerant to interruption of
medium flow, and even short periods of flow
stoppage will result in detrimental effects, notably complete dispersal of the biofilm. Hence,
it is important to plan ahead of the experiment.
For instance, if the growth of the biofilms is
carried out in a different room than the one
to be used for microscopic analysis, electrical
power must be available for the pump along
the route from one room to another, e.g., by
having a very long power cord. Also, when
the supply flasks are exchanged, it must be
done very quickly, with the pump running. The
small amount of air that enters the medium
lines at this point is removed by the bubble
traps.
Selection
The researcher should keep in mind that
flow cell experiments are selection experiments. Biofilms are dynamic systems, and mutants may efficiently out-compete wild-type
organisms. Biofilm experiments should therefore always be done in replicates, and biofilm
development properties of organisms from
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Current Protocols in Microbiology
Anticipated Results
Because many different aspects of biofilm
biology can be investigated, many different results can be expected. Some of the
more general results include: information
about biofilm formation steps including adhesion, microcolony formation, and further
structural biofilm development; information
about structure-function relationships in complex biofilms, including multispecies biofilms;
quantitative data to compare biofilms, e.g.,
those formed by a mutant and its wild-type.
Time Considerations
Assembly and sterilization of the flow cell
system will take 1 to 2 days. After that, the
system may run for several weeks, but many
biofilms reach a mature state within 5 to
7 days. Embedding and FISH labeling can
take up to two days, after which the sample
can be examined; however, live systems can
be observed instantly. Image analysis can take
several days.
Literature Cited
Caldwell, D.E. and Lawrence, J.R. 1986. Growth
kinetics of Pseudomonas fluorescens microcolonies within the hydrodynamic boundary layers of surface microenvironments. Microb. Ecol.
12:299-312.
Hentzer, M., Riedel, K., Rasmussen, T.B.,
Heydorn, A., Andersen, J.B., Parsek, M.R.,
Rice, S.A., Eberl, L., Molin, S., Hoiby, N.,
Kjelleberg, S., and Givskov, M. 2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone
compound. Microbiology 148:87-102.
Heydorn, A., Ersboll, B.K., Hentzer, M., Parsek,
M.R., Givskov, M., and Molin, S. 2000a.
Experimental reproducibility in flow-chamber
biofilms. Microbiology 146:2409-2415.
Heydorn, A., Nielsen, A.T., Hentzer, M., Sternberg,
C., Givskov, M., Ersbll, B.K., and Molin, S.
2000b. Quantification of biofilm structures by
the novel computer program COMSTAT. Microbiology 146:2395-2407.
Klausen, M., Aaes-Jorgensen, A., Molin, S., and
Tolker-Nielsen, T. 2003. Involvement of bacterial migration in the development of complex
multicellular structures in Pseudomonas aeruginosa biofilms. Mol. Microbiol. 50:61-68.
Growing and
Analyzing
Biolms in Flow
Cells
Key References
Wolfaardt et al., 1994. See above.
The first paper reporting the use of the flow cell
which is described here and widely used.
Lawrence et al., 1991. See above.
The first paper reporting the use of CLSM in biofilm
research.
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Internet Resources
http://www.im.dtu.dk/comstat
Web interface to the COMSTAT program and instructions.
http://www.itqb.unl.pt:1111/clsmip/
Web interface to CLSM biofilm imaging database
and image processing application.
http://www.erc.montana.edu/
Contains useful information for the biofilm researcher.
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UNIT 1B.3
The study of biofilms has stimulated the development of laboratory-based model systems which can be used to recreate various features of natural biofilm communities.
Whereas naturally formed biofilms are sufficiently complex that they are not experimentally tractable, model systems can provide controlled conditions and reproducible
sample sets. In a biofilm model, questions may be asked and conditions altered so that
specific elements of the biofilm structure, physiology, metabolism, and gene expression
can be described. Batch culture and flow-cell models are described in UNITS 1B.1 & 1B.2,
this unit describes two continuous culture laboratory systems, and UNIT 1B.4 describes the
study of natural biofilms in the field. Most biofilm researchers will find that different
model systems should be used, or standard methods optimized, to properly address their
questions.
In this unit, two models for the growth of biofilms under continuous culture conditions
using spinning discs (see Basic Protocol 1) and silicone tubes (see Basic Protocol 2) are
described. The spinning disc model consists of a plastic plate with an integrated stir bar
and slots for eighteen small glass or plastic chips. The spinning disc is submerged in
a reactor flask containing medium and incubated on a stir plate under continuous flow.
The silicone tube model consists of a length of silicone tubing through which a medium
is supplied under continuous flow. Both model systems provide multiple samples for
viable count analyses, and tube-grown biofilm samples may also be used for protein and
transcriptional expression profiling and biochemical analysis.
In comparison to the flow cells and static systems, the spinning disc and tube biofilm
models have advantages and disadvantages. For example, the biofilms which grow on the
chips of a spinning disc reactor are similar in structure and metabolic profile, providing
reproducible and statistically clean results. In contrast, more heterogeneous biofilms are
generated in flow cells and tube biofilms as nutrient and waste gradients are formed
along the length of the reactor and flow characteristics are altered. In static culture
biofilms, steep oxygen and nutrient gradients contribute to the formation of extremely
heterogeneous biofilms. Tube-grown biofilms are also advantageous in that they can
provide a homogeneous biofilm and large amounts of biomass for expression analyses.
Generally, spinning disc and tube-grown biofilms are not easily studied microscopically,
although tube biofilms may be grown in such a manner that nondestructive microscopic
examination is possible. For such studies, flow cell or static culture biofilms are more
appropriate.
CAUTION: Follow all biosafety requirements relevant to the microorganism under investigation. Refer to UNIT 1A.1 and other pertinent resources (see APPENDIX 1B) for instructions
on safe handling of microorganisms.
BASIC
PROTOCOL 1
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1B.3.1
Figure 1B.3.1 The spinning disc is made of polyvinylchloride into which eighteen slots have
been drilled, 4-mm wide and 1.5-mm deep. The biolm substrate (chips) are made of ground
glass and placed into the slots and held in place by red silicone rubber tabs. Also visible in the
top-down view are the screws that hold the star-head magnetic stir-bar in place to the underside
of the disc. The side view shows the placement of the stir bar relative to the chips.
Action
Steps
Day 1
117
Day 2
18
Day 3
1927
Day 4
28
protocol utilized porcelain discs, recent adaptations have utilized stainless steel, glass,
and plastic discs. The most important piece of equipment in this protocol is the spinning
disc. Made of Teflon or polyvinylchloride and silicone rubber, the disc is attached to a
magnetic stir bar on the underside, with chips of varying materials on the upper side (see
Fig. 1B.3.1). In the protocol described here, the disc is fitted with eighteen glass chips.
The biofilm reactor consists of a 1-liter glass beaker with an overflow port. Lastly, an
adjustable-speed peristaltic pump is required. Ideally, a multichannel pump is used to
allow multiple experiments to be run simultaneously. Because the bacterial culture within
the reactor is continually supplied with fresh medium, the culture is maintained in the
logarithmic phase, as in a chemostat. The chemostat allows the culture to approach steadystate so that biofilm growth is uncoupled from the planktonic growth phase. Therefore,
observed changes in the biofilms will be independent of growth phase phenomena. Chipgrown biofilms are particularly well-suited for antimicrobial susceptibility and viability
testing, and will yield highly reproducible results.
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Table 1B.3.1 presents a timeline for the procedures involved in this method.
1B.3.2
Current Protocols in Microbiology
Materials
Silicone glue
Starter culture (mid-log phase bacterial culture)
Rich medium (e.g., LB, TBS, or other; APPENDIX 2C)
95% ethanol
Antibiotic solution
PBS or KPBS (APPENDIX 2A)
Agar medium plates (for viable counting; see APPENDIX 2C)
1-liter glass (e.g., Pyrex) beaker with overflow effluent port (see Fig. 1B.3.2)
Rubber stopper (no. 15; 100-mm top diameter, 81-mm bottom diameter) with four
predrilled holes
Glass tubing, 5-mm o.d. or sized to fit holes in stopper, of appropriate length
Flexible silicone tubing, autoclavable: 12-mm o.d./6-mm i.d. and 6-mm o.d./3-mm
i.d., and of size appropriate to fit through peristaltic pump (Marprene Manifold
tubing from Watson-Marlow, cat. no. 978.0102.000, if Watson-Marlow
peristaltic pump is used)
0.22-m filter cartridges
Small test tube or vial with bottom removed
Medium reservoir and waste bottle: 2-liter, 4-liter, or larger-capacity autoclavable
plastic bottles (e.g., Nalgene jugs)
Elbow joint (Cole-Parmer), 0.25-in. (0.625 cm)
Peristaltic pump (e.g., Watson Marlow; http://www.watson-marlow.com)
Incubator with shaker
Aluminum foil
Sandblasted ground-glass biofilm chips
Spinning disc (Fig. 1B.3.1)
Magnetic stir bar
Tube clamps
Stir plate
96-well microtiter plate
Hemostats and forceps, sterile
1.5-ml microcentrifuge tubes
Bath sonicator or tissue homogenizer
Figure 1B.3.2 The reactor set-up consists of a 1-liter Pyrex jar with an overow spout and a no.
15 rubber stopper. The ports in the rubber stopper are tted with (1) a cap, (2-3) lter units, and (4)
medium supply tubing, as shown. When fully assembled, medium is pumped from the reservoir
(bottom left) through a peristaltic pump and into the reactor. The reactor is placed on a stir plate
with the spinning disc inside. Waste medium and culture uid ows through the overspill spout to
the waste bottle.
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Assemble reactor
1. Set up the spinning disc biofilm reactor using a 1-liter glass beaker that has an
overflow effluent port and a no. 15 rubber stopper with four pre-drilled holes (ports).
See Figure 1B.3.2 for a schematic diagram.
It is best to buy a solid stopper and drill holes in it. Three of the holes should be 5 mm
in diameter. The fourth port is optional, providing clean access to the planktonic culture
during the experiment (e.g., for OD readings); if such access is desired, create an access
port sized to fit a small capped vial or test tube.
2. Insert short segments of rigid tubing (e.g., 10-cm lengths of 5-mm i.d. glass tubing)
through the small ports in the rubber stopper to provide solid support for ventilation
and medium inlets. Using short segments of flexible, autoclavable tubing (silicone,
3-mm i.d.), connect 0.22-m filter cartridges to two of the inlets to allow for ventilation and prevent contamination. Fit the access port with a small test tube or vial
from which the bottom has been removed. Cap the tube/vial.
3. Prepare the medium reservoir as follows.
a. Create a port for medium delivery by drilling a hole in the lid and fitting it with a
segment of 6-mm i.d. silicone tubing that is long enough to reach the bottom of
the reservoir and is linked to the exterior of the lid with a 0.25-in. (0.625-cm)
elbow joint.
b. Create a port for ventilation in the lid of the medium reservoir by attaching a
0.22-m filter to the lid using rigid and flexible tubing (as described in step 2 for
the stopper of the reactor) or create a ventilation port in the shoulder of the medium
reservoir by directly inserting the filter, without the use of tubing adapters.
4. To the exterior of the medium outlet, attach silicone tubing long enough to reach
from the medium reservoir, through the peristaltic pump, and to the reactor vessel,
in three segments connected by barbed mini-tube fittings.
The first and third segments are of 3-mm i.d. and 6-mm o.d. The middle segment of tubing
must be of appropriate size to fit through the peristaltic pump. If a Watson-Marlow pump
is used, manifold tubing is available for this purpose.
The medium reservoir should be sized appropriately with respect to the amount of medium
required for the experiment, such as a 2-liter, 4-liter, or larger autoclavable Nalgene jug.
5. Use silicone glue to seal the tubing into the lid of the biofilm reactor and medium
reservoir. Cap and seal the fourth port on the stopper.
6. Fit another length of tubing (12-mm o.d./6-mm i.d.) to the outflow port on the side of
the 1-liter reactor flask. Use sufficient tubing to reach from the reactor to the waste
bottle.
Prepare culture
7. In the morning of day 1, inoculate a 5-ml starter culture from an isolated colony or
overnight culture, and incubate with shaking at the appropriate temperature.
The reactor, growth medium reservoir, and starter culture should be prepared on the same
day.
Growing and
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Fermenters
It is best to use a primary culture, such as an overnight culture or isolated colony grown
under antibiotic selection to inoculate the starter culture, as this will ensure culture
purity and growth phase when the culture is used later in the day to inoculate the reactor.
Using starter cultures of similar age/growth phase also helps ensure consistency between
experiments.
1B.3.4
Current Protocols in Microbiology
9. Replace the lid to the reservoir, with the supply tubing on the interior (tubing must
be long enough to rest on the bottom of the reservoir). Use aluminum foil to cover
the exterior end of the hard support tubing, as well as the filter units.
10. Wash eighteen biofilm chips one time with 95% ethanol and three times with distilled
water, then place the chips into the slots of the spinning disc.
Neither the discs nor the chips are commercially available to the specifications described in Fig. 1B.3.1. They can be easily constructed or fabricated by a university machine/instrument shop. Alternatively, a similar reactor can be purchased from Biosurface
Technologies.
11. Place the spinning disc into the reactor, making sure that the chip side faces up and
the stir-bar side is on the bottom.
The spinning disc may be autoclaved separately, wrapped in aluminum foil. It is best to
do this on the dry setting, but if the liquid setting is used, place the foil-wrapped disc in
an empty pipet tip box to keep it dry. Use extra caution when assembling the reactor if
this alternative method is chosen. An experimental timeline for this alternative technique
is described below.
12. Fill the reactor with 250 ml water and cover with the tube-fitted rubber stopper.
13. Place tube clamps on every tube to avoid backflow and contamination. Cover the
ends of the tubing and the filter units with aluminum foil.
14. Autoclave the reactor assembly and medium reservoir on the liquid setting.
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20. In row A, add the desired amount of antibiotic solution to the medium for a total
volume of 200 l. Mix the wells in row A and transfer 100 l from row A to row B.
Continue the serial dilution down the column to row G; transfer 100 l from row G
to waste, leaving row H with only 100 l medium.
For example, to achieve a final concentration of 1 mM for a given antimicrobial agent, in
row A, add 20 l of 10 mM stock solution to 180 l medium.
Modify the concentrations and the dilution schemes for specific experimental needs.
Maintain the 200-l volume for convenience and ease of use in the microtiter
plate.
21. Twenty four hours after starting the flow phase of the reactor culture (day 3), stop the
reactor and take a 1-ml sample of the planktonic culture and set aside on ice (source
of samples for susceptibility testing in parallel with the biofilm samples that will
be harvested). Measure the optical density (OD600 ) of this sample and/or perform a
viable count to determine the planktonic culture density.
It may be necessary to dilute this sample. To ensure comparable results, expose the
same number of planktonic and biofilm cells to the antimicrobial stress in the microtiter
plate.
22. Remove the disc by reaching into the reactor and pulling it out without touching the
biofilm chips. Remove the chips from the disc with sterile forceps and dip each one
into fresh medium to remove any loosely attached cells. Place one chip into each
well in columns 1 and 2 of the 96-well microtiter plate, and 10 l planktonic culture
into the wells of columns 3 and 4. Mix well.
23. Incubate the plate 5 hr at 37 C.
Temperature and incubation time may vary depending on the organism used and the
experiment at hand.
24. Transfer the chips into 1.5-ml microcentrifuge tubes containing 1 ml of 1 PBS or
KPBS. Likewise, transfer 100 l of the planktonic cultures into 1.5-ml microcentrifuge tubes containing 900 l of 1 PBS or KPBS.
25. Sonicate the tubes for 10 min, and then briefly vortex.
Alternatively, a homogenizer can be used to disperse the cells. Place the chips, cells, and
PBS or KPBS solution in sterile, capped tubes.
26. Serially dilute the cells from each tube by taking 10 l cells and mixing with 90 l
PBS or KPBS. Spot 10 l of each dilution in triplicate onto pre-dried agar plates of
appropriate medium.
27. Incubate the plates overnight at 37 C.
1B.3.6
Current Protocols in Microbiology
ALTERNATE
PROTOCOL 1
The reservoir and reactor are assembled and sterilized as described in Basic Protocol 1,
with one exception; a stir bar is placed in the reactor before autoclaving, and the spinning
disc is wrapped in an envelope of aluminum foil and autoclaved separately.
As described in Basic Protocol 1, steps 16 to 18, the reactor is inoculated and batch
culture incubation is begun. In this case, however, the culture is simply stirred with a stir
bar. On the morning of day 2, start the chemostat phase of the culture by attaching the
medium reservoir to the reactor, unclamping the tubing, and starting the pump. Allow
the chemostat to run for the day, and in the evening begin the biofilm culture. To do this,
aseptically remove the stir bar from the reactor, and replace it with the spinning disc.
This must be done very carefully, taking care not to touch the spinning disc as the foil
envelope is peeled away. The disc should be gently dropped into the tilted reactor, so that
the biofilm chips face upward. Replace the reactor on the stir plate, begin stirring and
restart the flow.
IMPORTANT NOTE: Remember to clamp the influent and effluent tubing whenever the
pump is stopped; this will prevent backflow in the system. Do not forget to unclamp the
tubing when restarting the pump.
Perform biofilm sampling and testing as described in Basic Protocol 1, steps 19 to 21.
BASIC
PROTOCOL 2
Materials
30% (v/v) hydrogen peroxide (H2 O2 )
1:8 diluted LB or other rich medium (APPENDIX 2C)
Mid-log phase bacterial culture
95% ethanol
PBS or KPBS (APPENDIX 2A)
Petri plates containing appropriate solid medium
30% H2 O2
Medium reservoir (see Basic Protocol 1)
Silicone tubing (2- to 3-mm i.d.)
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2. Set up parallel silicone tubing for the desired number of experiments (usually eight
or twelve, depending on the number of available channels on the pump in use).
The tubing should be long enough to accommodate the distance from the medium reservoir
to the peristaltic pump. The tubing that passes through the pump must be compatible with
the pump mechanism.
3. Cut 3- or 8-in. pieces of silicone tubing (2- to 3-mm i.d.) in which the biofilms will
be grown.
The length and inner diameter of tubing used depends on the experimental goals. These
lengths are simply recommendations, but the i.d. of the tubing can be 12 mm. Remember
that changes in tube diameter will change the flow dynamics: see Reynolds equation for
determining turbulent flow (step 16).
4. From the medium reservoir, run a piece of silicone tubing leading to the eight parallel
pump tubes. Connect the pump tubing with barbed mini tube fittings and segments
of silicone tubing to the bubble traps, which are directly connected to the biofilm
tubing. Connect the biofilm tubing to another length of tubing that leads to the waste
bottle.
If preferred, use self-sealing septa placed just upstream of the biofilm tubing. The septa
can be used for inoculation of the system, but it is equally straightforward to inoculate
directly into the tubing (see steps 15 and 16).
This experiment can be performed without the use of bubble traps. Be certain to seal all
joints completely to avoid the introduction of air into the system.
5. Wrap loose tubing ends in aluminum foil and autoclave the entire system.
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1B.3.8
Current Protocols in Microbiology
7. Flush the system with the 0.6% hydrogen peroxide solution for 2 hr at a pump setting
of 14 rpm. Be sure to fill the bubble traps, if used.
8. Using aseptic technique, switch from 0.6% hydrogen peroxide to sterile water, and
flush the system for an additional 2 hr at a pump setting of 14 rpm. Be sure to fill the
bubble traps, if used.
9. Again using aseptic technique, switch from sterile water to sterile medium. Flush
the system for 2 hr at a pump setting of 14 rpm, making sure to fill the bubble traps,
if used.
10. Stop the flow and clamp the tubing just upstream of the inoculation site.
Prepare inoculum
11. Prepare a 5-ml starter culture as described in Basic Protocol 1, step 7. Incubate with
shaking at 37 C to a culture OD600 of 0.1.
At this point, it is ready to be used as an inoculum in the tube reactor.
The medium used for this inoculum culture should be the same as that to be used in the
biofilm reactor.
Equation 1B.3.1
Equation 1B.3.2
14. Inoculate samples as quickly as possible, and allow the incubated tubing to sit
undisturbed for 30 min at experimental temperature.
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15. Adjust the rpm setting on the peristaltic pump before unclamping the tubes and
starting the flow.
Some researchers suggest that the biofilm tubing should be incubated in a vertical orientation to prevent settling over the duration of the experiment. This may be of particular
importance when using a low flow rate.
16. Check the flow rate after initiating the experiment, as well as before each sample
harvest, by collecting the effluent in a graduated cylinder for a predetermined amount
of time, then calculating the flow rate as milliliters per hour.
Although this step is not critical, it may serve as a useful gauge of the biofilm conditions,
particularly at later time points.
Calculate the flow characteristics of the system (laminar, transitional, or turbulent) using
the following Reynolds equation:
Equation 1B.3.3
where is the fluid density, V is the fluid velocity, D is the tube diameter, and is the
fluid viscosity. Flow is laminar at R values up to 2000. At R values >4000, the flow is
turbulent. R values between 2000 and 4000 are considered to reflect transitional flow,
where regions of both turbulent and laminar currents may be found.
17. When harvesting tube samples, place the end of the tubing from the bubble trap
(the inflow tubing) into a petri dish containing 95% ethanol (this will help maintain
sterility in the system).
19. Cut the tubing into two or three pieces using a sterile scalpel or razor blade. Wipe
the outside of the tube with 95% ethanol, and let evaporate to dryness.
20. If using a 3-in. piece of biofilm tubing, place the cut and surface-sterilized pieces
into a sterile 1.5-ml microcentrifuge tube. For an 8-in. piece of biofilm tubing, place
the pieces into a sterile 50-ml conical tube. Add 800 l of 1 PBS or KPBS.
21. Sonicate for 1 min and incubate on ice for an additional 1 min. Vortex vigorously
for 1 min. Repeat this step for a total of four times.
22. Remove the biofilm tube pieces, and place them in a fresh sterile tube.
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1B.3.10
Current Protocols in Microbiology
23. If the samples are for an early timepoint, combine multiple biofilm tubes into a single
sample.
This is useful if enough biomass is needed to perform OD measurements.
24. Thoroughly vortex the tube containing the cells for 1 min. Then sonicate, chill on
ice, and vortex, 1 min each, for a total of two times. Make sure the culture appears
homogeneous. If clumps are still visible, repeat the sonicate/chill/vortex cycle until
the culture looks homogeneous.
25. Record the total liquid volume of this tube.
Analyze samples
26. Remove 10 l of the cell suspension for cfu enumeration.
27. Prepare a 96-well microtiter plate with 90 l of 1 PBS or KPBS in each well (eight
wells/row/sample). Add the 10 l from step 26 to the first well, and serially dilute
the cells in a range from 101 to 108 .
Fewer dilutions may be required at early time points, when the biomass coverage is low.
Until the behavior of the bacterium is characterized, however, it may be best to use a
broad range.
28. Spot 10 l of each dilution, in triplicate, onto the appropriate solid medium. Incubate
the plates upside down overnight at 37 C.
Allow the spotted plates to dry before turning them over.
Using a small volume and multiple dilutions in this manner allows for viable count assays
to be performed very quickly and uses far fewer plates than the traditional viable count
assay, in which larger volumes are spread, i.e., one dilution per plate. Typically, up to six
10-l spots can fit on a standard petri plate.
Although LB medium has been referenced throughout these protocols, choose a medium
according to the needs of the organism and experimental design.
29. On the following day, count colony forming units (CFUs) for each dilution, where
possible.
For the limited spot size, 20 organisms is an ideal number.
30. Calculate the number of organisms per tube surface area. Average the triplicate
colony counts (N) for the dilution to be used:
Equation 1B.3.4
where voltotal is the total harvest volume (usually 1000 l), volspot is the volume of
the dilution that is plated onto the solidified medium (usually 10 l), dilution refers
to how diluted this sample is relative to the cell harvest (e.g., 105 ), and SA refers
to the surface area of the biofilm tube in square millimeters (this can be calculated
from the tube diameter and length, recorded in step 20).
31. At the very end of the experiment, run fresh hydrogen peroxide solution through all
of the tubing and bubble traps. Thoroughly wash the bubble traps and tubing with
mild soap and water. Rinse thoroughly with distilled water before autoclaving.
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COMMENTARY
Background Information
Growing and
Analyzing
Biolms in
Fermenters
Spinning disc
The biofilm culture method detailed here
has been used previously, with minor adaptations, to study the effects of various antibiotics and genetic mutations on biofilm formation, and on survival of biofilm populations
(Pitts et al., 2001; Teitzel and Parsek, 2003;
Lee et al., 2004). It is commonly referred to
in the literature as a rotating disc reactor, but
is not to be confused with the Rototorque and
the rotating annular reactor (Characklis, 1990;
Lawrence et al., 2000). The first iteration of the
spinning disc reactor model was developed to
study biofilm formation on ceramic surfaces
under intermittent flow conditions, as found
in toilets (Pitts et al., 2001). Biofilm growth
and susceptibility to chlorine were studied using a rotating Teflon disc carrying six ceramic
chips that was placed in an open-air reactor
through which alternate flushes of medium and
tap water were passed. The method was found
to yield highly reproducible and statistically
sound results, making it an excellent candidate for adaptation to other applications.
The Pitts protocol was adapted to the
study of Pseudomonas aeruginosa to test
the efficacy of photodynamic antimicrobial
chemotherapy (PACT; Lee et al., 2004). The
Teflon rotor in this case was fitted with 24
stainless steel chips and biofilms were incubated under both batch and flow conditions
before samples were removed and subjected
to treatment with photosensitizing agents and
subsequent irradiation. As demonstrated earlier by Pitts et al., the modified model provided statistically sound, reproducible results
(Pitts et al., 2001; Lee et al., 2004). The Pitt
model has been further adapted for the study
of P. aeruginosa, using discs carrying eighteen
glass chips (Hentzer et al., 2001; Singh et al.,
2002; Teitzel and Parsek, 2003; Boles et al.,
2004).
This method utilizes both batch and continuous flow conditions to establish biofilms on
small chips held in a disc fitted with a magnetic stir bar. The benefits of this method rest
in its adaptability and the reproducible results
it provides: rotating discs can be designed to
hold any number of chips of varying sizes and
materials, including glass, plastic, metal, and
porcelain. Spinning discgrown biofilms can
be inoculated at any point in the growth curve,
harvested in replicates at any time point, and
the resulting biofilm populations can be as-
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Anticipated Results
The experiments described here will yield,
at the minimum, viable counts which will reflect the amount of biomass in either the tubegrown biofilm or on the biofilm chips. In both
cases, biofilms may be subjected to treatments
with biocides and tested for survival. When
compared with the results of planktonic cells
processed in parallel, data can be obtained regarding the different susceptibilities of these
bacterial populations to various treatments.
The tube-grown biofilms can provide sufficient biomass to perform various expression
analyses. The cells may be processed for microarray analysis to determine wide-scale differences in expression between biofilm populations and their planktonic counterparts. More
simply, engineered reporter strains may be utilized in -galactosidase assays to examine the
expression levels of specific genes. Similarly,
reporter genes may be fused to GFP and the
bacterial populations examined microscopically for expression.
Time Considerations
A spinning disc biofilm experiment can be
run in a minimum of 4 to 5 days, from the
point of assembly through harvest. However,
the experiment may be run for a longer period, to obtain data over a time course, or for
a shorter period, if the data required are from
the earliest stages of biofilm formation.
The tube biofilm experiment will take a
minimum of 3 to 4 days, but may also run
longer if time course data is desired.
Literature Cited
Adair, C.G., Gorman, S.P., Feron, G.M., Byers,
L.M., Jones, D.S., Goldsmith, C.E., Moore,
J.E., Kerr, J.R., Curran, M.D., Hogg, G., Webb,
C.H., McCarthy, G.J., and Milligan, K.R. 1999.
Implications of endotracheal tube biofilm for
ventilator-associated pneumonia. Intensive Care
Med. 25:1072-1076.
Bagge, N., Hentzer, M., Andersen, J.B., Ciofu, O.,
Givskov, M., and Hoiby, N. 2004a. Dynamics
and spatial distribution of beta-lactamase expression in Pseudomonas aeruginosa biofilms.
Antimicrob. Agents Chemother. 48:1168-1174.
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Growing and
Analyzing
Biolms in
Fermenters
1B.3.14
Current Protocols in Microbiology
UNIT 1B.4
ABSTRACT
In their natural environments, microorganisms are under constant environmental selection to
form biofilms. Using aquatic biofilms as an example, this unit illustrates general concepts in field
biology and practical suggestions for designing, conducting, and analyzing biofilm experiments
at varying distances from the laboratory. The unit also addresses an example of a special situation
(space flight) where experimentation must be done by proxy through another individual or
C 2008 by John Wiley & Sons, Inc.
machine. Curr. Protoc. Microbiol. 10:1B.4.1-1B.4.14.
Keywords: biofilm r bacteria r field studies
INTRODUCTION
Field studies of biofilms present an exciting challenge to a microbiologist. Unlike in
the laboratory, microorganisms in nature exist
in mixed populations and are under constant
environmental selection to adhere to surfaces
and form biofilms. As a result, field experimentation would offer the most rigorous and
realistic conditions for the testing of materials
to attract or repel biofilms or for the isolation of
organisms best suited for biofilm growth. Conditions in nature are rarely predictable. Rapid
changes in weather, uncooperative wildlife, or
a support vehicles mechanical condition may
affect carrying out field studies, which, consequently, require a certain amount of foresight,
planning, and adaptability on the part of the
microbiologist.
There are a number of logistical issues that
may need to be addressed. These include accessibility of the sampling site; experimental
design in the field, including sample collection, storage of specimens, and transport to the
laboratory; and even issues related to investigator safety. Specimen analysis should also be
considered during planning. For viable counts
with dilution plating, samples are generally
collected and stored unaltered at lower temperatures, to restrict further growth. Ideally,
samples will be analyzed as quickly as possible. In the case of other types of analyses,
specimens may need to be manipulated on site
for initial preservation. This includes the use
of fixatives (for microscopy), addition of TE
buffer for a PCR-based detection such as denaturing gradient gel electrophoresis (DGGE;
UNIT 1E.1), or preservatives such as RNase inhibitors for RNA-based detection. Statistical
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be obtained through the National Park Service Research Permit and Reporting System (http://science.nature.nps.gov/research/
ac/ResearchIndex). Collecting permits may
or may not address permission for access.
Some specimen transport issues are addressed
by the National Park Service. However, in
cases where potential human, animal, or plant
pathogens are to be handled and transported,
other agencies will be involved. In the United
States, these include the Department of Agriculture (http://www.aphis.usda.gov/programs/
ag selectagent/index.html) and Centers for
Disease Control (http://www.cdc.gov/od/ohs/
default.htm). All such specific details should
be checked ahead of time. Public as well as
private lands often have restricted access and
regulations addressing specific activities that
may be conducted on the site. The importance
of communication and respect for the wishes
of the land owner or regulatory agency cannot
be overemphasized, particularly if one wishes
to have continued access to study sites.
IMPORTANT NOTE: The Web sites provided are current at the time of publication but
may change.
Safety
First aid
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Camping gear
Modern camping supplies (e.g., gas stoves,
water filters) are small and take up little space.
Efforts should be made to bring sufficient supplies to prepare meals at a campsite. This will
avoid the unnecessary time and expense of
traveling from the field to purchase a meal.
Accessibility
Vehicle accessible
Equipment and personnel can be moved to
research sites without problems. Emphasis can
be on taking a number of supplies, backup
equipment, and spare parts.
Inaccessible by vehicle
As equipment may have to be carried in by
backpack, pack animal, boat, or aircraft, the
emphasis must be on lightweight essentials.
Carrying duplicate equipment and spare parts
is usually prohibitive. These restrictions apply
especially to personal equipment.
Food
Keep meals simple. Limit perishable items,
particularly if backpacking, canoeing, or
camping in an area lacking refrigeration.
Personal gear
New field clothes, especially synthetics,
provide protection from the elements (e.g.,
sun, heat, cold, rain) to a remarkable degree.
The most important personal items are durable
but comfortable field shoes or boots, a good hat
for protection from sun or cold, suitable clothing (preferably layers of clothing that allow
adjustments for changes in temperature), and
a sleeping bag and mat.
Animal concerns
Animal interference can arise at a number
of environments. In North America, nuisance
animals can include raccoons, bears, or rodents. Check on these issues before going to
the field. As a general precaution to avoid unwanted guests, never store food in a tent.
Human concerns
In some instances, vandalism or some other
human activity (e.g., swimming in a sampling
area with possible inadvertent contact with scientific equipment) may represent a problem.
Aside from the loss of scientific data, there
is also concern for public safety and liability
(e.g., if a swimmer should get cut on a microscope slide).
Equipment
Tables provide a general description of
equipment (Table 1B.4.1) and a representative list of suppliers (Table 1B.4.2), as well as
checklists for general supplies (Table 1B.4.3),
microbiology supplies (Table 1B.4.4), and personal supplies (Table 1B.4.5). A brief explanation of some items is provided below.
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Item
Comments
Spare parts for repairs (generally applicable for larger pieces of equipment)
Fuses
Light bulbs
Power sources
Batteries
Chargers
Power converters
Electricians tape
Two-part pliable putty that cures in 5 min; useful for repairs to odd
shapes and locations on equipment; will not drip or run
Geologists hammer
Measuring tape
At least 6 m
Containers
Plastic containers
Documentation
Field book
Preparing for
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1B.4.4
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Item
Comments
GPS units
First aid
Standard first aid kit
Note any special medical needs of field team members and have
information readily available; especially important to medical
personnel in remote areas
Personal supplies
Insect repellent
Sun screen
Backpack
Binoculars
Labeling tape
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Item
Comments
Sample storage
Coolers
Preservatives
Sterile water
Scalpel blades
Aluminum foil
Microscope slides
Filters
Filter tweezers
Fresh filters
a These items are used for a variety of purposes. Some items may not be applicable for all trips.
b See Table 1B.4.2 for a representative list of suppliers.
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Supplier
Web site
Brigade Quartermasters
http://www.actiongear.com
Cabelas
http://www.cabelas.com
CampMor
http://www.campmor.com
Forestry Suppliers
http://www.forestry-suppliers.com
REI
http://www.rei.com
Items
Comments
Vehicle
Sampling equipment
Personal equipment
valuable sample, leakage can damage clothing or field notes. Permanent markers can be
used to label containers. For measuring liquids
in the field, one can use markings on a container or, if necessary, sterile plastic syringes
or pipets (along with a pipetting bulb). Due to
weight and breakage considerations, the use of
glass is strongly discouraged.
are especially useful for fluorescence and confocal microscopy applications because they
do not fluoresce under ultraviolet light. These
filters and filter holders are assembled in
the laboratory prior to the trip and wrapped
individually in aluminum foil, labeled, and
autoclaved. Labeling will permit easy identification of wrapped packages under field
conditions.
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Function
Item
Specimen manipulation
Scalpel blades
Tweezers
Scissors (if needed)
Ethanol (bring 95%)
Sterile water
Nitrile gloves
Aluminum foil (sterilized and folded)
Petri dishes
Matches or lighter for ethanol sterilization
Other
Camera
in the field; see Disinfection and Field Sterilization, below). Sterile petri dishes are very
useful for providing a small, sterile, working
area. Should a larger sterile working area be
necessary, we advise using sheets of aluminum
foil of a desired size (e.g., 0.5 m2 ), folding as
necessary, and putting a small amount of indicator tape onto the sheet. Several of these
sheets can be placed into an autoclave bag and
autoclaved, prior to going to the field. In this
fashion, a sterile work surface or wrapping
agent is readily available. The sterilized autoclave bag can also be used as a sterile container to transport larger specimens or to gather
waste.
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Item
Comments
Footwear
Protective clothing
Hat
Gloves
Cotton or leather
Swimsuit/shorts
For seining
Bedroll/sheets/pillow/cot
Tents
Field guides
Water container/canteen
Binoculars
Gatorade/water
Food
safety, and other needs, notably water. With respect to vehicle transport, materials may need
to be secured and insulated against damage
from vibration, particularly if the vehicle needs
to be driven over rough terrain.
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example, for a nucleic acidbased community profile, the sample should be collected
so as to preserve DNA or RNA. Preservation
of specimen viability may not be necessary,
and indeed, in some cases where viability is
maintained, differential growth and survival
of organisms in specimen containers (sometimes referred to as the bottle effect) may
actually be detrimental to the scientific analysis. Where feasible, the simplest approach
for DNA preservation is to freeze the sample (20 C or colder), and store frozen until
processed. Should vehicle access be available,
specimens can be placed in Styrofoam containers or coolers along with dry ice (frozen CO2 ).
Packaging materials, used to ship refrigerated
or frozen biochemical reagents, are often very
useful for this purpose. As a safety precaution,
these containers need to be in a well ventilated place so as to avoid asphyxiation of the
investigators by elevated CO2 levels.
For RNA analysis (and even DNA analysis), a stabilizing agent (e.g., RNAlater,
Ambion) is often used. This reagent
stabilizes RNA and genomic DNA,
but will denature many proteins (see
http://www.ambion.com/techlib/tn/113/6.html).
Once samples are placed in RNAlater, they
can be stored for several days at ambient
temperatures or up to one month at 4 C. Under
field conditions, storage in coolers along with
ice, will maintain temperatures close to 4 C.
The use of fixatives such as glutaraldehyde or
Preparing for
Biofilm Studies in
the Field
Figure 1B.4.1 Collection of biolms in streams. During eld studies of biolm growth in streams,
biological material such as a Platanus leaf (P) can be retained within a porous container such
as cheesecloth. A glass slide (arrow) can be used as a representative abiotic surface. Note the
presence of naturally occurring biolms on substrata such as a leaf (L) and the gravel sediment.
1B.4.10
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Figure 1B.4.2 Growth of biolms on glass microscope slides. Biolms readily form on glass
microscope slides that are placed in aquatic environments.
Depending upon the environment, colonization substrata can be placed onto a surface (e.g., river sediment) or suspended at a
selected depth in the water column by a paperclip or other clamping material, which is then
anchored to a string as shown in Figure 1B.4.1.
Such experimentally grown biofilms mimic
natural biofilms on the biotic (leaf) and abiotic (rock) surfaces naturally found in that environment (also shown in Fig. 1B.4.1). While
biofilms often form quite readily, surface coverage is typically quite variable. A representative biofilm grown on a glass microscope slide
is shown in Figure 1B.4.2.
Unique equipment
The local river environment presented several challenges. The river flow (typically 0.3
to 0.5 m/sec) caused leaves and other submerged materials to drift, and the grazing
Procedure
To maintain sterility in the field, nitrile gloves, disinfected by rinsing with 70%
ethanol were used. We have found these to be
much more comfortable than other materials
and are also suitable for people with allergies
to latex.
Approximately 50 leaves from several representative trees were collected by breaking
leaves at the base of the leaf stem. At least 10
of these should be used to determine size and
mass of leaves. One could also calculate area
of these leaves by placing individual leaves on
a scanner, obtaining an image, and then calculating the area using image analysis software
(e.g., NIH Image).
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Unique equipment
Cheesecloth bags were constructed as described above. As an alternative, nylon window screen mesh (available at any hardware
store) can be substituted for added strength.
These bags were filled with small rocks (1
to 2 cm3 ) as colonization substrata. A length
of rope was then securely attached to the bag.
The bags with the rocks were placed into an
autoclave bag, fastening one end of the rope to
the outside of the bag (permitting ready access
at the field site, yet preserving sterility of contents) and then autoclaved 30 to 60 min. This
extended autoclaving time is necessary to ensure that endolithic microorganisms within the
rocks are killed. Sterilized autoclave bags keep
the rocks sterile during transport to and from
the field site.
Procedure
Groundwater access can be obtained
by several means including wells, artesian
springs, and access caves. In this particular
study (Whiteley et al., 2001), access to the
groundwater was obtained through an access
cave named Rattlesnake Cave. Prior to entering the cave, precautions were taken to ensure this cave was not occupied by rattlesnakes
(Crotalus spp.) or other wildlife. Samples were
suspended below the water table for several
days to ensure the growth and maturation of
biofilms on the rocks. During specimen retrieval, autoclaved aluminum foil (to provide a
sterile working surface in the field) and autoclave bags (for specimen transport) were used
at the field site.
COLLECTION OF BIOFILM
SAMPLES REMOTE FROM THE
LABORATORY
It may be necessary to conduct biofilm studies some distance from the laboratory. Vehicle
accessibility will determine the amount and
types of supplies that can be taken. With respect to microbial samples, it may be a number
of hours or even days before the samples can
be returned to a laboratory. Sample storage
and transport are issues because changes in
microbial populations will occur; these issues
have been addressed in an earlier section (see
Technique Refinement and Testing).
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1B.4.12
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In the event of travel, especially across international boundaries, one should plan ahead
(often 2 to 3 months or more) and check with
appropriate government and courier agencies
for permits required for travel, access to field
sites, and shipment of any supplies or specimens. Given the political and security issues
that have arisen in the past few years, the
U.S.A. and other countries have adopted strict
regulations for the transport of biological materials. Often, it is best to ship a sample, along
with appropriate documentation, via a courier.
We have experience in conducting fieldwork in several remote sites involving lengthy
trips by vehicle (Big Bend National Park,
U.S.A.) or air (Viterbo, Italy and Yellowstone
National Park, U.S.A.). For remote trips, space
may be quite limited, particularly if supplies
need to be transported in luggage. In these
cases, the use of lightweight, sterile, plastic
containers and microscope slides can be quite
useful for a number of experiments. Often one
will need to improvise with available local
materials; bringing extra tape, pens, markers,
string, magnifying glass, a knife, camera, and
notebook is advisable.
Additional personnel
Are local support personnel, or alternatively, laboratory support personnel needed,
and does scheduling need to be coordinated?
Specimen transport
Plan procedures for shipment of materials
back to lab, along with necessary permits.
Documentation of location
In the field book, draw a map of the site
and location of experimental work. If possible,
enter GPS coordinates.
Documentation of environmental
conditions
Transportation
Accommodation
Is camping gear needed? This subject is
addressed under General Aspects of Field Research.
Scheduling
SAMPLING BY PROXY
For any number of reasons (e.g., safety, access to a particular environment or equipment),
it may be impractical for an investigator to personally conduct an experiment. As an alternative, the experiment will need to be conducted
by some other individual or device. Based on
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CONCLUSION
The techniques and approaches used for
field biofilm acquisition are quite variable
because they will relate to the questions
being addressed and the limitations of the
environment and available equipment. Nevertheless, several key issues arise which are
fundamental to any scientific investigation:
reproducibility, quality of data, and quality
of data analysis. These issues must be addressed during all stages of experimentation
from initial planning, through the actual fieldwork, to the final analysis and reports. Both
successes and failures must be recorded and
analyzed because they will provide invaluable
information for future work. Although practical issues have been addressed in this unit,
one may often need to improvise in the field
when presented with unexpected obstacles or
opportunities.
LITERATURE CITED
Cassanto, V.A. and Wood, B.J. 1998. Student experiments in space: A private-sector model. Earth
Space Rev. 7:25-28.
Dunn, K.A., McLean, R.J.C., Upchurch, G.R. Jr.,
and Folk, R.L. 1997. Enhancement of leaf
fossilization potential by bacterial biofilms.
Geology 25:1119-1122.
Friend, M. and Franson, J.C. 1999. Field Manual of
Wildlife Diseases: General Field Procedures and
Diseases of Birds. Biological Resources Division, U.S. Geological Survey, Washington, D.C.
McLean, R.J.C., Cassanto, J.M., Barnes, M.B., and
Koo, J. 2001. Bacterial biofilm formation under
microgravity conditions. FEMS Microbiol. Lett.
195:115-119.
McLean, R.J.C., Welsh, A.K., and Casasanto, V.A.
2006. Microbial survival in space shuttle crash.
Icarus 181:323-325.
Whiteley, M., Ott, J.R., Weaver, E.A., and McLean,
R.J.C. 2001. Effects of community composition and growth rate on aquifer biofilm bacteria
and their susceptibility to Betadine disinfection.
Environ. Microbiol. 3:43-52.
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UNIT 1B.5
Jonathan Pratten1
1
ABSTRACT
In order to grow organisms in such a manner as to mimic their physiological growth
state in vivo, it is often desirable to grow them as biofilms in the laboratory. There are
numerous systems available to accomplish this; however, some are more suited to the
growth of oral biofilms (dental plaque) than others. The operating parameters of one such
model, the constant depth film fermentor (CDFF), are given in this unit. This model is
particularly suited to studying the varied biofilms which exist in the oral cavity because
environmental factors such as the substratum, nutrient source, and gas flow can be altered.
C 2007 by John Wiley & Sons, Inc.
Curr. Protoc. Microbiol. 6:1B.5.1-1B.5.18.
Keywords: constant depth film fermentor r oral biofilms r dental plaque
INTRODUCTION
The mouth provides a large number of diverse surfaces, e.g., the soft shedding tissues
of the mucosa and hard nonshedding surfaces of the teeth, on which a wide variety of
complex biofilms form. These biofilms (dental plaque) consist of a microbial community
embedded in a matrix of polymers of bacterial and salivary origin. Dental plaque has high
species diversity and contains numerous microenvironments within gradients of nutrients,
oxygen, redox potential, and pH. For these reasons, mature dental plaque is a highly
dynamic community. Small alterations in the environment can lead to ecological shifts and
subsequent population changes, and in certain specific cases may result in predisposition
to a more pathogenic microbial community. Such biofilm-related oral infections are
implicated in the etiology of two of the most prevalent diseases affecting industrial
societies: caries and periodontal disease. Prevention of these conditions involves constant
mechanical removal of the plaque from the tooth surface, although there is often difficulty
in maintaining an effective mechanical oral regime. Hence, there is considerable interest
in the use of antimicrobial agents in the prevention and treatment of these diseases.
In order to study biofilm development and its perturbation, it is useful to generate steadystate systems. One approach to producing biofilms in a steady-state is to develop a
constant depth reactor where the surface growth is periodically removed to maintain
a constant geometry (Kinniment et al., 1996). Such a device, the constant depth film
fermentor (CDFF; Fig. 1B.5.1), was first described by Coombe et al. (1982), to investigate the growth of dental plaque organisms, and was further developed by Peters and
Wimpenny (1988). The CDFF consists of a glass vessel with stainless steel end-plates;
the top-plate has ports for the entry of medium and gas and for sampling, while the
bottom one has a medium outlet port. The vessel houses a stainless steel disk containing
fifteen polytetrafluoroethylene (PTFE) sampling pans which rotate under a PTFE scraper
bar that smears the incoming medium over the pans and maintains the biofilms, once
formed, at a constant predetermined depth. Each sampling pan has five cylindrical holes
containing PTFE plugs, which are recessed to a set depth to create a space in which the
biofilms form. The sampling pans can be removed aseptically during the course of an
experimental run.
Emerging
Technologies
Current Protocols in Microbiology 1B.5.1-1B.5.18, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc01b05s6
C 2007 John Wiley & Sons, Inc.
Copyright
1B.5.1
Supplement 6
Figure 1B.5.1
The constant depth lm fermentor complete with power unit and accessories.
Growing Oral
Biolms in a
CDFF
As well as being used for the study of bacterial perturbation (Pratten et al., 1998; Pratten
and Wilson, 1999; Deng et al., 2005), the CDFF has been used in studies evaluating
endodontic microleakage (Matharu et al., 2001), oral malodor generation (Pratten et al.,
2003), corrosion potential of dental plaque (Wilson et al., 1995), and gene transfer in
oral biofilms (Roberts et al., 2001). It is a sophisticated apparatus that can generate large
numbers of individual biofilms and has been used extensively to investigate factors that
may influence the growth of bacterial communities in the oral ecosystem. Figure 1B.5.2
provides a schematic representation of how the procedures detailed in this manuscript
interrelate.
1B.5.2
Supplement 6
STRATEGIC PLANNING
Nutrient Source and Operating Conditions
Supragingival plaque biofilms
Many studies use a simple mucin-containing artificial saliva (Pratten et al., 1998; Roberts
et al., 1999) modified from the studies of Russell and Coulter (1975) and Shellis (1978;
also see recipe); however, there are more defined growth media described in the literature (Wong and Sissons, 2001). The artificial saliva is delivered at a rate of 0.5 ml/min,
corresponding to the resting salivary flow rate in humans (Pratten et al., 1998). Additionally, an aqueous solution of 2% (w/v) sucrose can also be pumped over the biofilms
for periods of 30 min at the same speed via a second peristaltic pump. The sucrose
pulsing can be carried out four times a day; during this period the artificial saliva supply
should be maintained. The pH curve measured from the biofilm effluent under sucrose
supplementation conditions has shown to be typical of the Stephen curve, i.e., falling and
rising pH levels in response to sucrose supplementation (Zanin et al., 2005).
Subgingival plaque biofilms
Artificial gingival crevicular fluid (GCF; see recipe) is pumped into the CDFF at a rate
of 50 l/min, as an approximation of the flow rate of GCF during gingivitis. To model
the periodontal region, an anaerobic atmosphere of 5% CO2 , 95% N2 is provided by
connecting a gas cylinder to the fermentors air-inflow port. The pressure used should
be 1 bar (100 kPa) and the flow rate 100 cm3 /min. Reinforced 5/16-mm i.d. PVC tubing
is needed to connect the gas pressure gauge and the flow meter. Silicon tubing with an
8-mm i.d. connects the flow meter to the CDFF. The gas should be passed through air
filters to maintain sterility. A Y-branch tubing connector can be used to supply gas to
both the fermentor and the inoculum flask if required.
Perturbation
One of the main advantages of using this model is the ability to carry out antimicrobial
and antiplaque testing over time. An agent can be pulsed in via one of the free inlet ports
at appropriate times to mimic in vivo use, for example, pulsing twice daily (e.g., 9:00 a.m.
and 5:00 p.m.) for 1 minute with 10 ml of 0.2% (w/v) chlorhexidine gluconate. To carry
out pulsing at higher flow rates, wider bore pump tubing may be required. Additionally,
a timer can be added to the power supply for the peristaltic pump to deliver a challenge
to the CDFF without the operator being present.
With this setup, biofilms can also be removed from the CDFF and placed into an agent.
In this case it is advisable to place the whole PTFE pan into the solution (5 ml in a
25-ml conical skirted-base tube to cover the pan) in order to reduce disruption of the
biofilms. Using this method, it is possible to generate minimum inhibitory concentration
(MIC)-like data for the biofilms.
BASIC
PROTOCOL
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Materials
Bacterial growth medium (appropriate for the organisms being used)
10 ml single-species inoculum or 2 ml pooled saliva (see Support Protocol 5)
70% (v/v) ethanol
Phosphate-buffered saline (PBS; APPENDIX 2A), sterile
Air filters (e.g., Hepa-vent; Whatman)
Inoculum vessel: 500-ml Erlenmeyer flask with silicon rubber stopper having two
metal tubes going through
3- and 5-mm bore silicone tubing (depending upon bore of stainless steel tubing),
15-cm and other lengths (depending upon setup)
Connectors and clips for silicone tubing (e.g., Sigma-Aldrich Z12, 654-3 and Z12,
651-9)
Couplers for connecting silicone tubing of different size bores (e.g., Value Plastics
N220/210-6)
Nylon straps (for securing tubing)
Foil
Magnetic stir bar
Incubator, appropriate for inoculum used (see Support Protocol 5)
Magnetic stirrer
CDFF (sterilized; see Support Protocol 1), with motor and power supply unit
Peristaltic pump, precalibrated to desired flow rate
10- or 20-liter Pyrex glass bottles (for effluent collection and medium reservoir;
size depending on flow rate used) with silicone rubber stoppers having two metal
tubes going through
Glass grow-back traps (Hampshire Glassware)
Retort stand and clamp
Portable butane burner
Sample tool (wrapped in foil and autoclaved; provided with CDFF; see Support
Protocol 1)
Forceps, sterile (e.g., flame sterilized in 70% v/v ethanol just before use or
autoclaved)
Prepare CDFF inoculum vessel
1. Attach an air filter to the outside of one metal tube of the inoculum vessel stopper. To
the other metal tube, attach a length of silicone tubing reaching down to the bottom
of the flask (see Fig. 1B.5.3). On the opposite side of that metal tube connect a
short length of pump tubing, using appropriate sized silicone tubing and couplers, as
necessary to be able to pass the pump tubing through the peristaltic pump. Finally,
add a connector to the end of the silicone tubing.
2. For any unused ports, place a piece of silicone tubing on the metal tubing and clamp.
3. Cover all connectors in foil. Add 500-ml medium and a magnetic stir bar to the
vessel.
4. Autoclave the inoculum vessel with 500 ml bacterial growth medium 15 min at
121 C, leaving the stopper ajar. Also autoclave the effluent bottle with its attached
air filter and tubing with coupling insert (with the stopper ajar).
If the medium cannot be autoclaved, autoclave the vessel and tubing alone and then add
the medium aseptically.
The effluent bottle has two ports: one containing an air filter and the other a section of
tubing and a coupling insert, which needs to be autoclaved with the stopper ajar.
Growing Oral
Biolms in a
CDFF
For convenience, the continuous culture medium and reservoir could also be autoclaved
at this time (see steps 15 to 18).
1B.5.4
Supplement 6
Figure 1B.5.3
(CDFF).
Schematic of the setup required for inoculation of the constant depth lm fermentor
5. As soon as it is safe to open the autoclave, quickly add any supplements, push the
stopper firmly into place, and open the clamps to the air filters. Cool the medium to
22 C.
9. Place the peristaltic pump into the incubator; feed the electric power cord through
the hole in the top of the incubator.
10. Take the tubing from the inoculum vessel that has the pump tubing included and
pass it through the pump before connecting to one of the inlet ports in the top-plate
of the fermentor.
It is best to loosen the foil on both, then aseptically join the two.
12. When the inoculum starts to drip onto the turntable in the fermentor, switch on the
turntable motor at 3 rpm.
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13. At this stage, turn the pump down to achieve the desired flow rate and pass the
inoculum through the system for 8 to 24 hr at 37 C (see Support Protocol 5).
NOTE: The pump needs to be calibrated beforehand.
14. Finally, attach the effluent bottle (while the fermentor is being inoculated) prepared
in step 4.
The vessel can sit on the laboratory floor (within an appropriate spill tray) to allow flow
via gravity. If this is not possible an additional peristaltic pump may be required.
Figure 1B.5.4 Schematic of the constant depth rm fermentor (CDFF) system setup when
running continuously. The dotted line represents the incubator. The feed line required from the
peristaltic pump to the CDFF (including the grow-back trap) is described in the Basic Protocol.
Growing Oral
Biolms in a
CDFF
1B.5.6
Supplement 6
18. As soon as it is safe to open the autoclave door, push in the stopper and unclamp the
air filters.
19. Construct the feed line for the medium as follows by attaching a connector to a 30-cm
piece of silicone tubing and link to a short piece of pump tubing, using appropriate
couplers. Add an additional 15-cm piece of silicone tubing.
20. Add a glass grow-back trap between this piece of tubing and a further 15 cm of
tubing ending with a connector to attach to the CDFF. Ensure that the grow-back
trap is aligned so that the medium drips towards the CDFF.
Further tubing may be required if different bore lengths of tube do not fit together. For
example, a 5-mm bore tube will not fit the pump tubing connector, and in this case a
length of 3-mm bore will be needed to join the two.
21. Cover the connectors of each feed line with foil and sterilize.
22. Switch off the pump and disconnect the inoculum vessel. Remove the pump
from the incubator and place on top of the incubator together with the medium
reservoir.
23. Attach the feed line to a retort stand on top of the incubator by clamping onto the
grow-back trap.
24. Link the connector of the feed line (closest to the grow-back trap) to the connector of
the second fermentor inflow and the second connector of the feed line to the medium
vessel.
25. Pass the pump tubing of the feed line through the peristaltic pump.
26. Set the pump to maximum to prime the line until the medium starts to drip on to the
rotating turntable. After this time, turn the speed of the pump down to the desired
rate.
The continuous system is fully set up.
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SUPPORT
PROTOCOL 1
Materials
Spray lubricant (e.g., WD-40), optional
Silicon high-vacuum grease (VWR)
Constant depth film fermentor (CDFF; see Fig. 1B.5.1; contact Professor A. Peters
for purchasing information; APeters@UWIC.ac.uk) including:
Motor
Power supply
15 PTFE pans, each with 5 plugs (see Support Protocol 2 for preparation)
Recess tools
8-G 12 in. stainless steel needle tubing for CDFF inlet ports and vessels (e.g.,
SLS Scientific SRY700 8H)
3- and 5-mm bore silicone tubing (depending upon bore of stainless steel tubing),
15-cm and other lengths (depending upon setup)
Connectors and clips for metal tubing (e.g., Sigma-Aldrich Z12, 654-3 and Z12,
651-9)
Air filters (Hepa-vent; Whatman)
Nylon straps for securing tubing
Couplers for connecting pump tubing to 3-mm tubing (e.g., Value Plastics
N220/210-6)
Foil
Autoclave tape
Incubator with ports at the top and side (for media in and effluent out) and gas
tanks, as required (see Support Protocol 5) or 37 C room
Assemble CDFF
1. Place main fermentor tripod on the bench (Fig. 1B.5.6A).
2. Place the drive shaft of the stainless steel turntable through the hole in the base-plate
of the main fermentor tripod: it should rotate freely (Fig. 1B.5.6B).
A lubricant spray such as WD-40 can be used, if necessary.
Growing Oral
Biolms in a
CDFF
Figure 1B.5.5 Vertical schematic of the constant depth lm fermentor (CDFF) highlighting one
of the removable polytetrauoroethylene (PTFE) pans.
1B.5.8
Supplement 6
4. Liberally smear the convex side of one PTFE seal with silicone high-vacuum grease,
placing the grease downwards on the base plate of the main fermentor tripod (Fig.
1B.5.6C).
5. Liberally grease one end of the glass housing and place the greased end down onto
the PTFE seal (Fig. 1B.5.6D).
6. Liberally grease the other end of the glass housing and put the second PTFE seal
down on top of it (Fig. 1B.5.6E and F).
7. Liberally grease the PTFE seal and carefully place the fermentor top-plate down,
ensuring that the scraper blades do not touch any of the grease. Secure the top-plate
by tightening the screws using a wrench (Fig. 1B.5.6G).
8. Feed stainless steel tubing through the silicone stoppers of all three inlet ports of the
top-plate of the fermentor.
9. Attach to these metal tubes 15 cm lengths of silicone tubing with a connector
(coupling body) on the end (Fig. 1B.5.6H).
It is easier to have all the connectors on the CDFF the same; this way when the tubing is
connected, there will be no confusion about which connector type to use.
10. Attach silicone tubing with two air filters, with tubing between them, to the end of
the port in the middle of the top-plate, which is for air-inlet (Fig. 1B.5.6H). Clamp
off the filters at each end of the tubing with nylon straps before autoclaving.
All tubing joints need to be secured using nylon straps.
11. Connect a length of silicone tubing with a connector on one end to the single drain
hole on the base plate.
The length of the tubing will depend upon the distance between the CDFF and the effluent
bottle via the port in the incubator.
Sterilize CDFF
12. Cover all four connectors with tin foil and secure with autoclave tape (Fig. 1B.5.6H).
If the connectors being used are not the self-seal type, then tubing clips will need to be
added.
13. Place a piece of foil over the sample port, with the sample port open and the autoclave
probe inserted into the fermentor. Autoclave the fermentor 15 min at 121 C.
14. As soon as the autoclave door can be safely opened (i.e., ideally while everything
is still hot) remove the probe; quickly and aseptically screw the sample port tightly
shut.
15. Remove the fermentor from the autoclave and open the clips on the filter tubing.
Place into the incubator or 37 C room.
Opening the clips will allow the air to enter the fermentor as it cools. Once cooled, the
CDFF is ready for immediate use but could be stored until required.
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Figure 1B.5.6 (continues on next page) Pictures demonstrating the CDFF assembly procedure. The components are assembled in the following order: (A) fermentor tripod, (B) stainless steel turntable, (C) PTFE
(polytetrauoroethylene) seal, (D) glass housing, (E) application of silicone high vacuum grease, (F) second
PTFE seal, (G) top plate, and (H) silicone tubing with connectors.
SUPPORT
PROTOCOL 2
Growing Oral
Biolms in a
CDFF
1B.5.10
Supplement 6
Figure 1B.5.6
(continued)
Materials
Silicone grease
Plugs: PTFE (supplied with CDFF; see Support Protocol 1)
PTFE pans (supplied with CDFF; see Support Protocol 1): drilling a 2-mm small
hole in the bottom of the threaded hole recommended
Recess tool (supplied with CDFF; see Support Protocol 1)
Substratum (e.g., see Support Protocol 4)
CDFF turntable (supplied with CDFF; see Support Protocol 1)
Flat tool (supplied with CDFF; see Support Protocol 1)
1. Place the plugs into the holes in the pans (central threaded hole uppermost) with
their flat surface uppermost.
2a. For PTFE substratum: Push the plugs level with the top of the pan and use the recess
tool to push the plug down by the required depth.
The recess tool is simply a metal rod with a machined protrusion of known height. Two
recess tools are supplied with the CDFF, allowing depths of 100, 200, 300, and 400 m
to be made; however, custom tools can also be made.
2b. For other substratum: Place a small amount of silicone grease between the PTFE
and the chosen substratum to hold the material in place during autoclaving.
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Current Protocols in Microbiology
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Figure 1B.5.7 Sectional schematic showing the arrangement of PTFE pans within the stainless
steel turntable of the CDFF. Abbreviations: CDFF, constant depth lm fermentor; PTFE, polytetrauoroethylene.
Figure 1B.5.8
i. Starting with the plugs slightly raised above the pan, place a small amount of
silicone grease on the PTFE plug, place the disk on top of the plug, push down,
and wipe off excess grease.
ii. Push the plug up again slightly from underneath and then push down with the
appropriate recess tool.
3. Place the pans into the holes of the stainless steel turntable (Fig. 1B.5.8) using a flat
tool to ensure they are flush.
SUPPORT
PROTOCOL 3
Growing Oral
Biolms in a
CDFF
1B.5.12
Supplement 6
Materials
CDFF with biofilm-containing pans removed (Basic Protocol)
(0.01% v/v) Decon acid rinse (Decon Laboratories)
1. Switch off the turntable motor.
2. Switch off the pump and release the tubing from the pump head.
3. Disconnect from the effluent bottle and medium inflow tubing.
4. Remove the air filter and ensure that the sample port is open.
5. Autoclave the CDFF on a kill cycle.
6. Autoclave the effluent bottle on the same cycle as above, placing the temperature
probe (if available, set at 121 C) into the liquid.
7. Take apart the CDFF and leave to soak overnight in dilute (0.01% v/v) Decon acid
rinse.
After autoclaving, the tubing may be reused for future experiments; however, before setting
up a new system, be sure to examine the entire length of each piece of tubing for holes
that could cause leaks.
SUPPORT
PROTOCOL 4
Hydroxyapatite
Hydroxyapatite disks may be purchased from Clarkson Chromatography Products. Alternatively, they may be made by pressing hydroxyapatite powder into 5-mm diameter
disks (3 mm depth) in a 5-mm pellet die under a pressure of 250 kg. The disks are then
sintered 1 hr at 1000 C in a furnace.
Denture acrylic
Denture acrylic (polymethylmethacrylate cold-cure acrylic; e.g., Dentsply Limited) disks
can be prepared, according to the manufacturers instructions, using self-cure acrylic. In
order to produce disks of the correct size, the CDFF pans can be used as 5 1mm
molds.
Enamel and dentine
Enamel and dentine disks can be prepared using extracted teeth (sterilized and washed).
They are cut into 5-mm-diameter cylinders using a trepanning tool on a metal worker
lath/drill before being sectioned using a rotary diamond saw. Further information can be
found in Matharu et al. (2001).
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SUPPORT
PROTOCOL 5
Mixed-species inoculum
Inoculate 10-ml of medium (e.g., artificial saliva; see recipe) for each bacterial species
from a single colony and incubate for the desired time for each of the species being used.
Adjust the optical density of each culture to 1.0 at 600 nm and store separately in 1.0-ml
aliquots up to 3 months at 80 C. Prior to inoculating the CDFF, thaw two cryovials of
each bacterial suspension at room temperature, transfer the contents to a 10-ml sterile
universal, and vortex mix. Using a 10-ml sterile pipet, carefully add the suspension to the
rotating pans via the sample port of the CDFF. Allow to rotate an additional 1 hr. After
this time, connect the CDFF to a reservoir of sterile medium and culture as described in
the Basic Protocol, starting at step 15.
Microcosm plaque biofilms
Human saliva can be used as an inoculum to provide a multispecies biofilm consisting
of microorganisms found in the oral cavity. Collect saliva from at least ten healthy
individuals, preferably who have not taken antibiotics in the previous three months. Pool
equal volumes from each of the subjects. Store in 1-ml aliquots up to 3 months at 80 C
for subsequent use. To grow biofilms, proceed as in the Basic Protocol, using two 1-ml
aliquots to inoculate 500 ml sterile growth medium in step 6 and culturing 8 hr in the
CDFF at a flow rate of 1 ml/min in step 13.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Artificial saliva
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1B.5.14
Supplement 6
The artificial saliva formulation given is the same as "complete saliva" in Pratten et al.,
1998. Due to the light-weight nature of hog gastric mucin extra care should be taken when
weighing.
COMMENTARY
Background Information
Models of oral biofilms, including laboratory, animal, and human in situ models,
contrast noticeably in their microbiological
complexity. Animal models have proven unequivocally that formation of dental caries is
an infectious and transmissible disease. Use
of biofilm models has enabled comparisons to
be made of the cariogenic potential of different bacterial species, the role of the diet, and
the effects of potential anticaries agents. In
situ human models created using devices such
as the Leeds in situ plaque-generating device
have been designed to permit the development
of natural plaque on standardized enamel
surfaces freely exposed to the human oral environment (Robinson et al., 1997).
However, there are problems with the ethics
associated with animal and human models. In
addition, dental diseases are site specific, and
the plaque biofilm is heterogeneous, which can
lead to problems with sampling. Further, these
intra-oral models suffer from the inability to
use them for testing experimental products,
especially in terms of safety to the patient.
These restrictions have led to the development of laboratory-based models that simulate
oral biofilms in vitro. Such models range from
simple batch culture systems (e.g., the Zurich
Biofilm Model; Guggenheim et al., 2004), to
the development of artificial mouths.
In chemostat models there is a controlled
fluid environment and planktonic phase, with a
nutritionally limited growth rate. A number of
replicate samples and a variety of surfaces can
be placed within the chemostat, and properties
such as the pH can be controlled. However,
the large fluid phase is not consistent with the
in vivo situation where shear forces and a thin
film of fluid are present on the oral surfaces.
The flow cell (see UNIT 1B.2) is often the system of choice for short-term adhesion and colonization studies because it enables the nondestructive observation of biofilm development
in real time (Busscher and van der Mei, 1995).
The flow cell may be constructed in a number of ways but typically consists of a transparent chamber of fixed depth through which
the growth medium passes. When mounted
on a microscope stage, this system allows the
biofilm development inside the chamber to be
observed in real time. Other models which pro-
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Critical Parameters
The most debated parameter when carrying out studies on oral biofilms is the choice
of microorganism or microorganisms. This is
no different for the CDFF where, as already
described, the operator can use single-species,
multiple-species, or microcosm biofilms. Is-
Growing Oral
Biolms in a
CDFF
sues arise as to whether the studies are associated with biofilm formation (e.g., where primary colonizer such as Streptococcus sanguis
is studied) or a particular disease (e.g., caries
where only S. mutans is of interest). When
considering the criteria for a community study,
the organisms selected need to originate from
a pool of species common to the oral environment and important components of that community. Further, the group of species should
form a stable community where, under standard conditions, all members are retained over
the course of the experiment. To further produce biofilms which are more representative of
the in vivo scenario, the establishment of laboratory microcosms in association with a suitable substratum and nutrient source is useful.
Another important consideration is the
medium to be used. Examples of an artificial
saliva and artificial GCF are given in this unit.
Through its chemical and physical properties,
saliva exerts a profound influence on the adhesive interactions that occur in the mouth,
and thus choosing a suitable artificial saliva
is important. Due to the obvious difficulty in
using large volumes of human saliva, a synthetic substitute will be needed to reproduce
features of saliva which are considered important in cultural studies of plaque bacteria while
remaining relatively inexpensive and simple to
produce (see Strategic Planning).
1B.5.16
Supplement 6
Anticipated Results
It has been shown that using the CDFF
model, quasi steady-state biofilms can be generated after 4 to 7 days of growth, depending upon the organisms being studied. Such
biofilms typically contain 1 108 cfu/mm2 .
The reproducibility of the model has been
shown to be good, both between pans within
one experiment and between experiments.
This is particularly true with work on singlespecies biofilms. However, as might be expected in studies on microcosms where the
inoculum contains a diverse bacterial population, the resultant biofilms are far more heterogeneous. These results are less comparable, in
terms of the defined bacterial population, although this depends upon the sensitivity of
the technique used for quantification (McBain
et al., 2003).
The microstructure of oral biofilms, as
shown by confocal laser scanning microscopy
(CLSM), has revealed stacks of bacteria developing over time, separated by clear channels
(Fig. 1B.5.9). Additionally, the distribution of
viable bacteria in biofilms grown in the CDFF
has been shown to be similar to those in dental
plaque in vivo. This suggests that the CDFF
produces in vitro biofilms which are comparable to their in vivo counterparts in terms of
both structure and the spatial distribution of
viable bacteria.
When biofilms generated in the CDFF are
challenged with an antimicrobial, the results
are sensitive enough to be able to compare different treatment regimes. Such data has been
shown to be comparable, in terms of ranking
agents, to data from clinical trials.
Literature Cited
Allan, I., Newman, H., and Wilson, M. 2002. Particulate Bioglass reduces the viability of bacterial biofilms formed on its surface in an in vitro
model. Clin. Oral Implants Res. 13:53-58.
Busscher, H.J., and van der Mei, H.C. 1995. Use of
flow chamber devices and image analysis methods to study microbial adhesion. Methods Enzymol. 253:455-477.
Coombe, R.A., Tatevossian, A., and Wimpenny,
J.W.T. 1982. Bacterial thin films as in vitro models for dental plaque. In Surface and Colloid
Phenomena in the Oral Cavity: Methodological Aspects, (R.M. Frank and S.A. Leach, eds.)
pp. 239-249. IRL, London.
Dalwai, F., Spratt, D.A., and Pratten, J. 2006. Modeling shifts in microbial populations associated
with health or disease. App. Environ. Microbiol.
72:3678-3684.
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Internet Resources
http://www.imt.net/mitbst/Products.html
BioSurface Technologies produces several of the
biofilm reactors described, including flow cells,
annular reactors, and drip flow reactors.
http://www.tylerresearch.com/instr/biofilm.shtml
Tyler research produces several biofilm-generating
devices, including high-pressure and laminar
devices as well as chemostats.
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1B.5.18
Supplement 6
UNIT 1C.1
BASIC
PROTOCOL
Materials
Bacterial strain to be tested
Growth medium specic for bacterial strain
V. harveyi strain in BB152 (luxM::Tn5), frozen glycerol stock
V. harveyi strain in BB170 (luxN::Tn5), frozen glycerol stock
AB medium (see recipe)
Sterile syringe lters, 0.2-m pore size
1-ml syringes
Sterile 1.5-ml microcentrifuge tubes
Luminometer, scintillation counter, or equivalent device for measuring light
CAUTION: V. harveyi is a Biosafety Level 1 (BSL-1) organism. Such organisms are not
known to consistently cause disease in healthy adult humans, and are of minimal potential
hazard to laboratory personnel and the environment. Standard microbiological practices
should be followed when working with these organisms. See UNIT 1A.1 and other pertinent
resources (APPENDIX 1B) for more information.
NOTE: If measuring a large number of samples, use a 96-well microtiter lter plate with
a sterile 96-well plate for collection rather than individual syringes, syringe lters, and
microcentrifuge tubes.
NOTE: BB170 and BB152 strains (Bassler et al., 1993) are available at American
Type Culture Collection (http://www.atcc.org) with strain ATCC #BAA-1117 and ATCC
#BAA-1119, respectively.
2. Dilute the culture 1:1000 into fresh medium and resume growth.
Emerging
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Contributed by Michiko E. Taga
Current Protocols in Microbiology (2005) 1C.1.1-1C.1.8
C 2005 by John Wiley & Sons, Inc.
Copyright
1C.1.1
3. Remove 1-ml aliquots at time points of interest. Remove cells by passing through
0.2-m syringe lters into sterile 1.5-ml microcentrifuge tubes. Use a separate syringe
and lter for each sample.
If using a 96-well lter plate, remove 0.2-ml aliquots and lter directly into a sterile 96-well
plate.
If the AI-2 production prole of the organism has not been characterized, measure AI-2
production over a time course of growth. This is important because many bacteria produce
AI-2 only during a particular phase of growth. For example, maximal AI-2 accumulation
in V. harveyi occurs in the stationary phase, whereas in Salmonella typhimurium, AI-2 is
detectable transiently during exponential growth (Surette and Bassler, 1998). For bacteria
with rapid doubling times, collect cell-free culture uids every hour after the initial dilution,
and continue until the culture reaches the stationary phase.
In some cases, it is necessary to use the cell-free culture uids immediately or the day after
collection. The cell-free culture uids of some bacteria such as V. harveyi can be stored at
20 C without loss of AI-2 activity, while the AI-2 activity of other bacteria such as E. coli
diminishes during storage, especially with repeated freezing and thawing.
4. Collect cell-free culture uids from a culture of V. harveyi strain BB152 (i.e.,
AI-2+ ) grown overnight in AB medium to use as a positive control. Store this sample
indenitely at 20 C and use in future experiments.
7. Set up a series of duplicate mixtures containing 10% (v/v) cell-free culture uids
prepared in step 3 and 90% (v/v) diluted BB170 culture prepared in step 6. Incubate
at 30 C with aeration.
If using a 96-well microtiter plate reader for light measurement, set up 100-l samples per
well (i.e., 10 l cell-free culture uids and 90 l diluted BB170) in clear-bottomed black
microtiter plates such as Corning 3603 or clear assay plates such as Falcon 353911. If
using a luminometer that holds individual tubes, prepare 2- to 5-ml samples in glass test
tubes.
Methods for
Analysis of
Bacterial AI-2
Production
1C.1.2
Current Protocols in Microbiology
If using a 96-well microtiter plate reader, measure the light output from each well. If using
a luminometer that holds individual tubes, remove 100-l aliquots from each sample and
measure the light output.
Any one of a variety of light detection devices can be used in this assay, from a simple
photomultiplier tube to an automated microplate reader. The optimal device for the assay
will depend on the number of samples per experiment, frequency of use, and economical
considerations.
AB medium
Dissolve the following ingredients in 960 ml H2 O:
2.0 g Casamino acids, vitamin-free (0.2% w/v nal)
12.3 g magnesium sulfate heptahydrate (50 mM nal)
17.5 g sodium chloride (0.3 M nal)
Adjust pH to 7.5 with potassium hydroxide
Sterilize by autoclaving
When cool, add the following sterile ingredients to give the following nal concentrations:
1 mM L-arginine from 100 stock (pass stock through a 0.2-m lter and store
indenitely at 4 C)
1% (v/v) glycerol from 50 stock (sterilize stock by autoclaving and store indenitely at room temperature)
10 mM potassium phosphate buffer, pH 7.0 (APPENDIX 2A) from 100 stock (sterilize
stock by autoclaving and store indenitely at room temperature)
Store indenitely at room temperature
Never add glucose to the medium. Glucose inhibits bioluminescence of V. harveyi (see
Critical Parameters).
COMMENTARY
Background Information
Autoinducers are signaling molecules that
bacteria secrete and detect to regulate the expression of specic genes based on cell population density. This process, known as quorum
sensing, was initially discovered as the mechanism by which marine Vibrio species coordinately control bioluminescence of the population (Nealson and Hastings, 1979). Though
initially considered a curious phenomenon
limited to luminous marine bacteria, quorum
sensing has since been shown to regulate a variety of functions in >100 different bacterial
species, including virulence, sporulation, antibiotic production, and motility (Miller and
Bassler, 2001; Federle and Bassler, 2003).
The autoinducer termed AI-2 was identied
as one of two signaling molecules that control
bioluminescence in Vibrio harveyi (Bassler
et al., 1994). AI-2 is of particular interest
because it deviates from the quorum-sensing
paradigm in three signicant ways. First, most
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Current Protocols in Microbiology
Figure 1C.1.1 Proposed pathway for formation of AI-2 molecules. DPD cyclizes spontaneously to form two
stereoisomers, A and B. Spontaneous addition of water to A and B results in formation of C and S. typhimurium
AI-2, respectively. V. harveyi AI-2 is formed by addition of boric acid to C (Modied from Miller et al., 2004).
Figure 1C.1.2 Light production in the BB170 bioassay. Induction of bioluminescence is calculated
at the time point marked by the vertical line.
Methods for
Analysis of
Bacterial AI-2
Production
1C.1.4
Current Protocols in Microbiology
Problem
Probable cause
Solution
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Technologies
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Current Protocols in Microbiology
Problem
Probable cause
Solution
Methods for
Analysis of
Bacterial AI-2
Production
Critical Parameters
To perform the bioassay accurately, it is important to avoid conditions that can adversely
affect the growth or bioluminescence of the V.
harveyi reporter strain. Factors that inhibit the
growth or bioluminescence of V. harveyi include the presence of glucose, low pH, temperatures above or below 30 C, and various toxic
compounds. Additionally, the presence of
boric acid enhances light production of BB170
and limits its ability to distinguish between
different levels of AI-2 added. Therefore, it
is important to monitor the light production
of the medium control samples to ensure that
the reporter strain is not affected by factors
within it.
In order to calculate AI-2 activity, light must
be measured at the time point at which the
medium control produces the least amount of
light. Because the timing of the response to
AI-2 varies with each assay, it is important to
measure light production every 30 min to ensure that data can be obtained before all samples become bright.
Troubleshooting
A number of factors contribute to the overall success of the BB170 bioassay. A guide for
resolving problems with the bioassay is presented in Table 1C.1.1.
1C.1.6
Current Protocols in Microbiology
cpm at optimal
time point
Induction of bioluminescence
Medium control
15,288
N/Aa
0 hr
33,296
2.2
2 hr
92,954
6.1
3 hr
3,473,248
227.2
4 hr
8,061,968
527.3
5 hr
88,466
5.8
6 hr
26,842
1.8
Anticipated Results
Calculate the induction of bioluminescence
for each sample using data only from the optimal time point, i.e., the point at which the
medium control sample produces the lowest
amount of light (Figure 1C.1.2, vertical line):
induction of bioluminescence = light output
of the sample/light output of the control.
In the example shown in Figure 1C.1.2, induction of bioluminescence is calculated as
light output (B)/light output (A).
The light output is typically measured
as counts per minute (cpm). Sample data
from the optimal time point of an assay of
S. typhimurium cell-free culture uids collected over a time course of growth are presented in Table 1C.1.2. In this example, induction of bioluminescence is calculated as cpm
of the sample/15,288.
Time Considerations
The time required for the collection of cellfree culture uids varies depending on the
growth properties of the bacteria being tested,
with a minimum of 6 hr for bacteria with rapid
doubling times. The bioassay takes 6 to 8 hr,
including 30 min to set up the samples and 30
min for the analysis.
Literature Cited
Miller, M.B. and Bassler, B.L. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165199.
Emerging
Technologies
1C.1.7
Current Protocols in Microbiology
Surette, M.G. and Bassler, B.L. 1999. Regulation of autoinducer production in Salmonella
typhimurium. Mol. Microbiol. 31:585-595.
Methods for
Analysis of
Bacterial AI-2
Production
1C.1.8
Current Protocols in Microbiology
UNIT 1C.2
Many bacteria synthesize and secrete small molecules to the surrounding environment,
and as the cell density increases, accumulation of these molecules eventually reaches a
threshold concentration which, in turn, regulates gene expression. This process is called
quorum sensing. Many Gram-negative bacteria use acyl-homoserine lactones (AHL)
as the quorum-sensing signal molecule. Detecting AHLs from bacterial samples is an
important step in investigating and identifying novel cell-to-cell communication systems.
The protocols described in this unit can help to detect and identify unknown AHLs from
various spent bacterial media and environmental samples (Brelles-Marino and Bedmar,
2001). For detection of AI-2 (a non-AHL autoinducer molecule) using a bioluminescent
bacterial reporter strain, refer to UNIT 1C.1.
Basic Protocol 1 describes a simple method to quickly assay for AHL activity in a sample,
by quantitatively measuring -galactosidase activity of a reporter fusion gene of an AHL
bioassay strain grown in various concentrations of sample filtrates (assay filtrates should
first be tested at 10% to test for sensitivity and toxicity to the bioassay strain, then adjusted
as needed by dilution). This includes two parts; first, the assay samples are prepared and,
second, the bioassay strains with sample filtrates are incubated and the enzyme activity
assay is performed.
Basic Protocol 2 describes a method using thin-layer chromatography (TLC) to identify
AHL molecules. This protocol should be used if the initial AHL liquid assay is positive
and the AHL content and structure needs to be determined. Performing TLC separates
AHLs based on structure and polarity. Detection of AHLs on the TLC plate is performed
by coating the TLC plate with bioassay cells in agar medium containing Xgal and
examining for blue spots indicating lacZ-based Xgal hydrolysis induced by the presence
of AHLs. The relative Rf of the spots will allow for AHL identification.
The Support Protocol provides instruction for preinducing Agrobacterium tumefaciens
KYC55 (pJZ372)(pJZ384)(pJZ410), which is used as the AHL bioassay strain in both
basic protocols.
CAUTION: Follow all biosafety requirements relevant to the assay sample strain under
investigation. Refer to UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for instructions
on safe handling of microorganisms.
CAUTION: Agrobacterium tumefaciens is a Biosafety Level 1 (BSL-1) organism. Such
organisms are not known to consistently cause disease in healthy adult humans, and
are of minimal potential hazard to laboratory personnel and the environment. Standard
microbiological practices should be followed when working with these organisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
BASIC
PROTOCOL 1
This protocol consists of two basic steps. The first step requires the preparation of the
assay sample strains, which must be grown under the desired conditions. The culture
supernatant must then be collected at various time points and filtered to remove all
bacterial cells to ensure that no AHL molecules or -galactosidase from any native lacZ+
strains are being produced during the actual assay step. Alternatively, samples can be
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Contributed by Adam C. Joelsson and Jun Zhu
Current Protocols in Microbiology (2006) 1C.2.1-1C.2.9
C 2006 by John Wiley & Sons, Inc.
Copyright
1C.2.1
Supplement 3
extracted with organic solvents, such as ethyl acetate, to concentrate the AHL molecules
(see Basic Protocol 2).
The second part of the protocol consists of running the actual assay. First, AT medium,
with various concentrations of assay filtrates, are inoculated with the bioassay strains
(include necessary controls here, e.g., synthetic autoinducers and medium blanks) and
are grown for 12 to 16 hr at 28 C. After sufficient growth (OD600 reaches 0.2 to 1),
the -galactosidase assay is ready to be performed. This is done by lysing the cells and
introducing O-nitrophenyl--D-galactoside (ONPG), which is cleaved in the presence
of -galactosidase, turning the solution yellow. This assay is timed and care should be
taken to record the start time and stop times of each sample. The solution is centrifuged
after stopping to pellet cell debris and the OD420 of the supernatant is taken. Levels of
lacZ induction by AHL are then reported in Miller units.
Materials
Bacteria of interest
Medium appropriate to organism under study (e.g,. see APPENDIX 2C)
Preinduced cells (Support Protocol)
AT medium (see recipe) without antibiotics
Synthetic autoinducer
Z buffer (see recipe)
0.05% (w/v) SDS solution in H2 O
Chloroform
4 mg/ml O-nitrophenyl--D-galactoside (ONPG) in H2 O
1 M Na2 CO3
10-ml syringe and 0.2-m syringe filter
Prepare sample
1. Grow bacteria of interest to different time points in 2 ml appropriate medium.
Time points are best established experimentally, a good starting point would be low (early
log phase), medium (mid to late log phase), and high (stationary phase) cell densities to
pinpoint at what time the bacteria of interest may produce autoinducer and to test for
conditions favorable for AHL stability.
Occasionally, the production of lactonases or medium pH changes caused by some strains
may degrade the AHL produced. Therefore, it is necessary to collect samples at various
growth points.
Perform assay
3. Thaw one vial of preinduced cells on ice. For each filtrate to be assayed, inoculate 2 l
preinduced cells into 2 ml AT medium without antibiotics plus the desired amount
(up to 10%) of assay filtrate, synthetic autoinducer, or medium used to culture the
bacteria of interest. Grow 16 hr, until OD600 reaches 0.2 to 1 at 28 C.
Antibiotics are not needed as they may interfere with -galactosidase activity. In this
incubation time frame plasmids will not be lost.
LacZ-Based
Detection of AHL
Quorum-Sensing
Signals
Make a stock solution of cells in AT medium; for example, make a 20-ml solution with
20 l preinduced cells for ten assay samples to be analyzed simultaneously. This ensures
that all samples have the same starting concentration of the bioassay strain.
1C.2.2
Supplement 3
Controls should be included at this step as well. A positive control of synthetic AHLs or
cultures known to have AHL activity should be used, as well as media blanks (AT medium
and the medium used to grow assay strain).
4. Measure OD600 of the total cell suspensions. Combine 0.2 ml preinduced bacterial
cells in AT medium, 0.8 ml Z buffer, 2 drops (10 l) of 0.05% SDS solution, and
3 drops (15 l) of chloroform in 2-ml microcentrifuge tubes.
The 2 ml culture volume is to ensure optimal conditions for growth while shaking, and
to keep a consistent inoculation ratio between samples. For ten samples, 20 ml can be
inoculated and divided into 2 ml volume aliquots prior to addition of the filtrate.
5. Vortex vigorously for at least 10 sec and then add 0.1 ml of 4 mg/ml ONPG. Record
time (T0 ).
Add ONPG quickly to assay tubes to ensure a consistent T0 .
6. When the solution turns yellow, add 0.6 ml of 1 M Na2 CO3 to stop the reaction, and
record the time in minutes (Ts ).
If no appreciable yellow is seen, stop assay at 120 min.
7. Centrifuge cell debris 3 min at 16,000 g, room temperature, and measure OD420
of the supernatant.
8. Calculate results in Miller units as follows:
Time is in minutes.
For more information regarding -galactosidase assays and Miller units, refer to Miller
(1972). See Raleigh et al. (2002) for a more recent review of the Lac operon.
BASIC
PROTOCOL 2
This protocol describes a method developed by S.K. Farrand and colleagues to visualize
AHLs using thin-layer chromatography (Shaw, 1997; Meyers, 2001). AHLs in assay
samples are first separated on a C18 reversed-phase TLC plate. Agar medium containing
the lacZ-based bioassay strain and Xgal is then overlaid on the TLC plate. AHLs with
various retention factors (Rf ) on TLC plates then activate the lacZ reporter to produce
-galactosidase and hydrolyze the Xgal to form blue spots.
Materials
Bacteria of interest
Medium appropriate to organism under study (e.g,. see APPENDIX 2C)
Ethyl acetate
60:40 (v/v) methanol/water
Preinduced cells (Support Protocol)
2 AT buffer (1:10 dilution of 20 AT buffer; see recipe)
1.5% water-agar (see recipe), 45 C
20 mg/ml 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (Xgal) in
dimethylformamide (DMF)
10-ml syringe and 0.2-m syringe filter
2-ml glass vial
TLC tank (30-cm diameter 10-cm width 27-cm height)
C18 reversed-phase TLC plates (200-mm layer; Baker or Whatman)
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Current Protocols in Microbiology
Supplement 3
2. Microcentrifuge cells 1 min at 16,000 g, room temperature, remove the pellets and
filter supernatants using a 0.2-m syringe filter and 10-ml syringe. Use immediately
or freeze up to 1 month at 20 C until use.
3. Extract 5 ml filtered culture supernatants by adding 5 ml ethyl acetate to the supernatant in a 50-ml flask and agitate vigorously until the ethyl acetate is evenly
distributed. Let stand for 5 min to separate phases.
4. Transfer upper phase to a 25-ml beaker using a pipet. Repeat the extraction to the
bottom phase and add the new upper phase to the same 25-ml beaker, for a total
volume of 10 ml.
5. Evaporate or vacuum dry the collected upper phase solvent inside a hood. Resuspend
the dried sample in 50 l ethyl acetate to produce a concentrated AHL solution. Store
the AHL sample in a 2-ml glass vial up to 1 month at 20 C.
6. Add 200 ml of 60:40 methanol/water to the TLC tank. Close the lid and let equilibrate
2 hr.
7. Use a pencil to draw a line 2 cm away from the bottom of the TLC plates. Apply 1
to 5 l of the AHL samples along the line with a space of at least 1.5 cm between
samples, and let air dry (5 min).
Apply samples one at a time, and allow them to air dry between additions. This prevents
the sample from diffusing too much, creating a more contained elution.
Standards, such as synthetic AHLs, should be used here as a positive control. Culture
medium can be used as a negative control if desired.
8. Place the TLC plate in the tank and develop until the solvent front runs to the top.
Remove the plate and air dry.
Make sure the sample loading line is not immersed in the solvent.
9. Thaw one vial of preinduced cells on ice. For each filtrate to be assayed, inoculate
2 l preinduced cells into 2 ml AT medium without antibiotics. Grow 16 hr, until
OD600 reaches 0.2 to 1 at 28 C.
Antibiotics are not needed as they may interfere with -galactosidase activity. In this
incubation time frame plasmids will not be lost.
LacZ-Based
Detection of AHL
Quorum-Sensing
Signals
Make a stock solution of cells in AT medium; for example, make a 20-ml solution with
20 l preinduced cells for ten assay samples to be analyzed simultaneously. This ensures
that all samples have the same starting concentration of the bioassay strain.
1C.2.4
Supplement 3
10. Prepare the detecting culture mix by combining 50 ml of the overnight culture of
KYC55 (pJZ372)(pJZ384)(pJZ410) with 50 ml room temperature 2 AT buffer and
50 ml of 1.5% water-agar (maintained at 45 C). Add Xgal to a final concentration
of 60 g/ml. Mix thoroughly and maintain at 45 C.
Melt water-agar in microwave and maintain resulting solution in a 45 C water bath
before addition of 2 AT medium with the bioassay strain and Xgal. Upon addition of
these ingredients, immediately place solution back in 45o C water bath until use.
11. Seal the dried TLC plate with tape along edges forming a wall of tape 1-cm high
around the plate. Spread the culture mix over the surface evenly to a thickness of
0.5 cm.
Use burner flame to get rid of bubbles when overlaying the detecting agar.
12. After the agar has solidified, incubate the coated plate for 12 to 18 hr in a 28 C water
bath in a sealed plastic container and then examine for blue Xgal hydrolysis.
SUPPORT
PROTOCOL
Materials
AHL bioassay strain Agrobacterium tumefaciens KYC55
(pJZ372)(pJZ384)(pJZ410); see Zhu (2003)
LB medium (APPENDIX 4A) containing 1 g/ml tetracycline, 100 g/ml
spectinomycin, and
100 g/ml gentamycin
AT medium (see recipe) with and without 1 g/ml tetracycline, 100 g/ml
spectinomycin, and 100 g/ml gentamycin
15% (v/v) glycerol, sterile
28 C orbital shaker
2-ml microcentrifuge tubes
1. Grow AHL bioassay strain Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)
(pJZ410) in LB medium supplemented with antibiotics. Incubate on an orbital shaker
to late-log phase (OD600 of 1; 1 109 cfu/ml) at 28 C.
This bioassay strain is used as an example. It cannot produce AHLs and has three plasmids
containing PT7 -traR, a T7 polymerase gene, and a reporter traI-lacZ fusion.
Emerging
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Current Protocols in Microbiology
Supplement 3
AT buffer, 20
Per liter:
214 g KH2 PO4
Adjust pH to 7.3 with KOH
Autoclave then cool to room temperature
Store up to 6 months at room temperature
AT medium
50 ml 20 AT salts (see recipe)
50 ml 20 AT buffer (see recipe)
10 ml 50% (w/v) glucose (see recipe)
890 ml H2 O
Store up to 6 months at room temperature
Autoclave salts and buffer separately, then cool solutions to room temperature. Filter
sterilize glucose solution.
AT salts, 20
Per liter:
40 g (NH4 )2 SO4
1.56 g MgSO4
0.152 g CaCl2
0.1 g FeSO4 7H2 O
0.044 g MnSO4 H2 O
Autoclave, then cool to room temperature
Store up to 6 months at room temperature
Glucose, 50% (100)
Prepare a 50% (w/v) solution of glucose in H2 O
Filter sterilize
Store up to 6 months at room temperature
Water-agar, 1.5%
15 g agar per liter H2 O
Sterilize by autoclaving
Store up to 6 months at room temperature
Z buffer
Per liter:
16.1 g Na2 HPO4 7H2 O
5.5 g NaH2 PO4 H2 O
0.75 g KCl
0.245 g MgSO4 7H2 O
2.7 ml 2-mercaptoethanol
Adjust to pH 7.0 using HCl
Store up to 6 months at 4 C
LacZ-Based
Detection of AHL
Quorum-Sensing
Signals
1C.2.6
Supplement 3
COMMENTARY
Background Information
A number of bacterial processes, such as
antibiotic production, motility, and biofilm formation (see Section 1B), are regulated by
the exchange of chemical signals that permit a bacterial community to coordinate its
responses to environmental challenges or opportunities. The exchange of these signals enables all of the individuals in a community
to participate in reaching a consensus, and
is known as quorum sensing (Dunny, 1999;
Miller, 2001; Whitehead, 2001). In many proteobacteria, these chemical signals consist of
a set of diffusible acyl-homoserine lactones
(AHLs), also called autoinducers. The key regulatory components of these signaling systems
are LuxI-type proteins, which act as AHL synthases, and LuxR-type proteins, which serve
as AHL receptors and AHL-dependent transcription factors (Fuqua, 1994; Zheng, 2006).
All known AHLs contain invariant homoserine lactone moieties with highly variable fatty
acyl groups. These acyl groups range in length
from 4 to 18 carbon atoms. The C-3 carbon
can be fully reduced or can bear hydroxyl or
ketone substituents (Fuqua, 2001).
Detection and identification of cognate signal molecules has led to the discovery of new
bacterial cell-cell signaling systems. Specifically, the detection of AHL molecules involved in quorum sensing has been facilitated
by the development of a variety of bioassay strains. Such strains contain an easily
assayable reporter gene and lack all AHL
synthases, such that reporter activity requires
exogenous AHLs for activation. Various reporter genes have been described, including
lacZ, GFP, lux, and the production of an endogenous pigment. The Agrobacterium tumefaciens bioassay strain described here uses
the T7 expression system to strongly overexpress the regulator TraR and, thus, increases sensitivity and broadens the molecule
detection range in an A. tumefaciens AHLsynthase mutant. In the presence of exogenous AHLs, TraR can then activate a traIlacZ reporter fusion, resulting in production
of -galactosidase (Zhu 1998, 2003). Because
of the great sensitivity and broad range of
AHL detection with this bioassay strain, it
is a useful tool for detecting extremely low
concentrations of AHLs, or possibly other
Problem
Possible cause
Solution
Rich medium
Emerging
Technologies
1C.2.7
Current Protocols in Microbiology
Supplement 3
Critical Parameters
Troubleshooting
Commonly encountered problems with the
procedures outlined in this unit, along with
their possible causes and potential solutions,
are given in Table 1C.2.1.
LacZ-Based
Detection of AHL
Quorum-Sensing
Signals
Anticipated Results
The bioassay for AHL-like signals described here is quite sensitive. Figure 1C.2.1
shows an example of the use of this bioassay
to detect the AHL molecules from the supernatant of an unspecified bacterium. The liquid
-galactosidase activity assay showed over a
200-fold increase of traI-lacZ reporter induction as compared to the medium control. The
same supernatant contained at least six different AHL-like molecules when assayed using
the TLC plate. Practically, depending on the
concentration of the sample assayed, a minimum of a ten-fold increase over the medium
control is considered significant.
Time Considerations
Expect the complete autoinducer detection
assay procedure to take 2 days to complete.
Sample preparation time varies depending on
the rate at which the sample bacteria grow
and what time points the sample cultures are
removed. Expect the supernatant preparation
(cell pelleting and filtration) to take 5 to
10 min per sample. LacZ induction (overnight
growth of bioassay strain) takes 12 to 16 hr
to reach the proper concentration (OD600 of
0.2 to 1). Assaying for -galactosidase using
ONPG takes 2 to 3 hr depending on the rate
at which samples reach the desired endpoint.
The TLC assay will take 2 days to complete (Meyers, 2001). Preparing the TLC assay sample (if extract is evaporated under vacuum), tank, and plate will take 2 to 4 hr. The
1C.2.8
Supplement 3
Literature Cited
Brelles-Marino, G. and Bedmar, E.J. 2001. Detection, purification and characterisation of
quorum-sensing signal molecules in plantassociated bacteria. J. Biotechnol. 91:197-209.
Dunny, G.M. and Winans, S.C. 1999. Cell-Cell
Signaling in Bacteria. ASM Press Washington,
D.C.
Fuqua, W.C., Winans, S.C., and Greenberg, E.P.
1994. Quorum sensing in bacteria: The LuxRLuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176:269-275.
Fuqua, C., Parsek, M.R., and Greenberg, E.P. 2001.
Regulation of gene expression by cell-to-cell
communication: Acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35:439-468.
Meyers, C.L.F. 2001. Thin-layer chromatography.
In Current Protocols in Nucleic Acid Chemistry
(S.L. Beaucage, D.E. Bergstrom, G.D. Glick,
and R.A. Jones, eds.) pp. A.3D.1-A.3D.8. John
Wiley & Sons, Hoboken, N.J.
Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
Miller, M.B. and Bassler, B.L. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165199.
Raleigh, E.A., Elbing, K., and Brent, R. 2002. Selected Topics from Classical Bacterial Genetics.
In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. 1.4.1-1.4.14. John Wiley & Sons, Hoboken,
N.J.
Whitehead, N.A., Barnard, A.M., Slater, H.,
Simpson, N.J., and Salmond, G.P., 2001.
Quorum-sensing in Gram-negative bacteria.
FEMS Microbiol. Rev. 25:365-404.
Williamson, L.L., Borlee, B.R., Schloss, P.D.,
Guan, C., Allen, H.K., and Handelsman J. 2005.
Intracellular screen to identify metagenomic
clones that induce or inhibit a quorum-sensing
biosensor. Appl. Environ. Microbiol. 71:63356344.
Zheng, H., Zhong, Z., Lai, X., Chen, W.X., Li, S.,
and Zhu, J. 2006. A LuxR/LuxI-type quorumsensing system in a plant bacterium, Mesorhizobium tianshanense, controls symbiotic nodulation. J. Bacteriol. 188:1943-1949.
Zhu, J., Beaber, J.W., More, M.I., Fuqua, C., Eberhard, A., and Winans, S.C., 1998. Analogs of
the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J. Bacteriol.
180:5398-5405.
Zhu, J., Chai, Y., Zhong, Z., Li, S., and Winans,
S.C. 2003. Agrobacterium bioassay strain for
ultrasensitive detection of N-acylhomoserine
lactone-type quorum-sensing molecules: Detection of autoinducers in Mesorhizobium huakuii.
Appl. Environ. Microbiol. 69:6949-6953.
Shaw, P.D., Ping, G., Daly, S.L., Cha, C., Cronan, J.E. Jr., Rinehart, K.L., and Farrand,
S.K. 1997. Detecting and characterizing N-acylhomoserine lactone signal molecules by thinlayer chromatography. Proc. Natl. Acad. Sci.
U.S.A. 94:6036-6041.
Emerging
Technologies
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Current Protocols in Microbiology
Supplement 3
UNIT 1C.3
BASIC
PROTOCOL
Materials
Biosensor organism: Agrobacterium tumefaciens NT1(traR, tra::lacZ749; Piper
et al., 1993)
Emerging
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Contributed by Hai-Bao Zhang, Lian-Hui Wang, and Lian-Hui Zhang
Current Protocols in Microbiology (2007) 1C.3.1-1C.3.15
C 2007 by John Wiley & Sons, Inc.
Copyright
1C.3.1
Supplement 5
YEB medium with and without 50 g/ml kanamycin and 10 g/ml tetracycline
(see recipe)
Glycerol (optional)
Test organisms (e.g., bacteria from soil, water, and plant or animal tissues)
10 M N-3-oxo-octanoyl-L-homoserine lactone (3OC8HSL) and other AHLs (see
Zhang et al., 1993; Sigma-Aldrich; Cayman Chemical): dilute in YEB medium
Minimal medium agar plates supplemented with 50 g/ml X-gal (see recipe)
250-ml culture flasks
28 C incubator
Rotary shaker
96-well tissue-culture plates
UV chamber (Stratagene)
Marker pen
Ruler
Scalpel, sterile
Microcentrifuge with (optional) 96-well plate rotor
Plastic film (for sealing 96-well plates)
Prepare AHL biosensor and bacterial isolates
1. Grow AHL biosensor A. tumefaciens NT1(traR, tra::lacZ749) in a 250-ml flask
containing YEB medium supplemented with 50 g/ml kanamycin and 10 g/ml
tetracycline at 28 C, with shaking at 200 rpm to an OD600 of 1.5.
A. tumefaciens NT1(traR, tra::lacZ749) harbors two plasmids expressing TraR and the
reporter gene lacZ under the tra promoter, respectively (Piper et al., 1993).
The antibiotics are used to maintain the presence of the two plasmids, which also carry
genes for resistance to the antibiotics.
2. If not using the biosensor cell cultures immediately, add glycerol to 20% (v/v) and
store up to 1 year at 80 C.
3. Prepare individual bacterial isolates from soil, water, and plant or animal tissues
using standard techniques (see Collins et al., 1989).
4. Inoculate individual bacterial colonies into 150 l YEB medium in 96-well plates
and incubate overnight at 28 C, with shaking at 200 rpm.
Bacteria are the rich sources of novel enzymes. Incubations are generally at 28 C, and the
overnight cultures are usually ready for analysis of AHL inactivation, typically performed
in 96-well plates.
Fresh cultures of other living organisms such as fungi, plant, and animal tissues can also
be prepared using an appropriate medium (see Chun et al., 2004; Yang et al., 2005).
1C.3.2
Supplement 5
6 agar bars
sample loading area
biosensor strain
spotting area
Figure 1C.3.1 The AHL bioassay plate. Blue spot indicates that the diffusible AHL signals were
detected by the biosensor cells. Bar 1: the positive control (5 M 3OC8HSL in YEB medium) was
added. Bars 2 to 6: the reaction supernatants of ve bacterial isolates were added, separately.
The shorter distance of blue spots from the sample loading area in bars 2, 4, and 6 suggests that
the bacterial samples may have AHL-inactivating activity, whereas no blue spot in bar 5 indicates
a strong AHL-inactivating activity.
For the convenience of comparing different samples and measurement of distance, the
agar bars in plate can be prepared as rectangles of equal length by cutting off the top
and bottom parts. In general, six agar bars can be made from each 90-mm-diameter plate
(see Lin et al., 2003 and Fig. 1C.3.1).
The minimal medium agar bars are used as the supporting material for both AHL diffusion
and the growth of biosensor.
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1C.3.3
Current Protocols in Microbiology
Supplement 5
13. After incubation, examine the bioassay plates for the presence of blue spots and
measure their distance from the origin.
The biosensor spot will turn blue in the presence of detectable AHL signal (e.g., see Lin
et al., 2003 and Fig. 1C.3.1). Typically, the positive control with a final concentration of
5 M 3OC8HSL produces several blue spots with more than a 2 cm diffusion distance.
Any sample producing no blue spots or significantly fewer blue spots than the positive
control suggests potential AHL-inactivating activity and is considered a positive bacterial
isolate.
SUPPORT
PROTOCOL 1
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
1C.3.4
Supplement 5
2. Divide the reaction mixture into two 100-l aliquots in microcentrifuge tubes.
3. To one aliquot, add 10 l of 1 M HCl to acidify the reaction mixture (pH 1 to 2), and
add 10 l of water to the other aliquot as a control.
4. Incubate both tubes at room temperature.
5. Remove 5 l of reaction mix from each treatment and control tube at different time
points (5, 30, and 60 min after acidification) and assay as described in the Basic
Protocol, steps 10 to 13.
Progressive restoration of AHL activity in the acidified solution indicates a lactonase
enzyme. Typically, >90% of degraded activity can be restored within 60 min after acidification (e.g., see Yang et al., 2005). In contrast, if the AHL activity is not restored under
the acidic condition, then the enzyme that hydrolyzes AHL in the reaction mixture may be
an AHL-acylase or other enzyme that has not yet been identified.
SUPPORT
PROTOCOL 3
2. Load 5 l of each dilution onto a diffusion plate as described in the Basic Protocol,
step 9, and incubate 24 hr at 28 C.
Emerging
Technologies
1C.3.5
Current Protocols in Microbiology
Supplement 5
20
0.5
10 l of 4 mM
AHL in methanol
20 l of
200 M AHL
20 l of
20 M AHL
50 l of
2 M AHL
195
5% methanol (l)
180
180
150
160
100
200
200
200
200
200
200
AHL solution
Water (l)
0.1
0.05
40 l of
100 l of
0.5 M AHL 0.1 M AHL
3. After incubation, measure the distance from the last induced blue spot to the origin of
the 3OC8HSL spot in each agar bar to determine the diffusion distance of 3OC8HSL.
4. Establish the relationship between the concentration of 3OC8HSL loaded on the agar
bar (y, micromolar) and the 3OC8HSL diffusion distance (x, centimeters) by fitting
a set of x and y values to a logarithmic equation (Fig. 1C.3.2) using Microsoft Excel.
The relationship between 3OC8HSL concentration and diffusion distance has been empirically determined to be logarithmic, i.e., the coefficient of determination (R2 ) for the
logarithmic curve is higher relative to other curves (e.g., linear or exponential), indicating
the best fit.
A typical logarithmic equation describing the relationship between the concentration of
3OC8HSL loaded on agar bar (y, micromolar) and the 3OC8HSL diffusion distance (x,
centimeters) is as follows:
log y = 2.033x 4.382
The coefficient of determination (R2 ) of the above logarithmic equation is equal to 0.991,
indicating a highly correlated relationship between the concentration and diffusion distance on agar plate of the signal.
The above equation can be generally used to determine relative 3OC8HSL concentration
for any sample using 5 l of sample in the diffusion plate assay described above. However,
for accurate concentration determinations, it is recommended that each experimenter
build up a standard curve for each AHL signal of interest to take into account individual
differences in patterns of sample handling and measurement.
It is now possible to quantitatively determine and compare the relative enzyme activities
of the positive bacterial isolates identified.
6. Determine the concentration of remaining AHL in reaction mixture using the standard
equation. Calculate the amount of degraded AHL by deducting the remnant AHL
from the total amount of AHL used in reaction.
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
The enzyme activity can be expressed as AHL degraded (pmol) per minute per milliliter of
culture (OD600 = 1) or per microgram total protein or per nanogram enzyme, depending
on the nature of testing sample.
1C.3.6
Supplement 5
Figure 1C.3.2 Establishment of a standard curve for quantication of AHL. (A) Bioassay results
for 3OC8HSL dilutions. (B) The standard curve for 3OC8HSL concentrations (M) versus their
corresponding diffusion distances in an agar bar (cm) generated by the Microsoft Excel program.
SUPPORT
PROTOCOL 4
Emerging
Technologies
1C.3.7
Current Protocols in Microbiology
Supplement 5
3A)
and
6. Place supernatant aliquots containing 100 to 500 g total proteins into two 1.5-ml
microcentrifuge tubes for each sample. Keep one tube on ice. Boil the other tube for
5 min and then place on ice.
7. To each aliquot, add 3OC8HSL to a final concentration of 20 M and mix. Incubate
the reaction mixture at 37 C.
8. At different reaction time points (e.g., 0, 15, 30, 45, and 60 min), place a 20-l
aliquot of reaction mixture into a microcentrifuge tube. Heat the removed aliquots
in a boiling water bath for 3 min to terminate the reaction.
9. Centrifuge the timed samples and take 5 l supernatant from each tube to perform a
diffusion plate bioassay using the Basic Protocol.
10. Using Support Protocol 3, calculate the enzyme activity in the total protein samples
based on the amount of AHL inactivated, the amount of total proteins used in reaction,
and the reaction time.
SUPPORT
PROTOCOL 5
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
1C.3.8
Supplement 5
3. To 0.3 ml of the cell-free protein extracts containing 500 g total proteins (or
50 ng purified enzyme), add 3OC8HSL to final concentration of 1 mM in a 1.5-ml
microcentrifuge tube with a final reaction volume of 0.4 ml.
4. Add the same concentration of 3OC8HSL and 3OC8HS to KPBS buffer as a negative
and positive control, respectively.
5. Incubate the reaction mixture 30 to 60 min at 37 C with gentle shaking.
Typically, >80% of AHL should be degraded in the first 30 min of enzymatic reaction.
6. Denature the proteins in the reaction mixture by boiling in a water bath for 3 min.
7. Filter the reaction mixture through a 0.45-m PTFE filter. Collect the filtrate for
HPLC analysis.
The purpose of filtration is to get rid of undissolved particles, which could block or even
damage the HPLC system.
8. Inject 20 l of filtered reaction mixture and negative and positive controls, at separate
times, into the calibrated C18 reverse-phase column. Elute isocratically with 50:50
methanol/water at a flow rate of 1 ml/min.
The retention times of 3OC8HSL and 3OC8HS are 8.1 and 3.8 min, respectively.
If HPLC analysis shows that the enzymatic hydrolysis product has an identical retention
time as 3OC8HS, the AHL-inactivating enzyme under investigation should be a lactonase
(e.g., AHL-lactonase or PON).
The retention times of AHL and its degradation products are provided as reference only.
They may vary depending on the make and age of HPLC column even when using the
same solvent system.
Under the conditions used in this protocol, all AHLs can be measured with different
retention time from 4 min to more than 30 min, depending on the acyl chain of AHLs.
The retention time of these signals can be adjusted to 10 min by altering the composition
of the mobile phase, i.e., the percentage of methanol and water (see Table 1C.3.2). AHL
should be dissolved in methanol as a stock solution of 50 mM, which can be diluted with
water to an indicated concentration before analysis.
Unlike gradient elutions where a continuous change in the composition of the mobile
phase is employed, isocratic separations are performed with a constant concentration of
the eluent or mobile phase.
Emerging
Technologies
1C.3.9
Current Protocols in Microbiology
Supplement 5
Table 1C.3.2 Retention Times and Mobile Phase for Representative AHLs
Mobile phase
(methanol:water)
3-HO-C4-HSL
7.2
35:65
C4-HSL
7.7
40:60
3-oxo-C6-HSL
9.3
40:60
C6-HSL
8.6
45:55
3-oxo-C8-HSL
(3OC8HSL)
8.1
50:50
C8-HSL
9.6
50:50
12.5 or 15.3
60:40 or 50:50
C10-HSL
11.8
65:35
3-oxo-C12-HSL
9.9
75:25
AHL
3-oxo-C10-HSL
SUPPORT
PROTOCOL 6
Materials
N-3-oxo-octanoyl-L-homoserine lactone (3OC8HSL; Sigma-Aldrich) in KPBS
(see APPENDIX 2A for KPBS; for purified enzyme assay)
Purified enzyme (Lin et al., 2003)
3OC8HSL (solid; for cell debris assay)
Cell debris from 2 ml bacterial cultures (Support Protocol 4, step 4 pellet)
Homoserine lactone (HSL; Sigma-Aldrich) standard
2.5 mg/ml DANSYL chloride (Sigma-Aldrich) in acetone
0.2 M HCl
Additional reagents and equipment for HPLC (see Support Protocol 5)
1. Equilibrate a C18 reverse-phase column as described in Support Protocol 5, step 1.
2a. When using purified enzyme: Incubate 3 mM 3OC8HSL in 0.5 ml KPBS with
100 ng purified enzyme 3 hr at 37 C.
The final concentration of KPBS is not critical.
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
2b. When using cell debris: Incubate 3 mM 3OC8HSL in 0.5 ml cell debris 3 hr at 37 C.
Mix the reaction solution constantly by shaking until completion of the digestion
(3 hr), then centrifuge 10 min at 10,000 g, and collect the supernatants for step 3.
Complete digestion can be confirmed by using the Basic Protocol. Because the free
amino acids in cell-free protein extracts can interact with the dansylation reagent, i.e.,
DANSYL chloride, which increases the difficulty to resolve the DANSYL-HSL fraction,
1C.3.10
Supplement 5
it is recommended to use the purified AHL-inactivating enzyme (Lin et al., 2003) or the
KPBS bufferwashed cell debris because the cell membrane fraction (cell debris) contains
significantly higher AHL-acylase activity than cell-free protein extracts.
3. For DANSYL chloride derivatization, add 100 l of the digested mixture and a
1.5 mM HSL standard to an equal volume of 2.5 mg/ml DANSYL chloride,
respectively. Incubate 4 hr at 40 C.
4. After evaporation to dryness, add 50 l of 0.2 M HCl to the sample to hydrolyze any
excess DANSYL chloride.
5. Conduct HPLC analysis as in Support Protocol 5, steps 7 and 8; dilute the samples
when necessary before injection.
It is necessary to dilute the sample when the UV absorbance is >2.
If the enzymatic digestion mixture has a product peak with identical retention time as
the dansylated HSL standard (about 2.9 min under the above conditions), and shares a
similar UV spectrum, the AHL-inactivating enzyme under investigation is identified as an
AHL-acylase.
Emerging
Technologies
1C.3.11
Current Protocols in Microbiology
Supplement 5
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
1C.3.12
Supplement 5
se
tona
L-lac
N
H
AH
O
Cl
O
N
H
OH
OH
O
1N H
O H2O
1N H
Cl
O
AHL
-acy
lase
O
OH
H2N
O
Figure 1C.3.3 Enzymatic inactivation of AHL by AHL-lactonase and AHL-acylase. Unlike the AHL-acylase
hydrolysis products, the hydrolysis product of AHL-lactonase or paraoxonases (PONs) can reform AHL by
acidication.
Critical Parameters
AHL signals are not very stable in aqueous solutions; enzymatic reactions should not
proceed for more than 6 hr.
AHL biosensor strains other than those described in this unit may be used provided they
are sensitive to the low level of AHL signals
used in this unit.
For those biosensor strains with the reporter gene carried on a plasmid, it is important to supply appropriate antibiotics in growth
medium to prevent loss of the plasmid construct during propagation of cultures.
Certain enzymes such as AHL-acylase (Lin
et al., 2003) are membrane associated. When
AHL-inactivating activity is detectable using
bacterial cultures but not using cell-free total
protein extracts, one can use the cell debris
suspension to replace total protein extracts for
enzyme activity assay.
Support Protocol 3 is designed for quantitative analysis of relative enzyme activity
of different samples. The accuracy of analysis is dependent on the reliability of standard
curve and accurate determination of diffusion
distance.
Anticipated Results
The Basic Protocol is designed for rapid
and reliable screening of biological samples
with AHL-inactivating enzyme activities. Numerous bacterial isolates carrying either AHLlactonase or AHL-acylase have been identified
using this protocol or similar ones; however,
there may be other types of AHL-inactivating
enzymes. In theory, there could be at least four
types of enzymes that could degrade AHL signals, including lactonases and decarboxylases
that could hydrolyze the lactone ring as well as
acylases and deaminases that might separate
the homoserine lactone moiety and the acyl
side chain (for details, see Dong and Zhang,
2005).
The lactone ring broken by a decarboxylase
should not be restorable by acidification; the
product of an AHL-decarboxylase should have
an HPLC retention time different from that of
an AHL-lactonase because the products differ
in hydrophobicity. Therefore, both the acidification procedure (Support Protocol 2) and the
HPLC method (Support Protocol 5) described
in this unit should be sufficient for distinguishing a lactonase from a decarboxylase.
Degradation of AHL by a deaminase will
produce a fatty acid amide molecule, which
could also be dansylated by DANSYL chloride. The HPLC regime described in the Support Protocol 5 might not be able to resolve
short chain (less than six carbons) dansylated
fatty acid amide from dansylated homoserine
lactone. But the potential problem could be
resolved by analyzing several AHL signals of
different acyl side chain lengths.
By measuring the peak area of AHL and
its product using the built-in software in
the HPLC equipment, Support Protocols 5
and 6 can also be used to quantify quorumquenching enzyme activity. It is particularly
Emerging
Technologies
1C.3.13
Current Protocols in Microbiology
Supplement 5
Time Considerations
For the Basic Protocol, initiating growth of
biosensor and bacterial isolates can be done
in the morning of the first day; setting up the
reaction and bioassay will use the morning and
afternoon, respectively, of day 2; results will be
obtained on day 3. Confirmation of enzymatic
AHL inactivation (Support Protocol 1) and
testing AHL-lactonase (Support Protocol 2)
can be performed simultaneously and can be
completed within 3 days. Preparation of a
standard curve (Support Protocol 3) will take
2 days if the biosensor is ready. For HPLC
analysis (Support Protocols 5 and 6), growth
of bacterial cells and preparation of protein
extracts or purification of enzyme will take
2 days; the enzymatic reaction and HPLC
analysis can be accomplished within one day
for each type of enzyme.
Literature Cited
Byers, J.T., Lucas, C., Salmond, G.P.C., and Welch,
M. 2002. Nonenzymatic turnover of an Erwinia
carotovora quorum sensing signaling molecule.
J. Bacteriol. 184:1163-1171.
Chun, C.K., Ozer, E.A., Welsh, M.J., Zabner, J., and
Greenberg, E.P. 2004. Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by
human airway epithelia. Proc. Natl. Acad. Sci.
U.S.A. 101:3587-3590.
Collins, C.H., Lyne, P.M., and Grange, J.M. 1989.
Collins and Lynes Microbiological Methods,
6th ed. Butterworth, London.
Dong, Y.H. and Zhang, L.H. 2005. Quorum sensing
and quorum-quenching enzymes. J. Microbiol.
43:101-109.
Dong, Y.H., Xu, J.L., Li, X.Z., and Zhang, L.H.
2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and
attenuates the virulence of Erwinia carotovora.
Proc. Natl. Acad. Sci. U.S.A. 97:3526-3531.
Dong, Y.H., Wang, L.H., Xu, J.L., Zhang, H.B.,
Zhang, X.F., and Zhang, L.H. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature
411:813-817.
Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., and
Zhang, L.H. 2002. Identification of quorumquenching N-acyl homoserine lactonases from
Bacillus species. Appl. Environ. Microbiol.
68:1754-1759.
Detection and
Analysis of
QuorumQuenching
Enzymes Against
AHL QS Signals
Draganov, D.I. and La Du, B.N. 2004. Pharmacogenetics of paraoxonases: A brief review. Naunyn
Schmiedebergs Arch. Pharmacol. 369:78-88.
Draganov, D.I., Teiber, J.F., Speelman, A., Osawa,
Y., Sunahara, R., and La Du, B.N. 2005. Human paraoxonases (PON1, PON2, PON3) are
lactonases with overlapping and distinct substrate specificities. J. Lipid Res. 46:1239-1247.
Leadbetter, J.R. and Greenberg, E.P. 2000.
Metabolism of acyl-homoserine lactone
quorum-sensing signals by Variovorax paradoxus. J. Bacteriol. 182:6921-6926.
Lin, Y.H., Xu, J.L., Hu, J., Wang, L.H., Ong,
S.L., Leadbetter, J.R., and Zhang, L.H. 2003.
Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol.
Microbiol. 47:849-860.
McClean, K.H., Winson, M.K., Fish, L., Taylor,
A., Chhabra, S.R., Camara, M., Daykin, M.,
Lamb, J.H., Swift, S., Bycroft, B.W., Stewart,
G.S., and Williams, P. 1997. Quorum-sensing
and Chromobacterium violaceum: Exploitation of violacein production and inhibition for
the detection of N-acylhomoserine lactones.
Microbiology 143:3703-3711.
Molina, L., Rezzonico, F., Defago, G., and Duffy, B.
2005. Autoindution in Erwinia amylovora: Evidence of an acyl-homoserine lactone signal in
the fire blight pathogen. J. Bacteriol. 187:32063213.
Piper, K.R., von Bodman, S.B., and Farrand,
S.K. 1993. Conjugation factor of Agrobacterium
tumefaciens regulates Ti plasmid transfer by
autoinduction. Nature 362:448-450.
Wang, L.H, Weng, L.X., Dong, Y.H., and Zhang,
L.H. 2004. Specificity and enzyme kinetics of
the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase). J. Biol. Chem.
279:13645-13651.
Yang, F., Wang, L.H., Wang, J., Dong, Y.H., Hu,
J.Y., and Zhang, L.H. 2005. Quorum-quenching
enzyme activity is widely conserved in the sera
of mammalian species. FEBS Lett. 579:37133717.
Zhang, L.H. 2003. Quorum quenching and proactive host defense. Trends Plant Sci. 8:238-244.
Zhang, L.H. and Kerr, A. 1991. A diffusible compound can enhance conjugal transfer of the
Ti plasmid in Agrobacterium tumefaciens.
J. Bacteriol. 173:1867-1872.
Zhang, L.H. and Dong, Y.H. 2004. Quorum sensing and signal interference: diverse implications.
Mol. Microbiol. 53:1563-1571.
Zhang, L.H., Murphy, P.J., Kerr, A., and Tate, M.E.
1993. Agrobacterium conjugation and gene
regulation by N-acyl-L-homoserine lactones.
Nature 362:446-447.
Zhang, H.B., Wang, L.H., and Zhang, L.H.
2002. Genetic control of quorum-sensing signal
turnover in Agrobacterium tumefaciens. Proc.
Natl. Acad. Sci. U.S.A. 99:4638-4643.
1C.3.14
Supplement 5
Key Reference
Dong and Zhang, 2005. See above.
A comprehensive review on quorum-quenching
enzymes.
Emerging
Technologies
1C.3.15
Current Protocols in Microbiology
Supplement 5
UNIT 1D.3
BASIC
PROTOCOL 1
Emerging
Technologies
1D.3.1
or polypropylene test tubes. If possible, keep a separate set of pipettors used only for
RNA-extraction procedures. See Sambrook et al. (1989) for detailed instructions on
maintaining RNase-free conditions.
Materials
RNase-Away (Fisher Scientific)
Dry ice
Trizol reagent, stored at 4 C
RNAsecure resuspension solution (Ambion), stored at 20 C
Cryotubes containing B. burgdorferiinfected tissue, 80 C
Sterile, DEPC-treated, double-distilled water (APPENDIX 2A)
5% (w/v) SDS (prepared in sterile, DEPC-treated water)
1-Bromo-3-chloropane
2- Propanol (isopropanol)
75% ethanol (diluted in sterile, DEPC-treated water)
Powder-free exam gloves
Microcentrifuge tube racks
1-ml, 200-l, and 20-l micropipettors and corresponding RNase-free barrier tips
4 9/16in. (11.59-cm) blunt forceps
Ceramic crucible and pestle
Variable speed digital Dremel homogenizer (Fisher Scientific)
Ice buckets with lids
Aluminum foil cut into 2 2in. (5 5cm) squares
Sterile 12 75mm, 5-ml polystyrene round-bottom conical tubes
Sterile, RNase-free 5-ml conical tubes (Ambion)
Sterile, RNase-free 1.5-ml microcentrifuge tubes (Ambion)
60 C heating block
50-ml polypropylene conical tubes, sterile
Refrigerated microcentrifuge, 4 C
Freezer boxes
Prepare equipment and reagents for homogenization
1. Wearing gloves, spray work bench, tube racks, micropipettors, forceps, crucible and
pestle, and dremel portion of homogenizer with RNase-Away. Allow to air dry.
To ensure successful RNA extraction, all equipment and work areas must remain RNase
free for the duration of these experiments.
2. Fill one ice bucket with dry ice and place the 4 9/16in. (11.59-cm) blunt forceps,
crucible, and pestle into the bucket. Place one 2 2in. (5 5cm) square of
aluminum foil inside the crucible. Place lid on ice bucket and allow equipment to
equilibrate.
The items in the bucket will become sufficiently cold within 10 to 15 min. During equilibration, the remaining preparation steps listed below may be carried out.
3. Label 5-ml conical tubes (one per tissue) and 1.5-ml microcentrifuge tubes (two per
tissue) with a permanent ink pen. Change gloves.
Gloves must be changed frequently to maintain RNase-free conditions.
4. Using a 1-ml micropipettor, pipet 1 ml Trizol reagent into each labeled 5-ml conical
tube.
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
5. Fill the second ice bucket with ice and place the 5-ml conical tubes containing Trizol
reagent on the ice.
1D.3.2
Current Protocols in Microbiology
8. Working quickly over the dry ice bucket, remove the frozen tissue from cryotube
with forceps and wrap in the frozen aluminum foil square. Place wrapped tissue in
the frozen crucible on dry ice.
9. Using the frozen pestle, quickly pulverize the foil-wrapped tissue within the crucible.
For most frozen tissues, two to three applications of the pestle are sufficient to break the
frozen tissue into pieces suitable for homogenization. Do not allow the pestle or tissue to
thaw, as RNases can more easily degrade the RNA if samples or equipment become warm.
10. Quickly unwrap the foil. Using the forceps, quickly and carefully place all pulverized
tissue pieces into the 5-ml conical tube containing Trizol reagent and place tube back
on ice.
11. Keeping the tube on ice, use setting five on the tissue homogenizer to completely
homogenize the tissue pieces within the Trizol reagent.
This setting is sufficient to completely homogenize most tissues with only three to four
strokes. Due to the presence of bone, joints require an initial homogenization with the
seven setting. The five setting can then be used to complete the homogenization.
13. Dispense 30 ml of 5% SDS solution into a 50-ml centrifuge tube. Place the Dremel
end of the homogenizer into the SDS solution and homogenize on setting five for 5
to 10 sec. Remove the homogenizer.
14. Repeat step 12 three more times. Wipe off excess water from the Dremel end of the
tissue homogenizer with a Kimwipe.
15. Spray Dremel end of tissue homogenizer with RNase-Away. Wipe off excess RNaseAway with a Kimwipe.
16. To process another tissue sample, go back to step 7 and proceed with the remaining
protocol steps.
Extract RNA
17. Change gloves. Using the 1-ml micropipettor, transfer each homogenized sample
from its 5-ml conical tube to a 1.5-ml microcentrifuge tube. Incubate 5 min at room
temperature.
Emerging
Technologies
1D.3.3
Current Protocols in Microbiology
18. Change gloves. Add 100 l of 1-bromo-3-chloropane to each sample. Shake each
tube vigorously to mix contents. Incubate 3 min at room temperature.
Alternatively, 200 l of chloroform can be added to each sample. RNA extracted using
chloroform is suitable for northern blot analyses. However, chloroform inhibits the reverse
transcriptase reaction used to synthesize cDNA from RNA (Chomczynski and Mackey,
1995). For this reason, 1-bromo-3-chloropane is used in this protocol.
Do not vortex the sample as the RNA will shear.
21. Change gloves. Add 500 l isopropanol to each tube. Shake vigorously and incubate
10 min at room temperature. Centrifuge samples 10 min at 12,000 g, 4 C.
Isopropanol is added to precipitate the RNA out of solution. Following centrifugation, a
small white pellet containing the RNA is usually visible at the bottom of the tube. For some
very small tissues, such as bladder, this pellet may barely be visible.
22. Following centrifugation, carefully decant the supernatant into an appropriate waste
container. Change gloves. Add 1 ml of 75% ethanol to each tube to wash the pellet.
Centrifuge samples 5 min at 7500 g, 4 C.
The application of 75% ethanol helps wash impurities away from the pellet and makes it
easier to resuspend.
23. Carefully decant the supernatant from the pellet. Change gloves. Allow the pellet
5 min to air-dry by placing the open 1.5-ml microcentrifuge tube upside-down onto
a clean Kimwipe. Gently tap out all remaining drops of supernatant.
It is critical that the pellet does not over-dry, as it becomes extremely difficult to resuspend.
Do not allow drying time to exceed 10 min.
24. Change gloves. Using the appropriate micropipettor, resuspend each pellet in a suitable volume of RNAsecure resuspension solution that has been pre-warmed to 60 C
(Table 1D.3.1). Gently flick the bottom of the tube to aid resuspension of the pellet.
Immediately incubate all samples 10 min at 60 C.
Table 1D.3.1 Recommended Resuspension Volumes for RNA Pellets Obtained from Select Borrelia
burgdorferiInfected Mouse Tissues
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
Tissue
Bladder
1030
Heart
5060
Joints
5060
Spleen
100
Ears
5060
1D.3.4
Current Protocols in Microbiology
BASIC
PROTOCOL 2
Materials
RNase-Away (Fisher Scientific)
RNA extracted from B. burgdorferiinfected tissues, 80 C
10 Turbo DNA-free buffer (Turbo DNA-free kit) treated with RNAsecure
(Ambion)
25 RNAsecure solution (Ambion), stored at 20 C
DNase inactivation reagent (Turbo DNA-free kit) treated with RNAsecure
(Ambion)
10 U/l RNase-free DNase I (Roche Applied Science)
Agarose
1 TBE: dilute from 5 TBE stock (APPENDIX 2A; also see Sambrook et al., 1989)
in sterile, DEPC-treated water (APPENDIX 2A) and treat with RNAsecure
Ethidium bromide staining solution (APPENDIX 2A)
Emerging
Technologies
1D.3.5
Current Protocols in Microbiology
5 DNA loading dye (prepared in sterile, DEPC-treated water and treated with
RNAsecure; see Sambrook et al., 1989)
100-bp DNA ladder (New England Biolabs)
Takara 10 dNTPs (Fisher Scientific)
Takara 10 PCR buffer (Fisher Scientific)
30 M B. burgdorferi Fla1 oligonucleotide primer (Miller et al., 2005)
30 M B. burgdorferi Fla2 oligonucleotide primer (Miller et al., 2005)
Takara Taq polymerase (Fisher Scientific)
10-l micropipettor and barrier tips
37 and 60 C heating blocks
Sterile, RNase-free 1.5-ml microcentrifuge tubes
Refrigerated microcentrifuge
Agarose gel casting apparatus, gel trays, and combs
Agarose gel electrophoresis gel box
Electrophoresis power supply
UV light box
RNase-free, sterile, thin-walled PCR tubes
Thermal cycler
Remove contaminating genomic DNA
1. Wearing gloves, spray work bench and micropipettors with RNase-Away. Let air-dry.
2. Remove RNA samples from 80 C freezer and thaw on ice.
3. Thaw 10 Turbo DNA-free buffer and DNase inactivation reagent on ice. When
thawed, incubate 10 min in a 60 C heating block. Cool 5 min on ice.
Incubation at 60 C activates the RNAsecure (Ambion) previously added to these reagents
and inactivates any RNases present in these vials.
4. Change gloves. Remove 2 l of each RNA sample and place into labeled, sterile,
RNase-free 1.5-ml microcentrifuge tubes. Set these tubes aside on ice.
These dirty RNA samples will be compared with the corresponding clean (DNA-free)
samples by agarose gel electrophoresis, providing a method whereby the success of the
DNase I treatment is evaluated.
5. Change gloves. Add 0.1 vol of 10 Turbo DNA-free buffer and 3 l DNase I to each
RNA sample. Gently flick the bottom of each tube to mix its contents. Pulse spin
each sample in a refrigerated microcentrifuge so that all contents settle to the bottom
of the tube.
Alternatively, 1 l DNase I may be used. However, for the majority of samples, this
is not enough enzyme to digest all of the contaminating genomic DNA present. As a
result, repeated DNase I treatments are necessary. Repeated 37 C incubations increase
the potential for damaging RNase activity. For most samples, using 3 l enzyme ensures
that only one DNase I treatment is performed.
The DNase inactivation reagent also removes any divalent cations that are present within
the samples. This is important because some divalent cations act as co-factors for the
activation of RNases.
1D.3.6
Current Protocols in Microbiology
10. Prepare a 1% agarose gel in RNase-free 1 TBE. When the agarose solution has
cooled to 60 C, add ethidium bromide staining solution (2 l for small gels, 4 l
for large gels). Gently swirl the flask to mix contents. Pour the solution into the gel
tray set up in the casting apparatus and allow the gel to solidify.
Refer to Sambrook et al. (1989) for instructions on the preparation and casting of agarose
gels.
11. While waiting for the agarose gel to solidify, transfer 2 l of each DNase Itreated
RNA sample to labeled, sterile, RNase-free 1.5-ml microcentrifuge tubes and place
on ice.
12. Add 8 l RNase-free 1 TBE to each of the dirty RNA samples (set aside in step 4)
and DNase Itreated (clean) samples.
13. Add 2 l RNase-free 5 DNA loading dye to each RNA sample. Flick the bottom of
each tube to mix its contents and pulse spin to collect sample at the bottom of each
tube. Place all tubes on ice.
14. Place the solidified agarose gel and its gel tray into the gel box. Add enough RNasefree 1 TBE buffer to completely cover the gel.
15. Pipet 5 l of the 100-bp DNA ladder into one well of the gel.
16. For RNA-extracted samples, first pipet a dirty sample into a well, then add a clean
sample into the next well.
Applying the pre- and post-DNase Itreated aliquots of each RNA sample to the gel in
this specific order allows a side-by-side comparison of each dirty and clean aliquot,
therefore, assessing the efficacy of the DNase I treatment. In addition, if RNase-mediated
degradation of the RNA has occurred, this method allows the scientist to determine whether
this degradation occurred before or during DNase I treatment of the RNA.
17. Connect the electrodes of the gel box to the power supply and run the gel for 45 min
to 1 hr at 125 (small gels) or 145 V (large gels).
For more detailed instructions on the proper voltage to use for electrophoresis of agarose
gels of varying sizes, consult Sambrook et al. (1989).
18. After electrophoresis has concluded, check the integrity of each RNA sample by
illuminating the gel on a UV light box.
As illustrated in Fig. 1D.3.1, at least two sharp discrete bands should be visible for
each sample. Since RNA was extracted from mouse tissue, these two bands represent the
mammalian 28S and 18S ribosomal RNAs (rRNAs). If one or both of these bands are
missing or smeared, then RNase-mediated degradation of the RNA has occurred. Under
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Current Protocols in Microbiology
Figure 1D.3.1 Assessment of RNA integrity prior to, and following DNase I treatment. RNA was
isolated from bladder, heart, joint, spleen, and ear tissues obtained from a B. burgdorferi-infected
mouse. The integrity of each RNA sample was then assessed before (D = dirty) and after DNase I
treatment (C = clean). A volume of 2 l of each RNA sample was analyzed by agarose gel
electrophoresis and resultant bands were visualized with ethidium bromide. Mouse 28S, 18S, and
5.8S/5.0S rRNA bands are indicated by arrows. Note the presence and clarity of all three rRNA
bands.
these circumstances, the integrity of the sample is not good enough for Q-RT-PCR analysis.
A third, higher molecular weight band is sometimes visible in the dirty samples, which
represents the contaminating genomic DNA. This band should be absent in the clean
samples if the DNase I treatment was successful.
Occasionally, the 5.8S/5.0S rRNAs are visible on the gel. If visible, they will appear as
one very low molecular weight band, due to the limited resolution of the 1% agarose gel.
However, due to their small size, these rRNAs are often not visible on a 1% agarose gel.
21. Change gloves. Add 1 g of a DNase Itreated RNA sample and the appropriate
volume of sterile, DEPC-treated water to each tube (to bring volume up to 100 l).
Place the tubes back on ice.
For each sample of RNA, 1 g is used as the template for PCR reactions because this is
the amount of RNA that will later be reverse-transcribed into cDNA.
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.8
Current Protocols in Microbiology
23. Place the tubes into the thermal cycler. Using the following parameters, program the
thermal cycler according to the manufacturers instructions.
40 cycles:
1 min
1 min
1 min
94 C (denaturation)
50 C (annealing)
65 C (extension)
For this PCR reaction, 40 cycles are used because the LightCycler-based protocol (see
Basic Protocol 3) used for cDNA amplification requires 40 cycles.
24. Change gloves. Pipet 10 l of each completed PCR reaction and 2 l of 5 DNA
loading dye into clean, RNase-free microcentrifuge tubes. Analyze each PCR amplicon on a 1.5% agarose gel containing ethidium bromide. First load 5 l of the 100-bp
DNA ladder into one well of the gel, then load each PCR reaction. Run the gel for
1 hr at 135 V (for small gels) or 150 to 160 V (for large gels).
25. After electrophoresis, place the gel on a UV light box to examine the PCR results.
If the DNAse I treatment was successful, only the DNA ladder should be visible.
However, if genomic DNA is still present in the RNA samples, a band at 450 bp
will be visible. If such band is present, repeat the DNase I treatment. If no bands
are present, either store the RNA up to 1 year at 80 C or proceed directly to the
production of cDNA (see Basic Protocol 3).
SUPPORT
PROTOCOL 1
Materials
RNAsecure resuspension solution (Ambion), stored at 20 C
DNase Itreated RNA sample (see Basic Protocol 2)
RNase-free 1.5-ml microcentrifuge tubes
Spectrophotometer
Cuvettes
1. Using the RNA resuspension solution as diluent, prepare a 1:100 dilution of each
DNase Itreated RNA sample. Dispense into clean, RNase-free 1.5-ml microcentrifuge tubes. Flick the bottom of each tube a few times to mix its contents and pulse
spin to collect contents at the bottom of the tube. Place tubes on ice.
2. Pipet a suitable volume of RNA resuspension solution into a clean, RNase-free
1.5-ml microcentrifuge tube as the blank used to zero the spectrophotometer. Place
the tube on ice.
The blank and the diluent should always be the same solution in which the RNA was
resuspended.
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5. Change the wavelength on the spectrophotometer to 280 nm. Insert the blank cuvette
and strike the zero key, where the A280 should read 0.00 nm. Remove the cuvette and
set it aside.
6. Place the cuvette containing the first diluted RNA sample in the spectrophotometer.
Record the stabilized wavelength. Remove the cuvette and discard, if disposable. If
not disposable, rinse the cuvette out with water, followed by ethanol, and set it aside
to dry.
7. Repeat steps 3 to 6 for all additional samples.
8. To determine the purity of each extracted RNA sample, divide the A260 value by the
A280 value to obtain the A260 /A280 ratio. If the number obtained is 1.7, then the RNA
is of sufficient purity for RT-PCR (Sambrook, et al., 1989).
9. To calculate the concentration of each RNA sample, multiply the A260 value by 100
(the dilution factor), and then by 40 (the value of 1 l single-stranded RNA at an A260
of 1.0 is 40 ng). The resultant number is the concentration of RNA in nanograms per
microliter (Sambrook, et al., 1989).
The pathlength of the cuvette must also be considered in these calculations. Often, this
value is 1 cm, so no multiplication factor is necessary. If the pathlength is not 1 cm,
then the A260 must also be multiplied by this number. After the concentration has been
determined, the RNA can be stored at 80 C.
BASIC
PROTOCOL 3
Materials
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.10
Current Protocols in Microbiology
2 l 10 reaction buffer
4 l 25 mM MgCl2
2 l 40 mM (10 mM each) dNTPs
2 l 0.04 A260 U/l random primer p(dN)6
1 l 50 U/l RNase-inhibitor
0.8 l AMV reverse transcriptase
In this experimental setting, single-stranded cDNA is generated by the binding of the
random primer to numerous nonspecific RNA sequences (Sambrook et al., 1989).
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1D.3.11
Current Protocols in Microbiology
6. Flick the bottom of the tube a few times to mix its contents. Pulse centrifuge to collect
the contents at the bottom of the tube. Change gloves. Aliquot 11.8 l master mix
into each tube containing 8.2 l of RNA and DEPC-treated water (step 3).
The total volume in each tube should now equal 20 l.
7. Flick the bottom of each tube a few times to mix its contents. Pulse centrifuge the
samples to collect the contents at the bottom of the tubes. Incubate 10 min at room
temperature.
This first incubation step is performed to allow the random primer to anneal to the RNA.
13. Flick the bottom of the tube a few times to mix its contents. Pulse centrifuge to
recollect the contents at the bottom of the tube. Set the tube aside on ice.
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
14. Remove the LightCycler centrifuge adapters located within the aluminum cooling
block from 4 C. Change gloves. Place a 20-l LightCycler capillary tube into the
first adapter and aliquot 8 l master mix into it.
1D.3.12
Current Protocols in Microbiology
21. To set up the PCR reaction conditions that will be used for these samples, double
click the Add button.
The reaction conditions for these experiments have been previously reported (Gilmore
et al., 2001; Miller et al., 2005).
22. When prompted for a program name, enter denaturation. Enter Type as none.
Click the Ins button to insert the following cycling parameters:
Cycles: 1
Temperature Target: 95 C
Hold Time: 120 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
This program is needed to inactivate the hot-start enzyme that is bound to the Taq polymerase.
23. Double click the Add button and enter amplification. The Type is quantification. Prepare the program containing the cycling parameters for the PCR
reaction in the following manner.
a. Click Ins and enter the following information:
Cycles: 40
Temperature Target: 94 C
Hold Time: 5 sec
Slope: 20
2 Target Temp: 0
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Current Protocols in Microbiology
Step Size: 0
Acquisition Mode: none
b. Click Ins again and enter:
Temperature Target: 55 C
Hold Time: 5 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
c. Click Ins to add a third segment and enter:
Temperature Target: 72 C
Hold Time: 30 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
d. For flaB analyses, click Ins and enter a fourth segment with the following
parameters:
Temperature Target: 82 C
Hold Time: 1 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: single
To conduct PCR using primers specific for a gene of interest, delete this fourth segment
and change the acquisition mode on the third segment to single.
24. Double click the Add button and enter melting. The Type is melting curves.
Enter the following segemnents
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
a. Click the Ins button and insert the following cycling parameters:
Cycles: 1
Temperature Target: 95 C
Hold Time: 0 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
b. Click Ins and insert a second segment with the following parameters:
Temperature Target: 65 C
Hold Time: 15 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
c. Click Ins again and enter a third segment with the following parameters:
Temperature Target: 95 C
Hold Time: 0 sec
Slope: 0.1
2 Target Temp: 0
Step Size: 0.2
Acquisition Mode: continuous
1D.3.14
Current Protocols in Microbiology
This program is necessary so that the software will perform a melting curve analysis of
the data. One reason a melting curve analysis is important is because it indicates whether
the primers selected for analysis are specific, and whether the peak(s) present correlates
with the expected PCR amplicon(s) (visualized via agarose gel electrophoresis). Consult
Wittwer (2001) for a more detailed explanation.
25. Double click the Add button and enter cooling. The Type is none. Click the Ins
button to insert the following cycling parameters:
Cycles: 1
Temperature Target: 40 C
Hold Time: 30 sec
Slope: 20
2 Target Temp: 0
Step Size: 0
Acquisition Mode: none
26. Double click Edit Samples to be directed to the module where sample information is
entered. Type the sample name and indicate whether it is a negative, standard,
or unknown. Earmark cDNA replicates and enter the concentration of each genomic
DNA standard. At the bottom right of the screen, enter ng for concentration units.
Click Done.
27. Double click on Save Experiment File and name the file.
The protocol will now be saved in the protocol folder.
28. Carefully insert each capillary tube into the corresponding slot of the LightCycler
carousel. Place the carousel into the LightCycler and gently close the lid.
29. Click Run. When prompted, name and save the experiment to the Data folder.
The PCR assay will now be conducted.
Roche Applied Science publishes several references that provide more detailed information on the LightCycler and the use of LightCycler software. The LightCycler operators manual, version 3.5, and Principles and Applications of the LightCycler can
be found online (http://www.Roche-applied-science.com/lightcycler-online). Another description of the methods and applications of LightCycler-based PCR can be found in print
(Wittwer, 2001).
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Figure 1D.3.2 Quantitative analysis of B. burgdorferi flaB gene expression within cultured bacteria. cDNA was obtained from B. burgdorferi B31-MI-16 bacteria cultivated to mid-logarithmic phase
and 2 l was analyzed for expression of the housekeeping gene flaB. The fluorescence curves
(fluorescence versus the PCR cycle number) are displayed in the top right graph. A standard curve
(bottom right corner) generated using known quantities (ng) of B31-MI-16 DNA (top left panel) was
used by the LightCycler software to calculate the amount of flaB transcript present in each cDNA
triplicate (top left corner). Note the ideal regression value of 1.00 obtained in this assay.
However, due to the inherent user bias present with this module, the automated second
derivative maximum module is preferentially used to avoid this problem.
32. Three analysis steps will be now be present. Select step 3: analysis.
Two graphs will be shown. The top graph displays fluorescence curves represented as the
fluorescence versus the PCR cycle number. The bottom graph is the standard curve, which
is represented as the crossing point number for each genomic DNA standard versus the log
of the concentration (in ng) of each genomic DNA standard. Plotting of these data yields
a linear regression line, from which the concentration of each unknown cDNA sample is
calculated by the software. The slope of the line and an error value for the plotted data are
also reported (Fig. 1D.3.2). A regression (r) value of 1.00 is ideal, and values 0.95
are acceptable.
The other two analysis steps, step 1: baseline and step 2: noise band,
individually display the baseline and noise band lines determined for the fluorescent curve
graph by the software. As these features are not user-defined in the second derivative
module, and the baseline is also shown in the analysis step, these first two steps are not
as useful as the third step.
33. Although the quantification results for each sample are displayed in the panel to the
left of the graphs, not all the results are visible in the printable window. To view a
summary of all results, including the average and standard deviation values obtained
for each cDNA triplicate, click on Window and select Print Summary Report.
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.16
Current Protocols in Microbiology
35. Double click on Select a Program, and select Melting Curve is a . . . . Click the Melting
Curve button on the data screen.
The melting curve module is now open and two graphs should be visible. The top graph
displays the fluorescence versus the temperature and shows that the amount of SYBR
green binding to the DNA is decreased as the temperature increases. The bottom graph
replots this data as fluorescence (d(F1)/dt) versus temperature such that the data is now
displayed as a peak(s). Note how many major and minor peaks are present. If one PCR
product is expected then only one major peak should be present (Fig. 1D.3.3A).
Melting curve analysis is important as the major peak(s) represents a melting temperature(s) that is unique for a specific PCR product(s) (Roche Molecular Biochemicals,
2000; Wittwer, 2001). Thus, melting curve analysis indicates whether a PCR amplicon of
interest has been obtained and whether the PCR reaction was specific (minor peaks could
indicate primer-dimers, other major peaks imply the primers or other PCR conditions
utilized were not specific (see Troubleshooting).
36. To print the melting peaks, click on File and select Print Window.
SUPPORT
PROTOCOL 2
Materials
DNA-easy tissue kit (Qiagen)
5-ml mid-logarithmic phase culture of B. burgdorferi strain of interest (UNIT 12C.1)
0.7% agarose gel
RNase-free TE buffer (APPENDIX 2A)
Spectrophotometer
Gel electrophoresis equipment
Sterile, RNase-free, DNA-free siliconized 0.5-ml microcentrifuge tubes
1. Using the DNA-easy tissue kit (Qiagen), extract genomic DNA from a 5-ml
B. burgdorferi mid-logarithmic phase culture. Follow the manufacturers directions.
Remember to change gloves frequently to prevent DNA contamination of the kit
reagents.
Other methods for extracting genomic DNA can also be used (Sambrook et al., 1989), as
long as the resultant DNA is of high quality and purity. It is important that the B. burgdorferi
strain used to generate the genomic DNA standards is the same as the strain used to infect
the mice.
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3. Analyze the quality of the extracted DNA via 0.7% agarose gel electrophoresis.
One distinct band should be easily visualized on the agarose gel. If no band is present,
or the band is smeared, then DNase-mediated degradation of the DNA has occurred.
DNA that is partially or completely degraded is not suitable for LightCycler-based PCR
analyses.
4. Change gloves. Using sterile, DNA-free TE buffer as diluent and sterile, RNase-free,
DNA-free siliconized 0.5-ml microcentrifuge tubes, prepare at least six ten-fold serial
dilutions of the extracted genomic DNA. Set these tubes aside on ice.
To detect candidate genes which may be expressed at very low levels within B. burgdorferi
infected mouse tissues, a concentration range of 10 ng to 100 fg is recommended. These
samples will be the genomic DNA standards that will be used to generate a standard curve
during LightCycler PCR assays.
Other concentration ranges may also be used as the LightCycler is sensitive enough
to amplify templates of a wide range of concentrations. However, the level of detection
achievable with the LightCycler is dependent on numerous other reagents, including the
oligonucleotide primers utilized (see Critical Parameters and Troubleshooting and Table
1D.3.2).
5. Change gloves. Aliquot the remainder of the undiluted extracted genomic DNA into
small volumes and place in sterile, DNA-free microcentrifuge tubes.
6. For long-term storage (weeks to years), keep the DNA in a frost-free 20 C freezer.
If the DNA is to be used within 1 or 2 weeks, store at 4 C.
Avoid freeze-thaw cycles. These cycles can result in broken, degraded DNA samples, which
are not suitable for LightCycler-based PCR analyses.
BASIC
PROTOCOL 4
Materials
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.18
Current Protocols in Microbiology
1. Carefully remove the capillary tubes of interest from the LightCycler carousel and
place each into an open 1.5-ml microcentrifuge tube.
2. Carefully remove the cap from each capillary tube. Place the open capillary tube into
the microcentrifuge tube, with the cup portion of the capillary facing down into the
tube.
3. Centrifuge each sample 2 min at 1000 g, room temperature, to collect the sample
at the bottom of the microcentrifuge tube.
4. Discard empty capillary tubes into proper waste containers.
For agarose gel analysis, proceed to step 5a. For instructions on preparing the amplicon
for sequencing, go to step 5b.
Figure 1D.3.3 Melting curve analysis of B. burgdorferi flaB gene expression within cultured bacteria. (A) The fluorescence versus temperature is displayed in the top graph. This graph demonstrates the decrease in SYBR green binding to the DNA that occurs as the temperature increases.
The bottom graph replots this data and displays it as a peak(s). Note that one major peak, corresponding to the specific flaB amplicon, was obtained for each sample (except the negative control,
TE). The minor peak obtained at 89 C for the three cDNA samples is non-specific and does not
represent an amplification product (see Fig. 1D.3.3B). (B) Agarose gel electrophoresis analysis of
the LightCycler reaction products obtained in the above experiment (Figs. 1D.3.2 and 1D.3.3A).
Note that only one amplicon of the expected size was obtained for each sample (except the negative control, sample 1). Numbers above the lanes correspond with the numbers and samples listed
in the top left panel of A. PC = positive control flaB PCR product, 0.5 = 0.5-kb DNA size marker.
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To verify by sequencing
5b. Before the LightCycler amplicon can be sequenced, remove PCR buffers and other
reagents by using a Microcon-100 microconcentrator filter. Follow the manufacturers
directions to obtain purified DNA.
6b. Determine the concentration of the DNA using a spectrophotometer.
7b. Sequence DNA.
COMMENTARY
Background Information
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.20
Current Protocols in Microbiology
Critical Parameters
Prior to beginning any experiment, the
primers to be used for LightCycler-based PCR
analysis should be designed and ordered. It is
recommended that each primer length not exceed 24 bp, and that the target amplicon be between 350 and 500 bp. Amplicons that are 200
to 250 bp in size can be easily accommodated,
but products approaching 1 kb (or larger) will
require significant modification of the amplification protocol and/or MgCl2 concentrations used for PCR amplification. In addition,
oligonucleotide primers should be free of secondary structures, and should not exhibit melting temperatures (Tm ) below 60 C. Tm lower
than 60 C will require modifications of the annealing temperature used during the amplification program. Integrated DNA technologies
(IDT) has a Website that is very useful for assessing the Tm and secondary structures of potential PCR primers (http://www.Idt.com).
If RNA is not to be extracted from B.
burgdorferiinfected mouse tissues on the
day of animal euthanasia, the infected tissues
must be removed aseptically and immediately
snap-frozen in liquid nitrogen. Tissues stored
in this manner are good for 9 months. It is extremely important that RNase-free conditions
are maintained throughout the duration of
all RNA-based protocols. In addition, frozen
tissues must be kept cold on dry ice while
being pulverized to prevent RNase-mediated
degradation of the RNA. These experimental
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1D.3.21
Current Protocols in Microbiology
Protocol
Problem
Possible cause(s)
Solution(s)
RNA Isolation
Overdrying of pellet
Due to incomplete
homogenization,
proteinaceous particles may
be present resulting in a
gelatinous pellet
Improper storage/handling of
frozen tissues
Incomplete homogenization
of tissue
Insufficient removal of
aqueous layer
RNase-mediated degradation
of RNA
Improper storage/handling of
frozen tissues
RNase-mediated degradation
of RNA
Concentration of
RNA much lower
than expected
No RNA obtained
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
continued
1D.3.22
Current Protocols in Microbiology
Protocol
Problem
Possible cause(s)
Solution(s)
DNase I
treatment/
agarose gel
electrophoretic
analysis of RNA
RNA remains
contaminated
following DNase I
treatment
RNA is no longer
present, is smeared,
or only one rRNA
band is visible
following DNase I
treatment
RNase contamination of
Turbo DNA-free buffer
and/or inactivation reagent
RNase-mediated degradation
of RNA during Trizol
extraction
Prior to DNase I
treatment, RNA is
no longer present,
is smeared, or only
one rRNA band is
visible
LightCyclerbased
analysis
Capillary tube
breaks while
inserting into
instrument carousel
continued
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1D.3.23
Current Protocols in Microbiology
Protocol
Problem
Possible cause(s)
Solution(s)
Melting peak
analysis shows two
large peaks, but only
one was expected
Melting peak
analysis shows the
expected specific
peak, but a much
smaller peak is also
present at a lower
temperature
The quantification
The SYBR green is no longer
curves are very flat active
and the fluorescence
is extremely low
continued
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.24
Current Protocols in Microbiology
Protocol
Problem
Possible cause(s)
Solution(s)
Troubleshooting
There are several problems that can be encountered with each of the protocols described
within this unit. Possible causes and solutions
Anticipated Results
The following yields can be expected
upon extraction of RNA from B. burgdorferi
infected mouse tissues: 700 to 800 ng/l from
bladder, 2.0 to 2.2 g/l from heart, 1.2 to
1.4 g/l from joints, 5.0 to 7.0 g/l from
spleen, and 2.0 to 2.3 g/l from ear. The
conversion of RNA to cDNA by reverse transcriptase has a minimum efficiency of 30%,
resulting in at least 300 ng of cDNA being synthesized from 1 g of RNA (Sambrook et al.,
1989).
Time Considerations
RNA can be feasibly extracted from a maximum of twelve tissues per day, processed
in two groups containing six tissues each. If
a small number of samples are being analyzed, the DNase I treatment, agarose gel electrophoresis analysis, and diagnostic PCR reactions can all be performed in 1 day. RTPCR reactions and LightCycler-based PCR
analysis of a small number of samples can
be accomplished in 1 day, as the first-strand
cDNA synthesis reaction takes just over 1 hr
and LightCycler-based analyses only require
2 hr (1 hr to prepare 32 samples, and 1 hr
for LightCycler-based PCR of the samples).
To simultaneously analyze numerous samples,
more time will be required to complete each
phase of the protocol. Extracted RNA samples
are stable for 1 year at 80 C, and cDNA
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Current Protocols in Microbiology
Literature Cited
Akins, D.R., Bourell, K.W., Caimano, M.J., Norgard, M.V., and Radolf, J.D. 1998. A new animal
model for studying Lyme disease spirochetes in
a mammalian host-adapted state. J. Clin. Invest.
101:2240-2250.
Akins, D.R., Porcella, S.F., Popova, T.G.,
Shevchenko, D., Baker, S.I., Li, M., Norgard,
M.V., and Radolf, J.D. 1995. Evidence for in
vivo but not in vitro expression of a Borrelia
burgdorferi outer surface protein F (OspF)
homologue. Mol. Microbiol. 18:507-520.
Babb, K., El-Hage, N., Miller, J.C., Carroll,
J.A., and Stevenson, B. 2001. Distinct regulatory pathways control the synthesis of Borrelia burgdorferi infection-associated OspC and
Erp surface proteins. Infect. Immun. 69:41464153.
Caimano, M.J., Eggers, C.H., Hazlett, K.R.O., and
Radolf, J.A. 2004. RpoS is not central to the general stress response in Borrelia burgdorferi but
does control expression of one or more essential
virulence determinants. Infect. Immun. 72:64336445.
Carroll, J.A., Cordova, R.M., and Garon, C.F. 2000.
Identification of eleven pH-regulated genes in
Borrelia burgdorferi localized to linear plasmids. Infect. Immun. 68:6677-6684.
Carroll, J.A., El-Hage, N., Miller, J.C., Babb, K.,
and Stevenson, B. 2001. Borrelia burgdorferi
RevA antigen is a surface-exposed outer membrane protein whose expression is regulated in
response to environmental temperature and pH.
Infect. Immun. 69:5286-5293.
Carroll, J.A., Garon, C.F., and Schwan, T.G. 1999.
Effects of environmental pH on membrane proteins in Borrelia burgdorferi. Infect. Immun.
67:3181-3187.
Casjens, S., Palmer, N., van Vugt, R., Huang, W.M.,
Stevenson, B., Rosa, P., Lathigra, R., Sutton, G.,
Peterson, J., Dodson, R.J., Haft, D., Hickey, E.,
Gwinn, M., White, O., and Fraser, C. 2000. A
bacterial genome in flux: The twelve linear and
nine circular extrachromosomal DNAs of an infectious isolate of the Lyme disease spirochete
Borrelia burgdorferi. Mol. Microbiol. 35:490516.
Chomczynski, P. 1993. A reagent for the single-step
simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples. Biotechniques
15:532-537.
Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
Chomczynski, P. and Mackey, K. 1995. Modification of the TRI reagent procedure for isolation
of RNA from polysaccharide- and proteoglycanrich sources. Biotechniques 19:942-945.
Lederer, S., Brenner, C., Stehle, T., Gern, L., Wallich, R., and Simon, M.M. 2004. Quantitative
analysis of Borrelia burgdorferi gene expression
in naturally (tick) infected mouse strains. Med.
Microbiol. Immunol. 194:81-90.
1D.3.26
Current Protocols in Microbiology
McDowell, J.V., Sung, S.Y., Price, G., and Marconi, R.T. 2001. Demonstration of the genetic
stability and temporal expression of select members of the Lyme disease spirochete OspF protein
family during infection in mice. Infect. Immun.
69:4831-4838.
Miller, J.C. and Stevenson, B. 2003. Immunological and genetic characterization of Borrelia
burgdorferi BapA and EppA proteins. Microbiology 149:1113-1125.
Stevenson, B., Tilly, K., and Rosa, P.A. 1996. A family of genes located on four separate 32-kilobase
circular plasmids in Borrelia burgdorferi B31.
J. Bacteriol. 178:3508-3516.
Miller, J.C., Bono, J.L., Babb, K., El-Hage, N., Casjens, S., and Stevenson, B. 2000a. A second allele of eppA in Borrelia burgdorferi strain B31
is located on the previously undetected circular
plasmid cp9-2. J. Bacteriol. 182:6254-6258.
Wittwer, C. 2001. Rapid cycle real-time PCR: Methods and applications. In Rapid Cycle Real-Time
PCR: Methods and Applications (S. Meuer,
C. Wittwer, and K. Nakagawara, eds.). Springer
Verlag, Berlin.
Key References
Internet Resources
http://www.Roche-applied-science.com/
lightcycler-online
Excellent resource offering detailed technical notes
on the LightCycler and its applications. The
Emerging
Technologies
1D.3.27
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Q-RT-PCR
Analysis of
B. burgdorferi in
Mouse Tissues
1D.3.28
Current Protocols in Microbiology
UNIT 1E.1
Organisms within mixed culture biofilms are inherently difficult to study. Due to the wide
range of microenvironments within biofilms (Costerton et al., 1994), the distribution of
some organisms may be restricted to a few cells within a specialized microniche, whereas
others may be more broadly distributed. Although traditional culturing techniques are
widely used, they have an inherent bias in selecting those organisms capable of growing on
a particular laboratory medium. Molecular-based detection methods are frequently used
to detect bacteria in complex environments since they are unaffected by the limitations
of bacterial culturability (Amann et al., 1992; Muyzer and Smalla, 1998).
Several molecular tools are currently available to aid in the quest to describe and identify
the consortia within environmental biofilms. Fingerprinting assays like terminal restriction fragment length polymorphisms (t-RFLPs), which use PCR and restriction enzyme
digestion, create a banding pattern unique to each environmental sample, enabling descriptions of changes in bacterial community structure with time or treatment (Diez et al.,
2001). Cloning and sequencing of the PCR product of the 16S small ribosomal subunit
rRNA gene is typically used for sequence-based identification of bacterial community
members (Amann et al., 1992). However, fingerprinting assays are limited because they
fail to identify community members; cloning assays are likewise limited because the
number of clones that can be sampled and sequenced may fail to describe the overall diversity (Dunbar et al., 2002). Denaturing gradient gel electrophoresis (DGGE; see Basic
Protocol) both reveals a community fingerprint and provides sequence information for
identifying community members. This sequence information can then be used for fluorescence in situ hybridization (FISH) to determine the spatial distribution of individual
bacteria (Tonolla et al., 2005).
CAUTION: Follow all biosafety requirements relevant to the microorganisms and biofilm
substrates under investigation. Although the majority of organisms used with these protocols are environmental isolates, classified as biosafety level-1 (BSL-1) organisms,
additional safety precautions would be needed for BSL-2 and BSL-3 organisms. Refer
to UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for instructions on safe handling
of microorganisms.
BASIC
PROTOCOL
Emerging
Technologies
Contributed by Allana K. Welsh and Robert J.C. McLean
Current Protocols in Microbiology (2007) 1E.1.1-1E.1.17
C 2007 by John Wiley & Sons, Inc.
Copyright
1E.1.1
Supplement 4
Table 1E.1.1 Examples of General Eubacterial Primers Suitable for PCR and DGGE Analysisa
Primer name
Sequence 5 -3
Reference
GC 357F
907R
a Many other general and specific primers are available and described in the literature.
bands excised from the gel can give sequence-based identification of organisms detected
in the biofilm (see Critical Parameters and Troubleshooting).
DGGE procedures should be performed according to the instructions provided by the
equipments manufacturer; the following procedure was designed for the Bio-Rad DGGE
system. Successful DGGE requires careful optimization of the ideal denaturant concentration, time of electrophoresis, and voltage for each set of biofilm samples (Muyzer
et al., 1993; Lyautey et al., 2005). The conditions described below should work well
for biofilms from typical aerobic sites using the GC357F and 907R universal bacterial
primer set (Table 1E.1.1).
Materials
6% acrylamide solutions containing 30% (LO) and 50% (HI) denaturant (see
recipe)
0.5 TAE buffer: mix 100 ml 50 TAE (APPENDIX 2A) in a final volume of 10 liters
H2 O for at least 1 hr; store up to 3 months at room temperature
Ammonium persulfate (APS): store up to 1 year at 4 C
N,N,N,N -tetramethylethylenediamine (TEMED): store up to 1 year at 4 C
PCR-amplified samples and standards (Support Protocols 1 and 3)
2 DGGE gel loading dye (see recipe)
Ethidium bromide staining solution (see recipe)
TE buffer (APPENDIX 2A)
Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
DGGE System, e.g., DCode Universal Mutation Detection System (Bio-Rad) for
16-cm parallel gels, including:
Glass plates with spacers (grooved, 1 mm), two pairs
Sandwich clamps
Sponges
Casting stand
Alignment card
30-ml syringes, syringe sleeves, syringe cap screws, luer syringe locks, and luer
couplings
Tygon tubing
Model 475 gradient former
Y fitting
16-well combs
DCode lid stand
Electrophoresis/temperature control module
Electrophoresis tank
Sandwich core
Absorbent bench paper
50-ml conical polypropylene tubes
20-G needle
250-ml beakers
Micropipettor with gene sequencing tips
Power supply (e.g., Bio-Rad Power Pac 300)
1E.1.2
Supplement 4
Plastic mesh shelf liner (available from many stores, e.g., Wal-Mart)
UV transilluminator
Scalpel, sterile
1.5-ml microcentrifuge tubes, sterile
Digital or other camera for gel documentation
Additional reagents and equipment for sequencing DNA (e.g., Slatko et al., 1999;
optional)
Prepare gel sandwich equipment and denaturant gel solutions
1. Select one long plate (18 20 cm) and one short plate (16 20 cm) for each gel.
Wash the plates using deionized water and allow to air dry.
The Bio-Rad system is designed to fit two gels in the sandwich core to be run simultaneously. The authors typically run duplicates of the same samples on both gels for data
redundancy. It is possible to run only one gel at a time. In that case, the second position
in the sandwich core should hold the two glass plates with no spacer (see step 19 below).
The following protocols are for running two gels.
2. Cover the bench with absorbent paper. Pipet 20 ml HI denaturant (50%) acrylamide solution into each of two labeled 50-ml conical tubes and allow it to come to
room temperature in the dark. Pipet 20 ml of LO (30%) solution into each of two
additional labeled 50-ml conical tubes and allow it to come to room temperature in
the dark.
CAUTION: Unpolymerized acrylamide is a potent neurotoxin. Proper safety precautions
such as gloves and lab coat should be worn throughout the DGGE protocol (also see
UNIT 1A.3).
Bio-Rad recommends degassing the acrylamide solutions immediately before casting
the gel. The authors have eliminated this degassing step and have noticed no difference
in the time or quality of gel polymerization.
5. Place the aligned sandwich assembly on a sponge in the casting stand and lock the
assembly in place by securing the sandwich clamps in the stand.
6. Repeat steps 3 and 4 for the second gel to be cast, but set it aside. Do not place the
second sandwich assembly into the casting stand until the first gel has been cast.
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Current Protocols in Microbiology
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8. Place the Model 475 gradient former on the covered lab bench with the cam wheel
on your right side. Rotate the cam wheel counterclockwise to the vertical or start
position and tape the wheel in place so that it doesnt move when attaching the
syringes.
When first using this gradient former, filling both syringes with water, practicing putting
them on and off the former, and delivering the water into a beaker is highly recommended.
Getting a good feel for the gradient former will be critical to delivering the acrylamide
solutions quickly in later steps of this protocol.
9. Attach a 20-G needle to the luer coupling on 12 in. (30 cm) of Tygon tubing
which has a Y-fitting on the other end.
10. Place the needle (pointing down) at the top center of the sandwich assembly in the
casting stand and insert it between the two glass plates 1/4 in. (0.63 cm) beveled
side facing away. Tape the needle in place.
11. Prepare catalysts immediately prior to use:
a. In a 1.5-ml microcentrifuge tube, prepare a 10% ammonium solution (APS) by
adding 0.1 g APS and 1.0 ml deionized water. Vortex to dissolve.
b. Aliquot 90 l TEMED into another 1.5-ml microcentrifuge tube.
TEMED solution should be clear; if it is yellow replace it.
12. On the covered lab bench, place the rack with the 50-ml tubes with acrylamide,
gradient former, and casting stand with sandwich assembly such that the Tygon
tubes from the gradient former can attach to the Y-fitting on the needle. Prepare a
beaker of water to rinse the tubes and an empty beaker for waste water.
Steps 13 to 16 are time sensitive and need to be completed within 5 to 7 min. Ensure
that the sandwich assembly, syringes, and gradient former are completely ready before
proceeding.
14. With the syringe connected to the tubing, withdraw all of the high-density solution
into the HI syringe. Carefully release any air and a bit of solution back into the
tube with 19 ml remaining in the syringe. Place the syringe in the gradient former.
1E.1.4
Supplement 4
Parallel gels are a top-filling method, so the HI syringe is placed in the back position
on the gradient former farthest from you. The cap screw on the syringe slides into the
black groove on the gradient former, and the sleeve on the syringe is tightened into place
with the white plastic screw on the gradient former.
15. Repeat step 14 for the low-density solution into the LO syringe and place in the
front position on the gradient former.
16. Connect the tubing from each syringe to the Y-fitting with the needle. Rotate the
cam wheel slowly and steadily to deliver the gel solution.
17. When finished, remove the needle from between the glass plates. Carefully insert
the comb (16-well, 1.0 mm thick).
18. Place the tubing and needle into a beaker of water and reverse the cam to rinse any gel
solution. Rinse tubing and fittings thoroughly. Discard all acrylamide-contaminated
liquids into hazardous waste.
The residual casting mixture in the conical tube solidifies fairly quickly (within minutes).
If it remains liquid, or semisolid, there will be a problem with the gel. If the sandwich
assembly leaks there will be puddles of acrylamide in the corners of the sandwich
assembly, and air bubbles will form along the edges of the comb. Leaking disrupts the
gradient and the well integrity; the gel will need to be cast again.
19. Place the second gel sandwich assembly in the casting stand carefully. Prepare and
cast the second gel as in steps 7 to 18 above.
20. Let gels polymerize at least 60 min (up to 24 hr). If the gels are left overnight to
polymerize, place a layer of damp paper towels soaked in 0.5 TAE buffer on top,
then surround the top half of the sandwich assemblies in the casting stand with
plastic wrap (go around two or three times). Place in a dark cupboard.
22. Set the temperature controller to 60 C and set the ramp rate to 200 C/hr to allow
the buffer to reach the desired temperature most quickly. Turn on both the heater
and pump.
23. After the gels have polymerized, carefully remove one sandwich assembly from
the casting stand. Remove the comb.
24. Rinse the wells two or three times with buffer using narrow gene sequencing tips
that can slide between the glass plates or the 20-G needle and tubing on a syringe.
Repeat for the second gel.
25. Assemble the upper buffer chamber of the sandwich core while it lies flat on the
bench. Make sure the white core gaskets are clean and in place and lubricate the
entire gasket with running buffer to obtain a good seal.
26. Snap the sandwich assembly onto the core by sliding the grooved sandwich clamps
into the white posts on the core. Turn the core over and repeat with the other
sandwich assembly, forming an upper chamber dam.
The shorter gel plate should face the inside of the core buffer chamber.
Emerging
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Current Protocols in Microbiology
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If only one gel is to be run, assemble a set of glass plates without the spacers. Ensure
that the bottoms of the plates are flush. Attach it to the other side of the core to form an
upper chamber dam.
27. Pour 350 ml of 0.5 TAE running buffer into the upper buffer chamber. Check
for leakage. If it leaks, remove both sandwich assemblies from the core and repeat
steps 25 and 26.
28. Turn off the power on the electrophoresis/temperature module and remove it to the
DCode lid stand. Place the sandwich core in the tank with the red electrode post
on the right side of the tank and module. Replace the electrophoresis/temperature
module and turn on once again.
32. Load the samples into the wells. Load one or two wells with the DGGE standards.
Careful aim is critical while loading the samples and requires some practice. The sample
will rush out of the tip of the pipet upon contact with the hot buffer in the tank. The
authors find it helpful to allow a small air bubble at the tip of the pipet to allow a bit of
time to position the tip before the sample flows out.
33. Place the clear loading lid on top of the temperature control module. Attach the
electrical leads to the Bio-Rad Power Pac 300 power supply. Run the gel at 180 V
for 7 hr at 60 C.
Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
37. Cut a piece of plastic mesh shelf liner that is slightly larger than the gel. Lay the
mesh on top of the gel. Invert so that the mesh is now underneath and the gel is
above, still attached to the glass plate.
1E.1.6
Supplement 4
38. Release the gel from the plate using a spacer damp with buffer to pull one corner
of the gel loose from the plate while lifting that edge of the glass plate slowly.
39. Place each gel on its mesh into a plastic tub containing 500 ml ethidium bromide
staining solution. Stain 20 to 30 min in the dark. Destain in another tub with 500
ml of deionized water 20 to 30 min in the dark.
CAUTION: Wear gloves for protection from DNA stains. See UNIT 1A.3 for more information regarding ethidium bromide, including methods of disposal.
40. While staining and destaining, clean the DCode equipment. Pour out the running
buffer in the electrophoresis tank and fill the tank with deionized water. Place the
electrophoresis/temperature module back in the tank, turn on the pump, and run for
10 min. Rinse core, clamps, and other equipment with water and air dry.
41. Place the gel on a UV transilluminator using the mesh for transport, and photograph.
CAUTION: Wear UV eye protection and gloves.
46. If one band is confirmed, use another 2.5 l of the gel slice supernatant from step
43 as template in a 50-l reaction (see Support Protocol 1) to generate PCR product
for sequencing.
The 357F primer without the GC clamp should be used with the 907R primer. The
GC-clamp will interfere with the sequencing reaction.
47. Sequence the amplified DNA, e.g., as described by Slatko et al. (1999), or submit
samples to a sequencing laboratory.
SUPPORT
PROTOCOL 1
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Current Protocols in Microbiology
Supplement 4
denaturing in the DGGE gel (Muyzer et al., 1993). DGGE can not resolve PCR fragments
>600 bp (Muyzer and Smalla, 1998). To describe overall bacterial diversity, the authors
recommend using universal bacterial primers GC357 F and 907 R targeting the V3 to V6
regions of the 16S rRNA gene (Table 1E.1.1; Watanabe et al., 2001).
Materials
AmpliTaq GOLD Polymerase, 5 U/l (Applied Biosystems)
10 PCR buffer II (Applied Biosystems)
25 mM MgCl2 (Applied Biosystems)
10 mM dNTP blend (Applied Biosystems)
Primers (Table 1E.1.1): dilute to 20 M
DNA template: extracted from biofilm sample (Support Protocol 2)
Nuclease-free PCR water (Applied Biosystems)
Low mass DNA ladder (Invitrogen)
100% isopropanol
3 M sodium acetate pH 5.2 (APPENDIX 2A)
70% ethanol
TE buffer (APPENDIX 2A)
0.2-ml PCR tubes, sterile
Thermocycler with block for 0.2 ml tubes
80 C freezer
Vacuum centrifuge (Savant; optional)
Additional reagents and equipment for performing agarose gel electrophoresis and
ethidium-bromide staining (see Voytas, 2000)
Carry out PCR
1. Prepare the mix for PCR amplification as 100-l reactions in sterile 0.2-ml PCR
tubes containing the indicated final concentration of the components:
0.5 l 5 U/ml AmpliTaq Gold polymerase (2.5 U)
10.0 l 10 PCR buffer (1)
12.0 l 25 mM MgCl2 (3.0 mM)
8.0 l 10 mM dNTP blend (200 M each)
2.5 l 20 M GC 357F primer (0.5 M; see Table 1E.1.1)
2.5 l 20 M 907R primer (0.5 M; see Table 1E.1.1)
2.5 l DNA template (2.5% v/v)
62 l nuclease-free PCR water.
Volumes given are specific for 100-l reactions. If less product is needed (e.g., in reamplification of an extracted DGGE band or preparation of a particularly concentrated
biofilm DNA extraction), the reaction volumes can be reduced to 25 or 50 l. The
primary reason to reduce the volumes of some reactions is to save reagents and avoid
wasting Taq polymerase. In addition, the authors have found that contaminants in the
biofilm matrix interfere with PCR less when the least amount of template is added to the
PCR reaction.
The performance of the AmpliTaq Gold polymerase has been consistent in the authors
experience.
Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
This recipe is optimized with low stringency for the universal primer set. With a more
specific primer set it may be desirable to increase the stringency of the reaction by
decreasing the MgCl2 concentration or altering the primer concentration (Kramer and
Coen, 2001).
1E.1.8
Supplement 4
Final step:
10 min
45 sec
45 sec
60 sec
30 min
indefinitely
96 C
96 C
46 C
72 C
72 C
4 C
(denaturation)
(denaturation)
(annealing)
(extension)
(final extension)
(hold).
Concentrate sample
5. Pool all the PCR product required from one sample into a 1.5-ml centrifuge tube.
6. Add 1 vol isopropanol and 0.1 vol 3 M sodium acetate, pH 5.2. Place in a 80 C
freezer for 10 min. Centrifuge 15 min at 16,100 g, room temperature.
The PCR product should be in a tight white pellet.
7. Remove the isopropanol by decanting or aspirating with a pipet. Wash the pellet
with 70% ethanol by adding 500 l to the tube.
8. Centrifuge 3 min at 16,100 g, room temperature. Remove the ethanol with a
pipet. Repeat wash (steps 7 and 8).
The pellet may be loose and easily lost in decanting. The pellet should be nearly colorless
now.
9. Allow the pellet to air dry or place in a vacuum centrifuge to dry any remaining
ethanol.
10. Resuspend the pellet in 10 l TE if preparing sample for one gel and in 20 l TE if
preparing sample for two gels. Store the resuspended PCR product up to 3 months
at 20 C.
It is very difficult to load more than 40 l into a DGGE well. Loading dye should be
mixed with PCR product in a 1:1 ratio (see Basic Protocol). Therefore, precipitated PCR
product should be resuspended in a very small amount of TE (10 to 15 l).
SUPPORT
PROTOCOL 2
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1E.1.9
Current Protocols in Microbiology
Supplement 4
(McLean et al., 2005) and wastewater biofilms (A.K. Welsh and R.J.C. McLean, unpub.
observ.). DNA extraction methods may need to be modified for biofilms in other environments. To this end, one strategy employed for technique development is to add known
quantities of a test microorganism to an environmental sample and then use different
methods to extract and amplify 16S rRNA from the test microorganism. Ideally, the
test organism should not occur naturally in the particular environment being tested. In
this context, the organism will not be detected in negative control samples and will be
detected in positive control samples.
Pipets, dilution tubes, and media are used in large quantities during a field experiment.
The authors recommend preparing agar plates and tubes for dilutions at least 1 to 2
days in advance, and having 5% extra materials available in the event of unforeseen
circumstances. In this manner, any plates or tubes that become contaminated can be
identified and discarded. Types of substrata used in biofilm field studies are widely
variable and can be adapted to each unique environmental biofilm studied.
Materials
Biofilm sample (from the field)
95% or 100% ethanol
Sterile deionized H2 O
R2A agar medium (Difco): prepare agar plates according to manufacturers
instructions
TE buffer (APPENDIX 2A)
DNeasy tissue kit (Qiagen)
Enzymatic lysis buffer: 20 mM TrisCl, pH 8.0 (APPENDIX 2A)/2 mM EDTA
(APPENDIX 2A)/1.2% Triton X-100
Lysozyme (Fisher Scientific)
Writing paper
Petri dishes, sterile
Scalpel, sterile
Glass scintillation vials: Fisher brand 20-ml borosilicate vials with polypropylene
caps recommended, sterile
Bath sonicator (e.g., Branson Ultrasonic Cleaner Model 1510MT): fill to a depth of
1 cm prior to use; drain and clean after use
50-ml centrifuge tubes
Vacuum filter unit with 0.2-m filter
1. Optional: To cut a sample (e.g., a leaf) into a section prior to analysis, trace a 1-cm
square (or desired area) on a piece of writing paper. Lay a sterile petri dish on top of
the paper. Place the sample to be trimmed onto the petri dish and cut with a sterile
scalpel.
In this fashion one has an accurately measured area, yet can maintain aseptic technique.
2. Sterilize a pair of forceps by dipping in 95% to 100% ethanol and passing through
a flame. Use the sterile forceps to aseptically place the sample into a sterile glass
scintillation vial with enough sterile deionized water to completely submerge the
biofilm material.
Process biofilm samples from the field for molecular analysis as soon as possible (typically within one week of collection).
Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
The recommended glass vials are particularly useful for small objects because they
are inexpensive and can be readily autoclaved. Other similar type vials are perfectly
suitable. For larger objects, glass beakers covered with aluminum foil or bottles can be
substituted.
1E.1.10
Supplement 4
The use of plastic containers is strongly discouraged because the elastic walls dampen
the shear forces generated by the sonicator, which drastically reduces biofilm cell yield.
The containers used for sonication should be partially filled with a desired volume of
dilution medium (e.g., 10 ml water or physiological buffered saline in scintillation vials)
and autoclaved prior to use.
3. Place vials with biofilm material into a bath sonicator filled with water to just above
the level of liquid in the vials. Close sonicator lid tightly and sonicate for the optimal
time.
In the authors experience 10 to 15 min is optimal for most samples; however, optimizing
sonication conditions for the colonization substrata that are to be used is recommended.
For this purpose, perform a preliminary experiment by growing biofilms on a surface,
and then subjecting that particular surface to varying periods of sonication while periodically removing aliquots of liquid for dilution plating (McLean et al., 1999). As the
plate counts are plotted against sonication time, the time giving the maximum counts
is to be selected for the samples. Shorter times will not disrupt bacterial clumps (each
of which will plate as a single colony), whereas longer times may result in cell damage
and loss of viability, both conditions resulting in lower plate counts.
Some samples, notably limestone rock or plant leaves, will become cloudy or discolored
during this process. This cloudiness or discoloration should not cause any problems in
subsequent steps.
4. Decant the liquid containing the released biofilm bacteria from the scintillation
vials into 50-ml centrifuge tubes. Use the sonicated samples to perform plate
counts (step 5) and to extract DNA (step 7).
Ideally, the researcher should immediately proceed with DNA extraction. If necessary,
samples may be stored overnight at 20 C before proceeding with extraction.
6. Incubate for the appropriate time and at the appropriate temperature for the particular environmental sample. Count colony forming units (cfus).
Time and temperature of incubation for these plates greatly depends upon the environment from which the biofilm sample is obtained. Freshwater heterotrophs typically
require incubation for 72 hr at 25 C.
The number of cfu can give an overall indication of the concentration of heterotrophic
bacteria present in the biofilm. The data can be used to compare replicate samples or
samples with different treatments, in terms of the overall numbers of bacteria present.
DGGE can then be used to describe the overall diversity of bacteria present.
Extract DNA
7. Centrifuge the sonicated samples containing the bacteria 10 min at 5900 g, room
temperature. Decant or pipet off the supernatant.
8. Suspend the sample in 1 ml TE buffer to preserve nucleic acids during storage.
9. Extract the bacterial community DNA using 250 l of this sample and the DNeasy
Tissue kit following the manufacturers instructions for extracting Gram-positive
bacterial DNA, which includes an enzymatic lysis step using enzymatic lysis buffer
with 20 mg/ml lysozyme (see Internet Resources). Store the remaining sample at
4 C.
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The Qiagen kit uses an enzymatic lysis protocol, which will target active bacterial cells
(not dormant cells or spores) in the biofilm samples. The Gram-positive protocol will
readily lyse both Gram-positive and Gram-negative bacteria.
The authors have found the DNeasy Tissue kit to be a straightforward and reliable DNA
extraction kit. Other DNA extraction methods such as a freeze-thaw protocol (Gillan
et al., 1998) can certainly be used to extract bacterial DNA for DGGE analysis.
The authors have found that substances present in biofilm samples may interfere with
PCR. A DNA extraction and purification method is recommended, e.g., a Qiagen kit,
that captures DNA on a column and removes it from the biofilm debris.
SUPPORT
PROTOCOL 3
4. Mix the appropriate concentrations of PCR product for each of the strains in the
standard for use in 20 DGGE gels. Divide into 60-l aliquots (for four gels) and
store up to 1 year at 20 C.
1E.1.12
Supplement 4
COMMENTARY
Background Information
Denaturing gradient gel electrophoresis
(DGGE), originally developed to identify
point mutations, has now been successfully
used for evaluating microbial diversity in a
number of environments (Muyzer and Smalla,
1998). The banding patterns revealed by
DGGE for each biofilm sample yields two
kinds of information about the sample. First,
the banding pattern from DGGE can be used
as a community fingerprint which can be compared to other samples with similar or different treatments to evaluate overall community structure (Emtiazi et al., 2004; Sigler
and Zeyer, 2004; Webster et al., 2004). Unlike other popular community fingerprinting
techniques, bands can be extracted directly
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Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
Figure 1E.1.1 Representative DGGE gels. (A) Perpendicular DGGE gel of seven wastewater
biolm organisms used to establish gradient range for further analysis. Here changes in band migration occurred between 45% and 60% concentration of urea/formamide. (B) A parallel DGGE gel
analysis of mixed aquatic biolm communities. PCR product from three organisms: Pseudomonas
aeruginosa (P), Escherichia coli (E), and Chromobacterium violaceum (C) was used as standards.
The number of bands approximates the number of different organisms in each environment.
1E.1.14
Supplement 4
Anticipated Results
Figure 1E.1.1 shows the two types of gels
that are commonly used with DGGE: a perpendicular gel (Fig. 1E.1.1A) in which the
urea-formamide gradient is perpendicular to
the direction of electrophoresis, and the more
commonly seen parallel gel (Fig. 1E.1.1B). Although perpendicular gels are sometimes used
for identifying suitable gradients for analyzing a particular community, the authors have
found it to be more practical to use a parallel
gel with a wide gradient range (e.g., 30% to
70%) for range-finding, and a smaller gradient
range (e.g., a 35% to 45% gradient) to enhance
resolution of DNA fragments of similar G+C
content.
Time Considerations
Certainly transportation and other issues
will affect the time needed for field work.
Table 1E.1.2 provides a general guide for time
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Table 1E.1.2 Time Considerations for Field Sampling and DGGE Analysis of
Field-Grown Biolms
Procedure
Time required
1-2 days
Sonication of sample
30 mina
1 hr
PCR
2-3 hr
1-2 hr
1-2 hr
DGGE run
4-16 hrb
1 hr
a Sample can be stored overnight under suitable conditions (e.g., in appropriate buffer at 20 C).
b Specimens can be run at lower voltage for longer times (e.g., 16 hr at 80 volts, 60 C) or higher
Acknowledgments
This research in R.J.C.M.s laboratory
was sponsored by the U.S. Environmental Protection Agency (EPA) under grant
application GR825503-01-0, NIH grant R15
AI050638, and a subcontract from the U.S.
Air Force. The authors thank M.B. Barnes and
M.K. Windham for their help in developing
DGGE protocols.
Literature Cited
Characterization
of Bacteria in
Mixed Biolm
Communities
Using DGGE
1E.1.16
Supplement 4
Internet Resources
http://www1.qiagen.com/literature/handbooks/
PDF/GenomicDNAStabilizationAndPurification/
FromAnimalAndPlantIssues/DNY BloodTissue/
1037951 HB DNY Blood Tissue 062006 Web.
pdf
The Qiagen DNeasy tissue kit manual gives directions for extraction of DNA from Gram-positive and
Gram-negative bacteria.
Emerging
Technologies
1E.1.17
Current Protocols in Microbiology
Supplement 4
UNIT 1G.1
Michael Lea1
1
ABSTRACT
Approximately 1.1 billion people in rural and peri-urban communities of developing
countries do not have access to safe drinking water. The mortality from diarrheal-related
diseases amounts to 2.2 million people each year from the consumption of unsafe water.
Most of them are children under 5 years of age250 deaths an hour from microbiologically contaminated water. There is conclusive evidence that one low-cost household
bioremediation intervention, biological sand filters, are capable of dramatically improving the microbiological quality of drinking water. This unit will describe this relatively
new and proven bioremediation technologys ability to empower at-risk populations to use
naturally occurring biology and readily available materials as a sustainable way to
achieve the health benefits of safe drinking water. Curr. Protoc. Microbiol. 9:1G.1.1C 2008 by John Wiley & Sons, Inc.
1G.1.28.
Keywords: biofiltration r bioremediation r biosand filter r water treatment r
microbiological contamination r water quality r developing countries
INTRODUCTION
The burden of microbiologically contaminated water is borne most heavily by the rural
(largest, 80%) and peri-urban (fastest-growing) populations without access to safe water
in developing countriesall need microbiologically clean water to sustain their lives and
secure their livelihoods.
There is conclusive evidence that biological sand (biosand) filters are capable of dramatically improving the microbiological quality of drinking water. Biosand filters are based
on a centuries-old bioremediation concept: water percolates slowly through a layer of
filter medium (sand), and microorganisms form a bacteriological purification zone atop
and within the sand to efficiently filter harmful pathogens from microbiologically contaminated water. Household-scaled biosand filters are a small adaptation of traditional
large, slow sand filters such that they can uniquely be operated intermittently.
To use the simple, yet effective, on-demand biofiltration intervention, a person simply
pours contaminated water into the household biosand filter and immediately collects
treated water.
The purpose of the following comprehensive protocols is to facilitate knowledge transfer
with the goal to empower vulnerable, poorest-of-poor populations in rural and peri-urban
communities of developing countries, and to also promote using naturally occurring
biology and readily available materials that they already possess as a cost-effective
practical approach to combat poverty and inequality and achieve the health benefits of
safe water by developing their own household water security solutions.
Emerging
Technologies
Current Protocols in Microbiology 1G.1.1-1G.1.28, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc01g01s9
C 2008 John Wiley & Sons, Inc.
Copyright
1G.1.1
Supplement 9
CAUTION: Under ideal circumstances, a biosand filter can produce drinking water of
high quality. However, this cannot always be assured or guaranteed due to variations in
the construction and installation of the filter. This also applies to the consumption of
water from the biofilter. It should be noted that a biosand filter cannot be relied upon to
remove certain or all forms of water contamination.
CAUTION: If it is suspected that the community water source is contaminated with organic and inorganic industrial and agricultural toxicants, or in regions where the ambient
air reaches freezing temperatures, biosand filtration should not be recommended as an
appropriate water treatment technology.
STRATEGIC PLANNING
The performance efficiency of the biosand filter is limited by excessive turbidity, especially during the monsoon or rainy season where the performance of the filter will
be compromised with excessive clogging, requiring frequent recovery of flow rate (see
Basic Protocol 8). It is essential to obtain turbidity readings (see Basic Protocol 1) on the
raw water source where biofiltration is being proposed as a potable water solution.
When working in partnership with village health workers among the rural poor with the
challenge of providing affordable safe drinking water in every household using biosand
filters, it is recommended to implement a comprehensive community-based multiple
barrier approach (Nath et al., 2006). Education and training, emphasizing environmental
awareness, hygiene, and sanitation are thus first necessities. Reduction of excessive
turbidity and particulates by settlement or prefiltration or flocculation establishes a second
barrier (Basic Protocol 2). Removal of parasites, protozoal cysts, bacterial pathogens, and
in some situations removal of chemical contaminants (e.g., arsenic) by biosand filtration
is the third barrier (Basic Protocol 3). The fourth and final barrier, safe storage in a closed,
spigotted container and providing a disinfectant residual (see Basic Protocol 9) are all key
aspects to strengthen local capacity to carry out sustainable community-based primary
health care to prevent preventable illnesses and deaths caused by microbiologically
contaminated water.
Biological Sand
Filters
Strategically, the local availability of appropriate media (sand and gravel), parts, tools,
and their respective costs will be critical predetermining factors in making decisions on
the choice of construction method and corresponding materials used; therefore, stepwise
one-fits-all construction protocols cannot be standardized. This flexibility of construction choice based on locally available resources will also provide the lowest costan
important parameter for community acceptability and to ensure sustainability.
1G.1.2
Supplement 9
Figure 1G.1.1 Schematic diagrams of three common lters. (A) CAWSTs concrete (BioSand) lterhigh rates of user
acceptance (over 300,000 lters worldwide). (B) BushProofs Concrete (BioSand) Filterround shape provides additional
strength and requires less materials. (C) Barrel Filterlocally built biosand lters made from plastic barrels or metal
drums.
Figure 1G.1.2
1G.1.3
Current Protocols in Microbiology
Supplement 9
Figure 1G.1.3
1. Biosand filters can be built wherever there is a good source of sand and gravel and
where proper media preparation is undertaken (see Basic Protocol 4).
2. The most widely used version of the biosand filter is a concrete container filled with
16 to 20 in. (40 to 50 cm) of fine sand. This is the absolute minimum fine sand
requirement to ensure the best quality of water possible.
3. A layer of static standing water (the supernatant) is automatically maintained by
placing the outlet pipe 2 in. (5 cm) above the level of the top sand layer.
4. Allow 14 to 21 days for the biological zone to mature.
5. Water must be allowed to flow freely from the filternever plug or put a hose or tap
on the outlet spout.
Further discussion of specific detailed methods of construction are not required as there
are numerous methods and associated protocols readily available from various organizations for constructing the various featured configurations of biosand filters (see Key
References).
Biological Sand
Filters
1G.1.4
Supplement 9
Emerging
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1G.1.5
Current Protocols in Microbiology
Supplement 9
1G.1.6
Supplement 9
Sand and gravel should never come from a beach area. The grains are also too smooth,
round, and uniform in size. It may also contain salts that dissolve into the filtered water.
1G.1.7
Current Protocols in Microbiology
Supplement 9
The schmutzdecke merges with the deeper distinct biological zone, a continuation of the
area of biological action where microorganisms living below the schmutzdecke also help
to consume and trap other microorganisms. The bacterial activity is most pronounced in
the upper part of the filter bed and gradually decreases with depth as oxygen and food
becomes scarcer to sustain life.
The biofilm and biological zone typically develop within two to three weeks (it may take
up to 30 days) depending on the temperature and the biological content of the raw water.
The water from the filter can be used during the first few weeks while the schmutzdecke is
being established if a safer water source is not available, but chlorination is recommended
at least during this time period. Over time the filter flow rate may decrease when the
schmutzdecke becomes too thick and dense, requiring periodical maintenance (see Basic
Protocol 8).
1G.1.8
Supplement 9
11. Having a pause period between filter usages is important. The in-between time
when the filter is not actively filtering contaminated water is the pause period. A
pause period of 6 to 12 hr is a suggested time, which allows the biological zone to
remain vibrant by consuming the pathogens that have been introduced daily. The
percentage removal of biological contaminants is inversely proportional to the flow
rate through the filter because the greater biological removal of contaminants takes
more time. However, if the pause period is extended for too long, the microorganisms
will eventually consume all of the food supply and then die off. This will reduce the
filters treatment efficiency when it is used again.
12. One 5-gallon (20 liter) bucket of water should be poured into the filter every day
to maintain the schmutzdecke and the biological zone. A biofilter is most efficient
when operated consistently. The maximum volume of daily water that can be treated
amounts to a total of 15 to 20 gallons or 60 to 80 liters (CAWST, 2008) depending
on the schedule of the household, for instance, filter feeding can occur once in the
morning, noon, evening, and later with 6 hr pause periods in-between each feeding.
BASIC
PROTOCOL 1
Materials
Turbidity gauge (see Support Protocol)
Additional reagents and equipment for turbidity treatment (Basic Protocols 2
and 10)
1. Fill the tube with water until the black and white disk on the bottom of the newly
constructed gauge is no longer visible.
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Current Protocols in Microbiology
Supplement 9
Materials
1-in (2.5 cm) diameter PVC end cap
Waterproof marking pen
Glass or clear plastic tubing, 1-in (2.5 cm) diameter, 28-in. (70 cm) length
PVC cement (glue)
Measuring tape
1. On the inside of the PVC end cap, draw a secchi disk using the waterproof marking pen by splitting the PVC circle into 4 equal quadrants using the marking pen
(Catherman, 2006) as illustrated in Figure 1G.1.4.
2. Close one end of glass or plastic tube with the PVC end cap and cement in place.
This end should be leakproof.
3. With the marking pen and measuring tape, mark the levels of turbidity, as illustrated
in Figure 1G.1.4 (Rau, 2003).
Biological Sand
Filters
1G.1.10
Supplement 9
Materials
Collected water
Filter container
Sari cloth or other fabrics
1. Let collected water settle (sedimentation) in the container so that solids sink to the
bottom
In some situations, this may be all that is necessary, especially if water is allowed to sit
overnight. If so, settled water can be poured directly in the filter. If not, proceed with
fabric straining.
Figure 1G.1.4
Emerging
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1G.1.11
Current Protocols in Microbiology
Supplement 9
3. Fold the old sari cloth or other fabric four times and tie it over the mouth of the filter
container.
Old cloth is more effective because the worn fibers make the pores smaller and better for
filtering.
4. Slowly pour the high turbidity water through the cloth and into the filter.
5. When finished pouring the untreated water through the cloth, remove by untying the
folded cloth from the filter, wash the cloth, and expose to sunlight to dry.
Alternatively, in many countries, there are also indigenous coagulation solutions using
native plants, such as moringa seeds, known as drumstick or horseradish tree in India,
or benzolive tree in Haiti and the Dominican Republic, or malunggay in the Philippines.
BASIC
PROTOCOL 3
Figure 1G.1.5 Arsenic ltera simple adaptation for removing both pathogens and arsenic
based on biosand remediation and iron hydroxide adsorption principles.
Biological Sand
Filters
1G.1.12
Supplement 9
of Nepal, and Rural Water Supply and Sanitation Support Programme (RWSSSP) of
Nepal, based on slow sand filtration and iron hydroxide adsorption principles.
Anticipated results for the modified biosand filter are best summarized in the Centre for
Affordable Water and Sanitation Technology (CAWST) document entitled: A complete
summary of field and laboratory testing for the biosand filter is available for download
at the following link: http://www.cawst.org/assets/File/BSF Literature Brief.pdf.
Materials
Water (from the best source possible)
11 lb (5 kg) of nongalvanized iron nails, length <3/4 in. (20 mm)
Filter containing diffuser box (see Strategic Planning)
Small broken brick chips or stones, 2- to 4-in. (5 to 10 cm) diameter
1. Wash the nongalvanized iron nails.
2. Place all nongalvanized iron nails in the diffuser box that should already be in place.
Its important that the iron nails are evenly distributed and cover the diffuser.
5. The filter is now ready to remove arsenic from source water poured into the filter.
Over time the holes in the diffuser may clog due to exfoliated rust. If so, make the diffuser
holes bigger using a 1/4 -in. (0.6 mm) nail.
6. Once a year, remove the iron nails, break apart, wash thoroughly, and return the nails
back into the diffuser basin, covering again with the brick chips or stones.
The nails arsenic adsorption capacity will last years before new nails will need to be
added. The hypothesis is that as the iron nails get rusted, the rusted iron particles become
exfoliated and fall into the fine sand layer. This exfoliation exposes new iron surface,
allowing more arsenic to be adsorbed. Annual washing of the iron nails can help to
expose additional iron surface for improved adsorption capacity (Ngai et al., 2006).
BASIC
PROTOCOL 4
Next the sand and gravel will be washed to remove fine silt, clay, and other impurities
that the media may contain.
Refer to Strategic Planning for factors important in selecting a source of sand and gravel.
As a last resort, if biologically contaminated water has been used to wash the sand and
gravel, place the media in the sun to drythe solar radiation will inactivate any possible
attached pathogens. If this is not possible, remember that pathogens within the water or
attached to sand grains will be consumed by the filters normal biological processes or,
when exposed to an anaerobic environment within the filter, will not survive.
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1G.1.13
Current Protocols in Microbiology
Supplement 9
Materials
Mixed sand and gravel
Water (from the best source possible)
Materials for building three wooden sieves including:
1-in. 4-in. (2.5 cm 10.00 cm) lumber to construct three sieves, three 8 foot
(2.4 m) lengths
1-in. 1-in. (2.5 cm 2.5 cm) wood strapping, three 8 foot (2.4 m) lengths
1
/2 -in. (12 mm) screen, 1/4 -in. (6 mm) screen, and 1/16 -in. (1.5 mm) mosquito
screen, screens must be metal, not nylon or fiberglass
Tape measure
Hammer
Nails
Saw
Shovels
Tarp
5-gallon (20 liter) buckets
Sieve the mixed sand and gravel media
1. If sand and gravel is wet, dry in sun.
The solar radiation will inactivate many possible attached pathogens. Also, sieving is a
lot easier if the media is dry.
2. Construct three sieves, using the lumber and the 1/2 -in. (12 mm), 1/4 -in. (6 mm)
screen, and the 1/16 -in. (1.5 mm) mosquito screen (see Fig. 1G.1.6).
The suggested size is 16-in. (40 cm) 22-in. (56 cm) for the three sieves. For the
mosquito screen it is necessary to add a piece of 1/2 -in. (12 mm) screen under the finer
screen for additional support. The 1-in. 1-in. (2.5 cm 2.5 cm) strapping is measured
and cut to the same lengths as sieve frames. The strapping will be used to cover the screen
edges where the screens were nailed to the frames.
3. Using a shovel, pass the mixed sand and gravel through the constructed 1/2 -in.
(12 mm) sieve. Discard the media that doesnt pass through.
For a simplified understanding of size grading that is going to take place using the screens,
please reference Figure 1G.1.7.
4. Using a shovel, pass the mixed sand and gravel that passed through the 1/2 -in. (12 mm)
sieve through the 1/4 -in. (6 mm) sieve. Keep the media that doesnt pass through the
1/ -in. (6 mm) sieve.
4
Biological Sand
Filters
Figure 1G.1.6
1G.1.14
Supplement 9
Figure 1G.1.7
The underdrain (gravel) media layer is composed of gravel that passed through the 1/2 -in.
(12 mm) sieve but was held back by the 1/4 -in. (6 mm) sieve. The purpose of this media
layer is to allow the treated (effluent) water unrestricted flow out of the filter container
and into the standpipe.
5. Using a shovel, pass the mixed sand and gravel that passed through the 1/4 -in. (6 mm)
sieve through the mosquito sieve. Keep the media that doesnt pass through the
mosquito sieve (fine gravel), as well as the sand that passed through the mosquito
sieve.
The support (fine gravel) media layer is composed of sand particles that passed through
the 1/4 -in. (6mm) sieve but were held back by the mosquito screen sieve. The purpose of
this media layer is to prevent the fine filtering sand from mixing with the underdrain layer.
The filtering media layer is composed of fine sand that has been sieved through the fly
or mosquito mesh. It is this layer that is responsible for removal of pathogens and the
establishment of the biological zone, including the schmutzdecke. Please note that it takes
substantial sieving to reach the required volume of fine sand as this is the deepest layer
of the filter.
6. Store the sand and gravel in a protected dry area away from possible human or animal
contamination.
7. Cover the sieved sand and gravel with the tarp until needed.
Emerging
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Current Protocols in Microbiology
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12. Repeat steps 8 to 11 until the water in the bucket remains clear and contains no fine
particles.
13. Clean the remaining underdrain gravel using steps 8 to 12.
23. Quickly decant the mildly dirty water out of the bucket.
As you wash, count the number of times that you decant the bucket. It takes time and
practice with the following flow rate test (see Basic Protocol 5) to determine how much
the fine sand has to be washed.
24. Repeat steps 20 to 23 until all the remaining fine sand has been washed.
Once you have initially determined how much the fine sand has to be washed, this will
be used as a guideline for future media washes. The filtering sand is washed to ensure
an effective size (ES) of 0.10 to 0.25 mm and uniformity coefficient (UC) of 1.5 to 2.5
(CAWST, 2008).
26. Once dried, store the sand and gravel in a protected dry area away from possible
human or animal contamination. Cover the sieved sand and gravel with the tarp until
needed.
BASIC
PROTOCOL 5
Biological Sand
Filters
1G.1.16
Supplement 9
Materials
Water
Household chlorine bleach solution (see Basic Protocol 9)
Filter (see Strategic Planning)
Materials for correct media installation including:
2 in. (5 cm), 3 quarts (3 liters) of washed 1/2 -in. (12 mm) gravel
2 in. (5 cm), 3 quarts (3 liters) of washed 1/4 -in. (6 mm) gravel
20 in. (50 cm), 25 quarts (25 liters) of washed fine sand
Stick, 40 in. (100 cm) long
Measuring tape
Black magic marker pen
Diffuser plate or box (see Strategic Planning)
Materials for testing filter flow rate including:
Measured container (1 liter pop bottle is adequate)
Stop watch
Materials to disinfect the standpipe including:
3-foot (1 meter) garden hose
Hose clamps
Funnel
Bleach-soaked cloth
Place the media in the filter container
1. First, place the empty filter inside the home in an appropriate place (i.e., its final
location).
2. Make sure the drain hole, standpipe, or outlet pipe is clear and unobstructed.
3. Place a stick inside the filter.
4. Using a black magic marker, draw a mark across the stick where it meets the top
edge of the filter.
5. Measure and mark a second line 2 in. (5 cm) down from the first. Remove the stick.
6. Fill the filter container half full of water.
The water doesnt need to be uncontaminated; it can be from a raw water source that
eventually will be treated. Water is required to be inside the filter before adding sand
media to prevent pockets of air from being trapped within the media. If air pockets do
develop while placing media, this has the potential to slow the flow rate results.
7. Pour or place the lower underdrain gravel into the filter, level out the surface.
8. Place the stick back into the filter, placing the bottom of the stick on the newly placed
lower underdrain gravel.
When the second line matches up with the top edge of the filter container, enough gravel
has been added.
9. Measure and mark a third line 2 in. (5 cm) down from the second line.
10. Pour or place the middle support gravel into the filter, level out the surface.
11. Place the stick back into the filter, placing the bottom of the stick on the newly placed
middle support gravel.
When the third line matches up with the top edge of the filter container, enough middle
support gravel has been added.
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13. Ensuring that there is always water above the sand surface, quickly pour the majority
of washed upper filtering sand into the filter. Continue adding sand until water starts
pouring out of the standpipe.
A random distribution of different sand grain sizes is critical to the proper operation
of the filter. Adding sand quickly maintains the random distribution by not allowing the
different sizes of grains to settle into layers.
14. Wait until the water stops pouring out of the standpipe.
When the water stops pouring out of the spout, the water level is equalized.
The water level in the filter is determined by the spout. Due to a siphoning effect, the
water will stop coming out of the filter when the water is at the same level as the bottom
of the spout.
21. Place a waste water container (bucket) under the outlet pipe or standpipe.
The waste water captured can be reused.
22. Start pouring the cleanest water available into the filter (<50 NTU), continue pouring
water until the water starts running clear out of the outlet or standpipe.
This may take 10 to 20 gallons (40 to 80 liters).
If the water doesnt run clear after 25 gallons (100 liters), the gravel or sand was too
dirty to start with. It is probably easiest to take the media out, wash in pails, and then
place back in.
Biological Sand
Filters
1G.1.18
Supplement 9
a biofilter are found from measuring the time it takes to fill up a container of a known
volume with water.
23. Place the measured 1-liter container under the outlet or drainpipe spout.
24. Fill the filter container completely to the top with water.
The flow rate through the filter decreases as the height of the water added into the influent
reservoir drops.
25. With the stopwatch, time how long it takes the 1 liter container to be filled completely
with filtered waterideally, it should be 0.6 liter per min (or 100 sec per liter).
Experimentation may be necessary to achieve the desired flow rate.
If the flow takes a longer time (>100 sec) to fill the 1 liter container, the flow rate is
too slow. The filter is still functional, but it will require more maintenance than normally
required due to frequent clogging. Since flow rates are controlled by the screening and
washing of sand, the sand should be washed more to achieve the desired flow rate. The
slow flow rate can sometimes be improved by disturbing the upper sand layer with your
fingers and scooping out the dirty water. If this doesnt work, scoop out a few inches of
upper filtering sand which contains too many fine particles and rewash, and replace back
into the filter.
If the flow takes a shorter time (<100 sec), the flow rate is too fast, and the efficiency
of treatment will be compromised. Since flow rates are controlled by the screening and
washing of sand, the sand should be washed less to achieve a slower flow rate. In this
instance, the fine media should be taken out and replaced with finer media (less washed).
It can take up to 45 min for the 5 gallons (20 liters) of poured water to completely pass
through the filter. The flow rate of 0.6 liters per min is based on the top reservoir being
full of water. The actual flow of water will drop off as the water level (hydraulic head)
drops.
29. Hold the funnel higher than the spout for 2 min.
30. Remove the garden hose from the biofilter and drain the bleach solution from the
hose.
31. Wipe the spout with a bleach-soaked cloth.
32. Add 5 gallons (20 liters) of water to the top of the filter.
Never pour chlorine bleach solution into the top of the filter as chlorine will kill important,
purifying organisms.
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BASIC
PROTOCOL 6
Materials
Raw (untreated) water
Biosand filter including a diffuser plate (see Strategic Planning)
Buckets
1. Use a dedicated dirty bucket for fetching raw source water.
2. Place a dedicated safe storage container under the spout.
Place the container as close to the spout as possible to reduce dripping noise and prevent
recontamination.
5. Slowly pour raw (untreated) water into the filter daily, at least 5 gallons (20 liters),
twice per day to assist in the establishment of the biolayer [first 14 to 21 days (may
take up to 30 days)].
Based on the recommended flow rate of 0.6 liters per min and the time required for pause
periods, the biosand filter can effectively treat 15 to 20 gallons (60 to 80 liters) per day
(CAWST, 2008).
Thereafter, the family can establish a rate of usage that fulfills the familys daily water
needs. For example, the filter can be used in the morning, noon, and evening with 6 hr
pause periods in between each 5 gallon (20 liter) raw source water input.
The pause periods are very important because they allow time for the microorganisms in
the biological layer to consume the pathogens contained in the water, thereby increasing
the hydraulic conductivity of the filter. Consequently, the biosand filter is most effective
and efficient when operated intermittently.
Use the best source of water (least contaminated) availablethe better the raw water, the
better the treated water will be. Using the same source of water every day will establish
a constant biolayer and improve the filters effectiveness.
1G.1.20
Supplement 9
BASIC
PROTOCOL 7
Clean the entire outside surface of the filter regularly (only when dirty) with soap and
water or a chlorine cleaning solution.
IMPORTANT NOTE: Never pour chlorine cleaning solution into the top of the filter.
BASIC
PROTOCOL 8
Better water quality is actually produced at a reduced flow rate due to increased contact
time with the ripened biological zone. Thus, this cleaning procedure should only be
undertaken when the flow rate has become inconvenient to the familys daily needs.
Materials
Soap
Water
BioSand filter with a diffuser plate (see Strategic Planning)
1. Remove the lid, making sure that the diffuser plate is in place.
2. Pour enough water into the influent reservoir to the half-full mark, then remove the
diffuser plate.
3. Gently disturb the surface of the upper filtering sand layer with fingers, ever mindful
not to disturb the surface any deeper than 1 cm.
4. Remove suspended turbid water.
The water in the influent reservoir will now be holding inorganic and organic materials
in suspension that can be easily removed with a small container or cup.
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Current Protocols in Microbiology
Supplement 9
such as the zoonotic (referring to pathogens of animal origin that also infect humans)
protozoan Cryptosporidium. Furthermore, both SODIS and chlorination disinfection are
ineffective with higher turbidity in the water. A benefit of the SODIS method is that
its relatively inexpensive. Alternatively, chlorination requires the household to incur
additional costs. The following techniques can be used for effluent disinfection. If the
disinfection protocols are done properly, the filtered water will be of the highest quality.
Investigators will have to determine the most appropriate method consistent with their
purpose and needs and with the availability of resources.
BASIC
PROTOCOL 9
Materials
Household bleach
Clean water
Clean empty container
1. Add 1 cup (240 ml) of household bleach to a clean empty container.
Household bleach can contain different amounts of chlorine, 3.5% and 5% being the most
common. To get the correct measure of chlorine needed to disinfect water, a 1% (final)
stock solution is prepared (Conant, 2005).
Biological Sand
Filters
1G.1.22
Supplement 9
BASIC
PROTOCOL 10
Recent laboratory and field experiments indicate that a number of low-cost additives are
capable of accelerating the SODIS process in both sunny and cloudy weather. These
additives included 100 to 1000 mM hydrogen peroxide (both at room temperature and
at elevated temperatures), 0.5% to 1% lemon or lime juice, and copper metal or aqueous
copper plus ascorbate (with or without hydrogen peroxide; Fisher et al., 2008).
Materials
Biosand filtered water
1- to 2-liter clear bottles (e.g., soda bottles)
1. Obtain 1- to 2-liter clear bottle(s).
1- to 2-liter soda bottles have a better surface/volume ratio.
2. Wash the bottles well if its the first time using the bottles.
3. Fill one or many 1- to 2-liter clear plastic bottles 3/4 full with biosand filtered water.
Over time replace old or scratched bottles. Do not use green- or brown-colored plastic
bottlesthey do not transmit UV-A radiation. Use transparent bottles only.
6. Leave the bottle(s) in the sun for at least 6 hr; 2 days if the sky is cloudy. If the water
temperatures rises >50 C, the disinfection process is 3 faster.
7. Consume the water.
The UV radiation intensity is reduced by turbidity and water depth in the containers.
Water should have a low turbidity (<30 NTU) and the water depth in containers should
not exceed 10 cm (EAWAG, undated).
Solar disinfection is most effective in countries close to the equator.
Alternatively, in many countries, using UV lamp disinfection may be another option where
a source of electricity is available (see Internet Resources).
COMMENTARY
Background Information
Household-sized biological filters are simple in design; what is surprising are the complex purification processes that combine and
take place inside the filter to provide water
treatment. The principle processes are sedimentation, mechanical straining (filtering), adsorption (electrostatic), and most interestingly,
a biological process (NSFC, 1997).
Biological filtration
The bioremediation process starts when
raw turbid water is poured onto the filters
Emerging
Technologies
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Current Protocols in Microbiology
Supplement 9
Biological Sand
Filters
Critical Parameters
Multiple indicator tools required
Between 1972 and 1999, 35 new agents
of disease were discovered (WHO, 2003).
Waterborne pathogens of importance, such
as E. coli O157:H7 and Cryptosporidium are
amongst this group. A single traditional microbial indicator (i.e., Escherichia coli) primarily used for water quality monitoring is inadequate to determine Cryptosporidium cysts,
Giardia, and additional emerging water-related
diseases of unknown etiology. There is a need
for accurate, low-cost multiple indicator tools
that can be applied in remote areas of developing countries to extend the multiple barrier
approach to the potential risks of emerging
water-related microbial pathogens.
Further research
Two groups amongst the major causes of
diarrhea worldwide and a significant cause of
mortality amongst children are small roundstructured waterborne viruses (caliciviruses)
and rotaviruses. Presently, there is concern
that biosand filters may have a low rate of
virus inactivation, therefore further research is
required.
Affordability
Variations in regional conditions and availability of local materials contribute to the variability of trying to provide construction cost
estimates which are beyond the scope of the
provided protocols. As a general guideline,
concrete biosand water filters range from $12
1G.1.24
Supplement 9
Figure 1G.1.8 International Aids plastic HydraAid (BioSand) lterlighter weight, stores and
lters up to 47 liters (15 gallons) per hour.
Figure 1G.1.9 Schematic diagram of the JAL lter. No standpipe and only one layer of sand
required; lightweight and inexpensive to build.
to $30 US. A multibarrier approach as described increases costs and may limit use by
some of the worlds poorest people.
Drawbacks
There is no one-size-fits-all biosand filter.
Each has its own drawbacks, for example,
Emerging
Technologies
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Current Protocols in Microbiology
Supplement 9
Solutions
Uncertain whether the sand has been washed Place a small amount of sand, and stir in a clear glass of
clean
water, allow to settleif you cant see the sand surface
within 3-5 sec, the sand needs to be washed further
Suspicion that dissolved salts exist in filtered Ensure that media is not beach sand
water
Uncertain of gauge size for sieving screens.
Chlorine bleach was accidentally poured into Re-installation of sand and gravel will be necessary after
the top of the filter
being thoroughly washed and dried in the sun to ensure
sand is clear of chlorine. The schmutzdecke and biological
zone will need to be re-established with a ripening time of
14 to 21 days.
The standing water depth is no longer 2 in.
(5 cm)
Removal of arsenic
The holes in the arsenic diffuser box are
plugged with iron rust
Purchased nails for arsenic treatment seem to Ensure that nails are uncontaminated and nongalvanized
be oily
Biological Sand
Filters
1G.1.26
Supplement 9
Greatest obstacle
In most countries the greatest obstacle is
procuring sand and gravel to meet the media
specifications for the filter, and to a lesser degree the availability of components from local
suppliers (see Strategic Planning).
Troubleshooting
Table 1G.1.1 outlines some of the more
common problems that may be experienced
in performing the basic and supportive protocols from this unit. This is not an exhaustive
list; others may be encountered.
Anticipated Results
A fully established (ripe) schmutzdecke
will perform at 90% to 99% efficiency
(CAWST, 2008), removing biological
pathogens, but, it will take 14 to 21 days
(ripening period) of daily use to establish the
bioremediation zone and the schmutzdecke
(may take up to 30 days), depending on the
temperature and turbidity (biological content)
of the source water. Until the development
of the schmutzdecke, the filter will only be
performing between 30% to 70% efficiency
(CAWST, 2008). The water from the filter
can be used during the 14 to 21 day start-up
period, but, effluent disinfection (Basic
Protocols 9 and 10) is highly recommended
during this time period to ensure pathogen
free water quality.
Anticipated results under field conditions
with fully established schmutzdecke should
be: (1) E. coli bacteria removal of >97%;
(2) protozoa and helminths removal of >99%;
(3) removal of 50% to 90% of organic and inorganic toxicants; (4) removal of 90% to 95% of
iron; and (5) removal of 85% to 90% arsenic
with design modification known as Kanchan
Arsenic Filter (see Basic Protocol 3).
Under field conditions, the biosand filter
limitations are: (1) cannot remove some dissolved contaminants (e.g., hardness, salt, calcium, and magnesium); (2) cannot remove
some organic chemicals (e.g., pesticides and
fertilizers or color); and (3) cannot guarantee 100% pathogen-free waterit is recommended to disinfect the water after it has
passed through the biosand filter (see Basic
Protocols 9 and 10).
Additional laboratory and field testing results are best summarized in the Centre for
Affordable Water and Sanitation Technology (CAWST) document entitled: A complete
summary of field and laboratory testing for the
biosand filter (see Key References).
Time Considerations
The simple design facilitates procedures in
the unit to be completed in a timely manner.
In a developing country context, the longest
period required will be invested in procuring
uncontaminated sand and gravel and in finding
local suppliers for specific filter components to
ensure sustainability.
Literature Cited
Bruzunis, B.J. 1995. Intermittently Operated Sand
Filtration: A New Water Treatment Process.
Master of Engineering Thesis, Department
of Civil Engineering, University of Calgary,
Calgary, Alberta, Canada.
Catherman, R. 2006. Using Ultraviolet to Disinfect Household Drinking Water. MEDRIX,
Woodinvale, Wash.
Centre for Affordable Water and Sanitation Technology. 2008. Biosand Filter Manual: Design,
Construction, and Installation. CAWST.
Calgary, Ontario, Canada.
Conant, J. 2005. Water for Life: Community Water Security. Hesperian Foundation,
Berkeley, Calif. Available from: http://www.
energyandenvironment.undp.org/undp/index.cfm?
module=Library&page=Document&DocumentID
=5637.
EAWAG and SANDEC, undated. SODIS Technical
Note No. 7, Duebendorf, Switzerland.
Ellms, J.W. 1928. Water Purification, 2nd ed.,
McGraw-Hill, New York.
Fisher, M.B., Keenan, C.R., Nelson, K.L., and
Voelker, B.M. 2008. Speeding up solar
disinfection (SODIS): Effects of hydrogen
peroxide, temperature, pH, and copper plus
ascorbate on the photoinactivation of E. coli.
J. Water Health 6:35-51 IWA Publishing 2008
doi:10.2166/wh.2007.005. Available from:
http://www.iwaponline.com/jwh/006/jwh0060035.
htm.
Muhammad, N., Ellis, K., Parr, J., and Smith,
M.D. 1996. Optimization of slow sand filtration.
Reaching the unreached: Challenges for the 21st
century. 22nd WEDC Conference, pp. 283-285.
National Small Flows Clearinghouse. 1997.
Pipeline: Sand Filters Provide Quality, LowMaintenance Treatment. 8:1-7.
Ngai, T., Dangol, B., Murcott, S., and Shrestha,
R.R. 2006. Kanchan Arsenic Filter.
Massachusetts Institute of Technology
(MIT) and Environment and Public Health
Organization (ENPHO). Kathmandu, Nepal.
Nath, K.J., Bloomfield, S.F., Jones, M. 2006.
Household water storage, handling and point-ofuse treatment. A review commissioned by IFH;
published on http://www.ifh-homehygiene.org.
Rau, R. 2003. Complex simplicity: A slow sand
filter for drinking water. Home Power 93:52-56.
Smethurst, G. 1992. Basic Water Treatment:
For Application World-Wide. Thomas Telford,
London.
Emerging
Technologies
1G.1.27
Current Protocols in Microbiology
Supplement 9
Key References
Centre for Affordable Water and Sanitation Technology. 2008a. Source of Square Concrete
Biosand Filter Construction Manuals. CAWST.
Calgary, Ontario, Canada.
Provides the most comprehensive concrete filter
construction materials.
Centre for Affordable Water and Sanitation Technology. 2008b. Source of Summary of Field
and Laboratory Testing for the Biosand Filter.
CAWST. Calgary, Ontario, Canada.
Provides the most comprehensive summary of research results. Available from http://www.cawst.
org/assets/File/BSF Literature Brief.pdf
Internet Resources
http://www.who.int/household water/network/en/
The International Network to Promote Household
Water Treatment and Safe Storage.
http://manzwaterinfo.ca/
Dr. David Manzs internet-based cooperative discourse for the Biosand Water Filter.
http://www.who.int/water sanitation health/
monitoring/jmp2005/en/index.html
Link to the report Water for life: Making it happen.
This report, prepared by the WHO/UNICEF Joint
Monitoring Program, makes it clear that achieving
the International Decade for Action Water for Life
20052015 target of access to safe drinking water
and basic sanitation will bring health and dignity
to millions of the worlds poorest people.
http://www.medrix.org/water.html#UVWater
Treatment
Medical Education and Development of Resources
through International Exchange (MEDRIX)
provides a UV lamp water treatment (with optional
sand filter) system handbook available upon
request.
http://www.biosandfilter.org
A very useful Website, which contains detailed technical information (guidelines) on how to build the
metal mould, how to produce the round concrete,
and provides description and drawings for plastic
or metal drum biosand filter construction.
http://web.mit.edu/watsan/worldbank summary.htm
Massachusetts Institute of Technology-Kanchan
Arsenic Filter (KAF). Project promotes KAF technology.
http://www.hydraid.org/
International Aids new plastic HydrAid BioSand
Water Filter initiative.
http://www.jalmandir.com/filtration/biosand/biosandfilters.html
Clearinghouse for Low-cost Household Water
Treatment Technologiesprovides overview of
biosand filtration technology.
http://www.jalfilter.org
Provides description, photographs, and schematics
drawings for JAL filter construction.
http://www.safewaterintl.org/
Safe Water Internationals development initiative
toward a lightweight collapsible filter container and
safe storage pouch combination.
http://www.cawst.org
The Centre for Affordable Water and Sanitation
Technology (CAWST) is a Canadian humanitarian
organization that provides training, education, and
technical consulting in water and sanitation to organizations working with the poor in developing
countries.
http://www.biosandfilter.org/biosandfilter/index.php/
item/229
BushProof, Biosand Filter home page. (n.d.).
Source of Round Concrete and Metal Drum Biosand
Filter Construction Manuals. Retrieved March 15,
2007. Presents the most comprehensive online reference material.
Biological Sand
Filters
1G.1.28
Supplement 9
UNIT 2A.1
2A.1.1
Figure 2A.1.1
Diagram of the major component parts and centering screws for a research upright light microscope.
Protocol 2 deals with calibrating image magnification. Several procedures (see Support
Protocols 3, 4, and 5) are given for testing the optical performance of the microscope;
these also describe test specimens for microscope performance and their sources. Finally,
Support Protocol 6 deals with the care and cleaning of microscope optics. The Commentary refers the reader to references that provide more comprehensive treatments of the
basic modes and methods of light microscopy as well as advanced microscope imaging
methods like multiwavelength, confocal, or multiphoton imaging, and electronic imaging
techniques, including video and digital microscopy.
Familiarizing oneself with the components of the light microscope is best done using the
manual for the microscope as reference. Figure 2A.1.1 provides a diagram of an upright
research compound light microscope equipped for both transmitted light and epifluorescence microscopy. Locate the following components on the microscope, identify their
2A.1.2
Current Protocols in Microbiology
adjustment screws (if applicable) and movements (e.g., condenser focus), and ascertain
that they appear correctly mounted. In this and the following sections, z is a direction
along the microscope axis; x and y are perpendicular directions.
Image-Forming Components
Specimen stage and focus
A mechanical carrier on the stage holds the specimen slide. Knobs control movement in
the xy direction. Look for the vernier scales that mark the x and y positions. Rotatable
stages are typically used for DIC microscopy. The objective is usually fixed and the
specimen is focused by moving the stage along the z axis using coarse- and fine-focus
knobs on the microscope body. Check the scale on the fine focus; on research microscopes,
this is usually is 1 m/unit.
Body tube
In the modern research light microscope, the imaging light leaving the objective and
nosepiece is unfocused and the image is projected to infinity (Fig. 2A.1.2, left). This
infinity space above the objective allows insertion of filters without changing the focal
position of the image at the intermediate image plane. A positive focusing lens, the
tube lens, above the infinity space, is used to bring the imaging light into focus for the
eyepieces or camera.
Microscopy
2A.1.3
Current Protocols in Microbiology
Check for the following possible inserts in the infinity space of the microscope.
1. DIC prisms for the DIC objectives. These are usually located right above the objective.
2. Analyzer. This is used for DIC microscopy and should be removed from the light path
for bright field, phase-contrast, and fluorescence microscopy.
3. Epi-illuminator filter-cube changer. These devices usually hold two to four filter
cubes for fluorescence microscopy, each of which contains an excitation filter, an
emission filter, and a dichroic mirror (Fig. 2A.1.5) designed specifically for different
fluorophores. Check the numbers on the filter cubes and mark the corresponding
positions on the outside of the filter changer to identify the proper cube position for a
given fluorophore.
4. Magnification changer and Bertrand lens. Body tube magnifications are selected from
a turret, and possible values are 1.0, 1.25, 1.5, and 2.0. Often one position of
the turret contains a Bertrand lens. This lens is used in combination with the eyepieces
to produce a telescope view of the objective back focal plane (Fig. 2A.1.2, far right).
This is an important device for checking that the lamp image is centered and in
focus during alignment for Kohler illumination, and for adjustment of the condenser
diaphragm (see Transillumination Components, below).
Beam switch
This device switches the light between the binocular and the camera port. Check for the
percentage of light in each direction. For fluorescence microscopy, it is important to be
able to send 100% of the imaging light either to the eye or to the camera.
Eyepieces
Eyepiece magnification to the eye is marked on the barrel (e.g., 10). Note that there
are two and maybe three adjustments. The interpupillary distance for the eyes is adjusted
by grasping the bases of the eyepiece tubes and moving them closer or further apart. At
least one of the eyepieces is adjustable so that the eyes are parfocal.
Camera adapters
Check the type of camera adapter and whether it matches the detector. There are several
different types of camera adapters. One type uses an eyepiece tube (not the binocular
tube) and a projection eyepiece combined with a camera lens to project an image onto
the camera detector. This method is common in photographic film cameras (which use
35-mm film) and for some video cameras with large-size detectors (e.g., 1 in., equivalent
to 2.54 cm). The recent video and cooled slow and progressive scan charge-coupled device
(CCD) cameras have small detectors ( 23 in., equivalent to 1.7 cm). These cameras
require smaller projection magnifications or nonein the latter case the detector is
mounted at the intermediate image plane, the focal plane of the objective (Fig. 2A.1.2),
and no eyepiece or projection lens is used.
Transillumination Components
Lamp and housing
Proper
Alignment and
Adjustment of
the Light
Microscope
The lamp is typically a low-voltage 100-W quartz halogen bulb with a tungsten-filament
light source and variable control. Some lamp housings have no adjustments for centering
the bulb; the socket is prefixed. Most lamp housings have xy adjustments for the bulb
2A.1.4
Current Protocols in Microbiology
socket, while an advanced lamp housing also has a mirror in the back for reflecting an
image of the lamp back along the microscope axis. This mirror usually has adjustments
for x, y, and z positions of the mirror image of the lamp. There is usually a knob on the
side of the lamp housing for moving the collector lens back and forth along the z axis to
focus the tungsten-filament image onto the condenser diaphragm plane.
Field diaphragm
This is usually located just above or beneath the mirror that deflects the light up to the
condenser lens. It controls the specimen area illuminated by the condenser (Fig. 2A.1.2,
left).
Polarizer
This is inserted below the condenser diaphragm for polarization and DIC microscopy.
Otherwise, it is removed from the light path.
Condenser diaphragm
The condenser diaphragm is located on the bottom of the condenser. It controls the angle
(NA) of the condenser cone of illumination of the specimen (Fig. 2A.1.3).
Condenser turret
Condensers have turrets with inserts for special image-contrast techniques such as phasecontrast, DIC, and dark-field microscopy. Each insert matches certain objectives. In
phase-contrast microscopy, the inserts are annuli of different diameters designed to match
the phase ring in phase-contrast objectives (Fig. 2A.1.6). For DIC, the inserts are typically
DIC prisms designed for certain objectives (Fig. 2A.1.8). For dark-field microscopy, the
NA of the condenser must exceed that of the objective. To see these inserts, as well as
the opening and closing of the condenser diaphragm, remove the condenser from the
condenser carrier by loosening the locking screen and turning it upside down.
Microscopy
2A.1.5
Current Protocols in Microbiology
Condenser lens
The condensers main function is to provide bright, uniform illumination of both the
specimen field (Fig. 2A.1.2, right) and objective aperture (Fig. 2A.1.3) for objectives
with different NAs. Check the markings on the condenser to see if it is designed for air
(dry) or oil immersion with the glass slide. Dry (no oil-immersion) condensers have NA
values of 0.9 and should not be used with immersion oil. Oil-immersion condensers
usually have NA values of 1.0 to 1.4 when using immersion oil.
Epi-illumination Components
Lamp and housing
The light source for the epifluorescence illumination is typically an HBO 50-W or HBO
100-W mercury arc lamp, or a xenon lamp of similar wattage. These lamps need to be
handled with care because they can blow up if mistreated. Their glass envelopes should
be carefully cleaned with 70% ethanol before installation to prevent fingerprints or other
materials from inducing hot points that may result in fracturing of the bulbs. Look for
the adjustment screws to adjust the xy position of the lamp. Look also for the rear
mirror and its x, y, and z adjustment screws. Identify the focusing knob for the lamp
collector lens. Do not turn the lamp on until the bulb has been installed according to the
manufacturers instructions, the collector lens has been inserted properly, and the lamp
housing has been installed on the back of the microscope. These lamps produce intense
light, and the mercury bulbs have large peaks in the UV range. Therefore, UV-protective
glasses should always be worn when handling and installing these lamps. The light
intensity decreases and the probability of explosion increases with the number of hours
of operation and lamp starts. Check the timer on the lamp power supply to see that the
recommended limits are not exceeded.
Shutter
Photobleaching is a major problem in fluorescence microscopy. A shutter is used to block
the light from the specimen when not taking exposures on a camera or viewing by eye.
Filters
A heat-reflecting filter is used to prevent infrared illumination of the specimen and to
keep infrared scattered light from reaching the camera. Neutral-density (ND) filters are
used to reduce the light intensity of fluorescence illumination by the amount indicated
on each filter.
Filter cubes
See Transillumination Components, above.
2A.1.6
Current Protocols in Microbiology
Imaging-Ray Paths
The upper left section of Figure 2A.1.2 shows ray paths for the image-forming light
from the objective. The objective, in combination with the tube lens, produces a real,
magnified image of the specimen at the intermediate image plane. The eyepiece provides
a second stage of magnification to the eye. The magnification to the eye is the product
of objective magnification and eyepiece magnification multiplied by any magnification
provided by additional lenses in the body tube of the microscope between the objective
and the eyepieces.
When a camera detectorfilm, video, or charge coupled device (CCD)is mounted
at the intermediate image plane, the magnification to the camera is the magnification
produced by the objective and body tube. When a camera is mounted above an eyepiece,
the unfocused light leaving the eyepiece must be focused onto the camera detector by
a camera lens, which modifies the total magnification to the detector. Special adapters
(see Image-Forming Components, Camera Adapters) are also available for projecting the
objective image onto a camera detector with magnification, but without the need for an
eyepiece.
Microscopy
2A.1.7
Current Protocols in Microbiology
Figure 2A.1.2 The imaging and illuminating light paths for a bright-eld microscope aligned for
transmitted light Kohler
illumination. Modied from Keller (1998).
Proper
Alignment and
Adjustment of
the Light
Microscope
2A.1.8
Current Protocols in Microbiology
Figure 2A.1.3 Numerical aperture (NA) of objective light collection and condenser illumination.
The objective numerical aperture (NAobj ) depends on the angle of the cone of light from the
specimen, which is accepted by the objective aperture while the numerical aperture of condenser
illumination (NAcond ) is controlled by the condenser diaphragm and limited by the maximum NA of
the condenser when the condenser diaphragm is wide open.
microscopy (see Support Protocol 3). Note on the right side of Figure 2A.1.2 that the
image of the condenser diaphragm is in focus where the lamp image is in focus along
the microscope axis, at the objective back focal plane, and the exit pupil of the eyepiece.
Trace the imaging light rays from the field diaphragm through the microscope
(Fig. 2A.1.2, left). Note that the field diaphragm controls the specimen area illuminated by the condenser. Note also that the field diaphragm is placed between the lamp
collector lens and the condenser lens in a plane where the lamp image is out of focus
(compare Fig. 2A.1.2, left and right). When the condenser lens has focused the image of
the field diaphragm onto the specimen, then the field diaphragm will be in focus with the
specimen at the intermediate image plane and at the eye or detector.
Microscopy
2A.1.9
Current Protocols in Microbiology
Figure 2A.1.4 The illuminating light paths for dark-eld microscopy using (A) a multifunctional condenser
tted with a dark-eld stop or (B) a cardioid condenser.
stop must be of a specific diameter to block the direct light, but it is designed to be open
around the outer perimeter, letting light pass around the dark-field stops circumference.
The aperture diaphragm of the condenser must be fully opened so that some light can
pass through the dark-field stop. These rays are refracted in the condenser at an oblique
angle such that they form a hollow cone of light that passes just outside the objective lens.
Compare this to the illuminating light path for a bright-field microscope in Fig. 2A.1.2,
in which all of the direct light passes through the specimen and up into the objective lens.
When a mounted specimen is placed on the stage, it is located at the apex of the hollow,
inverted cone of light. As the light passes through the specimen it is scattered in all
directions. Some of the light is reflected or refracted up into the objective lens, so the
specimen appears as a luminous object against a dark background. Only the rays of light
reflected, refracted or diffracted by the specimen will enter the objective. The remaining
light forms a hollow cone of light that passes outside the objective (Fig. 2A.1.4).
BASIC
PROTOCOL 1
Proper
Alignment and
Adjustment of
the Light
Microscope
2A.1.10
Current Protocols in Microbiology
2. Center and focus the lamp filament near the condenser diaphragm plane. Remove
the condenser lens. Place a sheet of lens paper on the microscope stage. Close down
the field diaphragm and adjust the intensity of the lamp so that the lens paper is
moderately illuminated. Use the lamp-focusing knob to move the condenser lens (or
lamp) along the z axis until an image of the filament is in focus on the lens paper.
Roughly center the lamp on the microscope axis with the adjustment screws on the
lamp housing, then adjust the mirror image (if there is a mirror) using its adjustment
screws on the lamp housing.
3. Place the lens paper on the condenser carrier and raise the condenser carrier until it
is 20 mm from the top of the stage.
This is the approximate position of the condenser diaphragm when a condenser is installed
and in focus.
4. Refocus the lamp and mirror images at this position of the lens paper.
If there is no mirror image, center the lamp image. If there is a mirror image, then position
the images so that they sit side by side to fill the condenser aperture. Remove the lens
paper and replace the condenser.
6. Move the condenser lens up close to the lower surface of the slide and open the field
and condenser diaphragms all the way. Look down the eyepieces, and use the coarseand fine-focus knobs to bring the specimen into sharp focus.
7. Focus the image of the field diaphragm centered on the specimen by adjusting
the condenser xy screws and the condenser focus. Initially, close down the field
diaphragm until an edge of the image can be focused by the condenser, then close the
field diaphragm further as the image is centered, using the xy translation screws.
Usually, the field diaphragm is opened just enough to match the field of view in the eyepiece
or the camera. However, the best image contrast is obtained if the field diaphragm is
opened just enough to illuminate the region of interest. This eliminates the presence of
scattered light from outside the region of interest.
8. Center an in-focus image of the lamp and mirror images at the objective back focal
plane using the focus and adjustment screws on the lamp housing. View the objective
back aperture (the position of the back focal plane; Fig. 2A.1.3) by using the Bertrand
lens in the magnification changer in combination with the eyepieces, by replacing
one eyepiece with a telescope, or by simply removing one eyepiece and peering down
the body tube. Open the condenser diaphragm all the way. Readjust the collector
lens and the mirror so that the lamp images are in focus and sit side by side to fill the
objective aperture.
This is a rough adjustment with the low-power objective and will need to be fine-tuned
using the objective with the highest NA.
Microscopy
2A.1.11
Current Protocols in Microbiology
9. Adjust the opening of the condenser diaphragm so that the diameter of its image at
the objective back focal plane (see far right of Fig. 2A.1.2; also see Fig. 2A.1.3) is
slightly less than the diameter of the objective back aperture.
Some research condensers also have centering adjustments for the condenser diaphragms.
The image of the condenser diaphragm should be centered in the objective back aperture.
10. Adjust the light intensity for comfortable viewing of the specimen by adjustment
of the light-source intensity, either using the power-supply rheostat or by inserting
neutral-density filters in the illumination light path.
The microscope is now adjusted properly for the low-power objective.
12. Focus and center the field diaphragm with the condenser focus and the condensercarrier xy adjustment screws.
13. View the objective back aperture (Fig. 2A.1.3).
Notice that the image of the condenser diaphragm adjusted for the low-power objective
is only about half the diameter of the 40 back aperture. The ratio of the diameter of
this image to the diameter of the objective back aperture is equal to the ratio of the NA of
the condenser illumination to the NA of the objective (Fig. 2A.1.3). When the condenser
illumination NA equals the objective NA, the aperture of the objective is filled with light
and maximum resolution will be achieved for the objective NA (see Commentary).
14. Adjust the condenser diaphragm so that the condenser illumination NA just about
matches the objective NA.
Each time objectives are changed, these procedures should be repeated.
16. Complete the rotation of the nosepiece so that the oil-immersion objective clicks
into place. Lower the objective nosepiece if appropriate.
IMPORTANT NOTE: Use only the fine adjustment knob when working with the oilimmersion lenses.
The space between its front lens and the coverslip is now filled with immersion oil.
17. Remove the eyepiece (or use the telescope or Bertrand lens) and inspect the back
aperture of the objective. Open the condenser diaphragm as much as possible to try
and match its image with the objective aperture (Fig. 2A.1.3).
Proper
Alignment and
Adjustment of
the Light
Microscope
This will be impossible if the condenser is dry and not designed for oil immersion, since
dry condensers have NA 0.9 and the relative size of the image of the condenser aperture
or condenser diaphragm seen in the objective back focal plane is given by the following
ratio (see Fig. 2A.1.3).
2A.1.12
Current Protocols in Microbiology
18. View the objective back aperture, and tune up the focus and position of the lamp
images so that they fill the objective aperture with light as uniformly as possible.
19. Replace the eyepiece (or remove the Bertrand lens) and examine the specimen.
Adjust the field diaphragm until its margins just match the field of view.
With inexpensive condensers, the image of the edge of the field diaphragm will not be
in good focus, even at the optimum position. If a condenser designed for oil immersion
is being used, enormous improvement in the field iris image can be achieved by oil
immersion.
20. Oil the condenser (if possible) to achieve the highest resolution and image quality
using the following procedure:
a. Rotate the objective out of the way and remove the slide.
b. Oil the condenser.
c. Replace the slide and refocus the objective.
Before oiling the condenser, make sure it is not designed to be a dry condenser by
checking that it is marked with an NA > 0.9. It takes much more air-bubble-free oil than
the objective.
The image of the field diaphragm should now be much sharper when in focus and centered.
Before oiling the condenser, make sure it is not designed to be a dry condenser by
checking that it is marked with an NA >0.9.
The image of the field diaphragm should now be much sharper when in focus and centered.
21. Look at the objective back aperture. Again, tune up the focus and position of the lamp
images so that they fill the objective aperture with light as uniformly as possible.
After oiling, the aperture should be filled by the condenser illumination when the condenser diaphragm is wide open.
Notice also that viewing the objective back aperture is the best way to see if air bubbles
have become trapped in the oil. If so, carefully wipe off the oil with lens paper and repeat
steps 15 through 21.
23. When finished for the day, use lens paper to remove excess oil from the objective
and condenser lens surfaces to prevent dripping on specimens.
It is not necessary, however, to completely clean oil from the optics after every use (see
Support Protocol 6).
BASIC
PROTOCOL 2
Microscopy
2A.1.13
Current Protocols in Microbiology
3a. If the microscope has a target reticle: Rotate the reticle into the field of view. Close
the left eye and use the diopter-adjustment ring on the right eyepiece until the target
is in sharp focus for the right eye. Then close the right eye and use the diopteradjustment ring on the left eyepiece until the target is in sharp focus for the left eye.
Remove the target to view the specimen.
3b. If the microscope has no target reticle: Set the right eyepiece tube to the inter-eyepiece
setting (if possible). Close the left eye and bring into sharp focus a structural detail
near the center of the field of view with the right eye. Close the right eye and use
the diopter-adjustment ring on the left eyepiece tube to bring the specimen detail
into sharp focus for the left eye. With both eyes open, fine tune the focus of the left
eyepiece tube.
BASIC
PROTOCOL 3
Proper
Alignment and
Adjustment of
the Light
Microscope
Figure 2A.1.5
2A.1.14
Current Protocols in Microbiology
Some microscopes have a special device for lamp alignment. It screws into the objective
nosepiece and projects an image of the epi-illumination arc and electrodes onto a small
diffusion screen within the barrel of the device.
2. Install the mercury bulb in the lamp housing and attach the lamp housing on the back
of the microscope according to the manufacturers instructions.
3. Turn on the power source and ignite the lamp.
CAUTION: It is usually a good idea to make sure computers in the vicinity are turned
off before starting the lamp because the high voltage pulse used to ignite the arc plasma
may damage the electronics. Laboratory personnel should protect their eyes against UV
light from the lamp.
It takes 10 min for the arc to brighten.
4. Close down the field diaphragm and open the condenser diaphragm (if there is one)
all the way.
5. On the diffusion glass screen or on the white card on the stage, the image of the
arc between the tips of the two electrodes should be visible. Use the lamp focusing
knob to obtain an in-focus image of the lamp arc and electrodes. Use the lamp xy
adjustment screws to approximately center the image of the arc and electrode tips
(see Figure 2A.1.2).
6. If there is a lamp mirror, use its adjustment screws to focus the mirror image of the
arc and electrode tips, centered on the target.
Often the primary image and the mirror image of the arc are adjusted side-by-side and
slightly overlapping, but centered on the microscope axis.
10. Switch to a high-power objective. For an oil-immersion objective, place a small drop
(2 mm diameter) of immersion oil directly onto the coverslip above the region of
Table 2A.1.1 Suppliers for Items Used in Light Microscopy
Test specimen
Supplier
Stage micrometer
Fisher
Microscopy
2A.1.15
Current Protocols in Microbiology
interest. Carefully swing in the objective. Open the shutter and focus on the specimen.
Readjust the size and centration of the field diaphragm.
This oil drop must be free of air bubbles and dirt.
11. To prevent photobleaching, be sure to close the epi-illumination shutter when not
viewing or taking camera exposures.
BASIC
PROTOCOL 4
Proper
Alignment and
Adjustment of
the Light
Microscope
Figure 2A.1.6 Illumination light path through the condenser annulus and objective phase ring in
a microscope aligned for phase-contrast microscopy.
2A.1.16
Current Protocols in Microbiology
passes through the phase ring in the objective when the annulus is selected and aligned
properly. Light scattered by the specimen mainly passes through the objective aperture
outside the phase ring. Light scattered from a thin transparent specimen is 14 wavelength
retarded from the unscattered illumination light. The additional 14 wavelength retardation
between the scattered and illumination light produced by the objective phase ring makes
the scattered and illumination light 180 out of phase. They destructively interfere with
each other at the image plane to produce the dark contrast of structural detail typical
of phase contrast images of biological specimens.
Because the phase annulus and the phase ring reduce the intensity of the background
light, a bright illuminatore.g., a 100-W quartz-halogen illuminatoris necessary at
high magnifications (because image intensity decreases as 1/magnification2 ). For living
cells, heat reflection and green illumination filters should be used.
To examine the objective phase ring and matching condenser phase annulus, remove the
low- and medium-power phase objectives and the phase condenser from the microscope.
View the phase ring (phase plate) within the objective by looking in the back end. The
phase ring is located at the position of the objective back focal plane; its diameter is
usually 23 that of the objective aperture. Note that it is situated within the objective
body and is visible because it absorbs light. Locate the annulus for each objective in
the condenser turret. The annulus is located at the condenser diaphragm plane, which
is situated at the condenser front focal plane. Notice that as the objective NA increases,
the diameter of the corresponding annulus in the condenser turret increases. The NA of
condenser illumination from an annulus is designed to match that of the phase ring in the
corresponding objective.
Replace the condenser on the microscope and illuminate with white light. Hold a piece
of tissue paper near the top of the condenser and examine the illumination cone. Notice
that it is an annular cone of illumination. Change the annulus from the setting for the lowpower objective to the 40 setting. Note that the NA or angle of illumination increases.
Points of illumination further from the central axis in the condenser diaphragm plane
produce higher-aperture rays passing through the specimen.
Perform alignment using the following steps.
1. Align the microscope for bright-field Kohler illumination using the low-power phase
objective (10 or 16) and a cheek cell preparation for the specimen (see Support
Protocol 4).
The cheek cell specimen is an excellent transparent test specimen for alignment of the
microscope for phase contrast, dark field, and DIC. The cheek cells are transparent and
only barely visible by bright-field illumination. To find the plane of focus in bright field,
initially close down the condenser diaphragm and look for the edges of air bubbles in the
preparation. These edges scatter much light and appear dark in the image. When using
dark-field configuration, the cheek cells will appear white against a dark background.
2. Align the condenser annulus with the phase ring by rotating the condenser turret
to the position where an annulus matches the phase ring in the objective; use the
telescope, your eye, or the Bertrand lens to view the objective back aperture.
As the condenser turret is rotated, notice in the objective back focal plane the images
of the different-diameter annuli designed for the different numerical aperture objectives.
The correct annulus may not be properly aligned with the objective phase ring.
3. Use the adjustment screws in the condenser (and the special tools if necessary) to
move the annulus in the xy plane to achieve alignment with the phase ring in the
objective.
Microscopy
2A.1.17
Current Protocols in Microbiology
Figure 2A.1.7 DIC images of a human cheek cell test specimen. (A) Low magnication of cheek cell preparation
with a 20 objective. Bar = 20 m. (B) High-resolution image of the surface of the cell at the top of (A) using a
60/(NA = 1.4) Plan Apochromat objective and matching condenser ilumination. The ridges on the cell surface
are often diffraction limited in width. Bar = 5 m. From Salmon and Tran (1998), reprinted with permission from
Academic Press.
Note that the phase ring is slightly wider than the image of the correct annulus. It is
critical that the image of the annulus be within the phase ring, but it is not critical if it is
very slightly off center.
4. When the objective phase ring and condenser annulus are aligned, view the specimen
and properly adjust the focus and centration of the field diaphragm.
Notice that if the phase ring and annulus are slightly misaligned (rotate the turret slightly),
the background light intensity goes up. This is because the phase ring is designed to absorb
much of the illumination light. The unscattered illumination light becomes closer to the
intensity of the light scattered by the specimen, which passes through the objective aperture
outside of the phase ring. Minimizing the background light intensity while viewing the
specimen image can also be used to align the annulus with its phase ring or to touch up
the alignment done by viewing down the body tube.
5. Switch to the 40 phase objective and then the high-power 60 or 100 phase
objective (NA 1.25 to 1.4) and repeat steps 1 to 4.
It is necessary to increase light intensity at the higher magnifications. It should be possible
to see the ridges on the cell surface (see Fig. 2A.1.7 for a DIC image of the ridges). Notice
the phase halos at discrete edges, a problem that limits conventional phase contrast in
high-resolution imaging.
BASIC
PROTOCOL 5
Proper
Alignment and
Adjustment of
the Light
Microscope
2A.1.18
Current Protocols in Microbiology
Figure 2A.1.8 The optical system for DIC microscopy. From Salmon and Tran (1998), reprinted with permission from Academic Press.
Microscopy
2A.1.19
Current Protocols in Microbiology
Figure 2A.1.8 outlines the principles of DIC image formation and contrast. The DIC
beam-splitting prisms are located at the condenser diaphragm plane, just above the
polarizer in the condenser turret. Objectives of different magnification and/or NA usually
require condenser prisms of different optical characteristics.
The condenser prism splits the light coming from the polarizer into divergent polarized
light wavefronts whose planes of polarization are orthogonal to each other and at 45
with respect to the initial plane of polarization (Fig. 2A.1.8, left). The divergent beams
are converted by the condenser into two wavefronts, which pass through the specimen
separated laterally from each other in the direction of the prism wedge (the shear direction,
Fig. 2A.1.8, middle) by a tiny amount that is usually less than the resolution limit of
the condenser-objective lens combination (Fig. 2A.1.8, right). These two wavefronts are
recombined just above the objective by a beam-combining prism. Often, each objective
has its own prism so that it accurately matches the action of the condenser prism. In
some microscopes, there is one beam-combining prism for all objectives and a different
condenser prism for each objective. Check which is the case for your microscope.
DIC image contrast depends on the compensation or bias retardation () between
the two wavefronts along the microscope axis (Fig. 2A.1.8, right). When the objective
beam-combining prism is perfectly aligned with the condenser beam-splitting prism and
there is no compensation ( = 0), the background light is extinguished and the edges
of objects are bright against a black background (Fig. 2A.1.8, upper right). When one
wavefront is retarded relative to the other by , this increases the optical path (OP)
between the wavefronts (Fig. 2A.1.8, middle right) and brightens the background light.
One edge of an object becomes brighter than the background while the opposite edge
becomes darker (Fig. 2A.1.8, upper right). This produces the shadow cast appearance
of DIC images. Reversing the sign of retardation reverses the contrast of the specimen
edges.
In some microscopes, the objective beam-combining prism (Fig. 2A.1.8, middle) is used
as a compensator by translating the prism in the direction of prism wedge away from the
position of background light extinction. One direction produces positive while the other
produces negative retardation (). In other microscopes, both the objective and condenser
prisms are fixed at positions that produce background light extinction, and typically a
deSenarmont compensator (a birefringent quarter-wave retarder in combination with a
rotatable polarizer or analyzer; Inoue and Spring, 1997) is inserted just above the polarizer
or beneath the analyzer.
Bright illumination sources are required for high magnification because of the crossed
polarizer and analyzer; at least the 100-W quartz-halogen illuminator is usually needed.
To examine the DIC prisms, remove the objective prisms and the DIC condenser from the
microscope. Examine the objective prism. Notice that it is very thin and wide enough to
cover the objective aperture. Also notice if there is a screw that can be used to translate the
prism back and forth in one direction across the objective aperture. Examine the prisms
in the condenser turret. Notice that there are different prisms for low- and high-NA
objectives. The highest-resolution (NA) objectives often have special prisms designed
only for the optical properties of that objective. In aligning the microscope for DIC, be
careful to use the condenser Wollaston prism that matches the objective in use. Reinstall
the condenser.
Proper
Alignment and
Adjustment of
the Light
Microscope
Steps 1 to 6 in the following procedure are for the initial alignment of the microscope.
Once this is done, begin at step 7 for routine use.
2A.1.20
Current Protocols in Microbiology
4. Insert the objective DIC prism (observe caution as the image will be very bright).
Observe the extinction fringe in the middle of the prism (you must use the telescope,
remove an eyepiece, or insert the Bertrand lens to focus on the objective focal plane).
Make a drawing of your view of the fringe in the back aperture.
The fringe should be at 45 with respect to the analyzer-polarizer transmission azimuths.
5. Remove the objective DIC prism and rotate into place the condenser prism that
matches the objective. Observe the objective back focal plane.
The orientation of the fringe should match the orientation of the extinction fringe for the
objective prism in your drawing.
6. Insert the objective prism and observe the objective back focal plane. Rotate in the
other condenser prisms and notice that the extinction fringe is no longer spread across
the objective aperture. Rotate in the correct condenser prism.
If the condenser and objective prisms are properly matched and oriented in the same
direction, the fringe should become spread across the objective aperture (a dark cross
will still occur in high-power, unrectified objectives; Inoue and Spring, 1997).
Microscopy
2A.1.21
Current Protocols in Microbiology
contrast, then change illumination intensity to provide the camera with enough light for
good image quality.
Due to the blockage of the majority of the light passing through the condenser of a
microscope configured for dark-field microscopy, a 100-W tungsten-halogen bulb is
required as a minimum for both low- and high-power magnification. Microscope slides
must be 1 0.1 mm thick, and slides used for preparation of specimens for dark-field
illumination should not be reused. All slides and optical surfaces in the microscope
2A.1.22
Current Protocols in Microbiology
Figure 2A.1.9 Dark-eld image of the Lyme disease spirochete, Borrelia burgdorferi with a 20
objective and a turret condenser with dark-eld stop included.
must be spotlessly clean since dark-field microscopy will detect every speck of dust and
particulate matter. Dilute particulate preparations if necessary.
A bright-field microscope can easily be converted for use in dark-field microscopy with
the addition a turret condenser with a dark-field stop included. If a turret is not available,
any multifunction condenser will work. Dark-field stops can be purchased or handmade.
Hayden (2002) provides an excellent resource for instructions on how to devise dark-field
patch stops.
BASIC
PROTOCOL 6
1. Align the microscope as for bright-field Kohler illumination using the lower-power
phase objective, but ignore the step used to optimize the opening of the condenser
aperture.
Dark-field conditions require that the condenser aperture be fully opened to its maximum
setting to let in as much light as possible. Kohler illumination provides a default position
for the user to reference and return to while changing the optics for optimal dark-field
settings.
2. Align the condenser turret for dark-field settings using the default dark-field stop. If
the turret does not include a dark-field stop, insert the appropriate diameter patch stop
(16 to 18 mm stop for use with the 10 objective) by either positioning it into the
substage condenser filter holder or by taping it onto the underside of the condenser.
The turrets usually possess five or six openings, one opening for bright-field, one opening
has a dark-field stop (labeled DF), and the remaining openings house several phase stops.
3. Open both the field and the substage condenser aperture iris diaphragms fully to their
maximal setting.
This allows the condenser to operate at its highest NA.
Microscopy
2A.1.23
Current Protocols in Microbiology
4. Check the dark-field stop diameter by removing the eyepiece and looking down into
the focal plane of the objective.
The dark-field stop diameter should eclipse the bright disk of light that can be normally
viewed at the objective rear focal plane under bright-field conditions. If the dark-field stop
does not completely mask the bright disk, use the substage screws to center the condenser
until the bright disk is fully blocked by the stop. Replace the eyepiece.
5. Place a low-contrast specimen that is suitable for dark-field illumination on the stage.
The specimen image should be bright against a black background view. Dust and small
contaminants on the slide may create artifacts that could obscure the object. Be sure
to use premium quality microscope slides and clean them thoroughly with ethanol and
Kimwipes Delicate Task Wipers before observing specimens under dark-field illumination.
If the specimen image is too dark, increase the light source to its maximum voltage.
6. Focus the condenser by adjusting up or down until a single bright ring of light is
central in the field of view.
When using the condenser dry, the top of the lens should almost come into contact with
the bottom of the microscope slide. If a small dark region lies within the bright ring of
light, then the condenser is too low. The bright ring of light will appear off-center in the
field of view if the microscope is not properly aligned for Kohler illumination.
7. When the condenser is properly adjusted, view the specimen and properly adjust the
focus.
If the field of view has a dark spot in the center obscuring the image, but objects in the
periphery appear normal, then the substage condenser may need to be re-positioned or
the inappropriate diameter stop is being used or the NA of the objective. Re-establish
alignment for Kohler illumination (see Basic Protocol 1) and repeat the steps for dark-field
illumination.
BASIC
PROTOCOL 7
3. Adjust the condenser until it is <2 mm from the bottom of the specimen slide.
4. Place a low-contrast specimen that is suitable for dark-field illumination on the stage.
5. Use the 10 objective to center the high NA dark-field condensers.
While viewing the specimen with the 10 objective, focus the condenser as described in
step 6 for low magnification dark-field illumination.
Proper
Alignment and
Adjustment of
the Light
Microscope
6. If using a high NA immersion condenser, apply oil to the lens of the condenser by
first lowering the condenser below the mechanical stage and placing a drop of oil on
the lens. Next, slowly raise the condenser until it makes contact with the underside
of the specimen slide. Make sure to eliminate any air bubbles trapped between the
slide and the condenser top lens.
Note that when an oil immersion condenser is used without oil, regardless of the magnification of the objective used, light will not emerge from the condenser.
2A.1.24
Current Protocols in Microbiology
7. Switch to the 40 DF objective and then the higher power 100 objective. Position
the objective to be used for viewing the specimen.
Some objectives can be used dry or with oil immersion. For oil immersiononly objectives,
place a drop of oil on the specimen slides cover slip before positioning the objective.
Observe the back focal plane of the objective to check for air bubbles. If bubbles are
detected, move the objective to the next detent stop on the microscope and remove the oil
with lens paper. Then re-apply oil again and re-position the objective.
SUPPORT
PROTOCOL 1
This procedure uses the diatom test slide (see Table 2A.1.1 for supplier information). For
more details on matching a camera to the microscope see Hinsch (1998) or Inoue and
Spring (1997).
1. Look down the eyepieces and obtain an in-focus image of the frustrule pores of the
diatom Pleurosigma angulatum (Fig. 2A.1.10, number 6 in panel A) using 40/(NA
0.7) phase-contrast or DIC and proper Kohler illumination.
The pores should be clearly visible by eye.
2. Project the diatom image onto the faceplate of a video or CCD camera.
3. Adjust the gain and contrast camera controls for optimum image brightness and
contrast.
Figure 2A.1.10 (A) The diatom test plate. The rows of pores are spaced in the silica shell by
0.25 m in Amphipleura pellucida (panel B; also number 8 on panel A), 0.41 m in Surrella
gemma (panel C; also number 5 on panel A), and 0.62 m in Pleurasigma angulatum (panel D;
also number 6 on panel A). Bar: panel A, 10 m; panels B, C, and D, 2.5 m. From Salmon and
Tran (1998), reprinted with permission from Academic Press.
Microscopy
2A.1.25
Current Protocols in Microbiology
As you increase contrast, you will need either more camera gain or brighter illumination.
Usually, a better signal-to-noise ratio is achieved by leaving the gain control in the middle
of its adjustment and increasing illumination intensity.
If the projection magnification to the detector is too small, the frustrule pore lattice will
be invisible in the video image, although it will be clearly visible when viewing the image
by eye.
3. Use the 100- or 10-m scales to calibrate distance in images taken with your objectives.
The magnifications on the objective barrel and projection lenses are only approximations;
accurate measurements of distances in images require a calibration scale.
4. Acquire images of the scale in both the horizontal and vertical directions to check if
the camera has square pixels.
Proper
Alignment and
Adjustment of
the Light
Microscope
where is the wavelength of light, NAobj is the objective NA, and NAcond is the condenser
NA (see Fig. 2A.1.3). The lateral resolution, r, is equal to 0.195 m for the highest
objective NA, which is equal to 1.4, with NAcond = NAobj and 546 nm green light. As
2A.1.26
Current Protocols in Microbiology
seen in Figure 2A.1.10B, this objective is capable of resolving the rows of pores in the
shell of Amphipleura, but not the individual pores, which are slightly <0.19 m apart.
Use the above equation to calculate the diffraction limit of resolution for your other
objectives and use the diatoms to test whether they achieve their theoretical limits.
SUPPORT
PROTOCOL 4
Cheek cells are a convenient specimen for testing the performance of phase-contrast,
DIC, and dark-field microscopes. As seen in the low-magnification view (Fig. 2A.1.7A)
they are large and flat, 3 m thick except near the cell center which contains the nucleus.
The upper and lower surfaces have fine ridges which swirl around much like fingerprints.
Many of the ridges are <0.2 m in width and separated by <0.5 to 1.0 m.
To prepare the cheek cells, gently scrape the inside of your mouth with the tip of a plastic
pipet or similar soft tool and spread the cells and saliva on the center of a clean no. 1.5
coverslip. Invert the coverslip quickly onto a clean slide and press down to spread the
cell preparation into a thin layer. Seal the edges with a thin layer of nail polish.
Testing Fluorescence Using a Red, Green, and Blue Fluorescent Tissue Culture
Cell Test Slide
SUPPORT
PROTOCOL 5
The cells in Figure 2A.1.11 are triple labeled: DAPI stained nuclei and chromosomes
(blue fluorescence; Fig. 2A.1.11A); Alexa 488-phalloidin labeled actin filaments (green
fluorescence; Fig. 2A.1.11B); and X-Rhodamine immunofluorescently labeled microtubules (red fluorescence; Fig. 2A.1.11C). The microtubules and fine actin filamentous
arrays are <100 nm in width, but they should appear sharp and in high contrast in the microscope. There should be no bleed-through from one fluorescence channel to another
if the filters are chosen properly (Taylor and Salmon, 1989).
In fluorescence microscopy, the diffraction-limited lateral resolution is given by (Inoue
1989; Inoue and Spring, 1997):
where Iex is the excitation light intensity entering the objective and Mp is the projection
magnification from the objective to the eye or camera. The rate of photobleaching, Rp ,
of a specimen depends on:
Microscopy
2A.1.27
Current Protocols in Microbiology
Figure 2A.1.11 Epiuorescent images of xed tissue culture cells stained with (A) DAPI, making DNA uorescent blue;
(B) Alexa 488 bound to phalloidin to label actin laments uorescent green; and (C) X-rhodamine labeled antibodies
against tubulin to label microtubules uorescent red. Bar = 20 m. Images recorded with a 40/(NA = 1.4) Plan Fluor
objective, 1.5 magnication, to a cooled CCD camera and the multi-modem multiwavelength microscope described by
Salmon et al. (1998).
square of objective NA (a measure of the excitation light collected by the objective) and
magnification (which concentrates the excitation light on the specimen).
Verify these equations by imaging the fluorescent specimen (the microtubules in the
spread cells are a good choice) for objectives with different magnifications and numerical
apertures. It will quickly be seen why in fluorescence one wants to use the minimum
total magnification necessary to resolve the structures of interest in the detector, as well
as use the maximum NAobj in order to maximize light intensity for the least amount of
excitation light (and the least amount of photobleaching).
SUPPORT
PROTOCOL 6
5. When cleaning lens surfaces, avoid touching the lens surface with anything (even
lens paper if possible).
IMPORTANT NOTE: Never use Kimwipes or commercial facial tissue, because they may
contain a filler that is part diatomaceous material (glass). One pass of a Kleenex could
ruin an objective.
2A.1.28
Current Protocols in Microbiology
6. Remove dust by gently brushing with an oil-free (ether-washed) camels hair brush
or by using a low-velocity stream of purified air.
7. Remove water-soluble contamination using distilled water with a small amount of
detergent, such as Kodak Photoflow solution.
Much (modern) immersion oil can also be removed by washing with detergent.
8. Remove most immersion oil by passing a high-quality lens tissue over the objective
or condenser front element.
IMPORTANT NOTE: Do not rub. No area on the tissue should come in contact with
the lens twice. This prevents dust and dirt removed from the lens from coming back and
possibly scratching it. This is easily accomplished by passing the tissue over the lens in
a Z pattern or by making parallel passes.
9. Clean objective lenses by holding a piece of doubled lens paper over the objective
and placing a few drops of solvent on the paper. Draw the paper across the lens
surface so that the solvent flows rapidly in a circular pattern over the recessed lens
surface (see Inoue and Spring, 1997). Finish the stroke with a dry portion of the
paper. Repeat as necessary.
In this way, the solvent contacts the lens but the paper does not, because the lens is
recessed. For solvent, first use a 1% solution of Kodak Photoflow in distilled water to
remove much of the oil and water-soluble material. Then use a small amount of oil solvent
like ethyl ether or xylene to clean all the oil from the surface. Avoid soaking a lens with
solvent, to prevent damage to lens cements.
10. To clean recessed front elements of dry objective lenses or to remove stubborn dirt,
use a cotton-tipped applicator that has been soaked in cleaning solution and then
shaken to remove excess fluid. Rotate the cotton tip over the lens surface to clean.
Again, first use a 1% solution of Kodak Photoflow in distilled water to remove much
of the oil and water-soluble material. Then use a small amount of oil solvent like
ethyl ether or xylene to clean all the oil from the surface.
11. Use a detergent solution or ethanol to clean the surfaces of the eyepiece lenses.
Do not use xylene as it may solubilize enamel surfaces.
COMMENTARY
The development of lasers, electronic cameras, digital image analysis, and specific fluorescent molecular probes have recently made
light microscopy an enormously powerful tool
in the biomedical sciences. There are a number
of excellent books and review articles about
these applications, as well as the optical principles of light microscope design and image
formation. Listing of all these references is beyond the scope of this unit; only a few of these
books and reviews are given here, and these
can be used to find other useful references.
Spencer (1982), Zernike (1958), and Keller
(1995, 1997, 1998) provide excellent introductions to the principles of image formation and contrast in the light microscope for
beginners, while Pluta (1988, 1989) provides
a more comprehensive treatment. Inoue and
Oldenbourg (1995) also review the basic con-
Microscopy
2A.1.29
Current Protocols in Microbiology
Proper
Alignment and
Adjustment of
the Light
Microscope
Literature Cited
Keller, H.E. 1998. Proper Alignment of the Microscope. Methods Cell Biol. 56:135-146.
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Current Protocols in Microbiology
Pawley, J. (ed). 1995. Handbook of Biological Confocal Microscopy, 2nd ed., Plenum, New York.
Pluta, M. 1988. Advanced Light Microscopy, Vol. I:
Principles and Basic Properties. Elsevier/NorthHolland, Amsterdam.
Pluta, M. 1989. Advanced Light Microscopy Vol. II:
Specialized Methods. Elsevier/North-Holland
Amsterdam.
Rizzuto, R., Carrington, W., and Tuft, R. 1998. Digital imaging microscopy of living cells. Trends
Cell Biol. 8:288-292.
Rost, F. and Oldfield, R. 2000. Photography with
a Microscope. Cambridge University Press,
Cambridge.
Salmon, E.D. and Tran, P. 1998. High resolution
video-enhance differential interference contrast
(VE-DIC) light microscopy. Methods Cell Biol.
56:153-183.
Salmon, E.D., Shaw, S.L., Waters, J., WatermanStorer, C.M., Maddox, P.S., Yeh, E., and Bloom,
K. 1998. A high-resolution multimode digital
microscope system. Methods Cell Biol. 56:185215.
Sluder, G. and Wolf, D.E. (eds). 1998. Video microscopy. Methods Cell Biol. vol. 56.
Smith, C. L. 1997. Basic confocal microscopy.
In Current Protocols in Neuroscience (J.N.
Crawley, C.R. Gerfen, R. McKay, M.A.
Rogawski, D.R. Sibley, and P. Skolnick, eds.)
pp. 2.2.1-2.2.13. John Wiley & Sons, New York.
Spencer, M. 1982. Fundamentals of Light
Microscopy. Cambridge University Press,
Cambridge.
Svoboda, K. and Block, S.M. 1994. Biological applications of optical forces. Annu. Rev. Biophys.
Biomol. Struct. 23:247-285.
Taylor, D.L. and Salmon, E.D. 1989. Basic fluorescence microscopy. Methods Cell Biol. 29:207237.
Taylor, D.L. and Wang, Y.-L. (eds). 1989. Fluorescence Microscopy of Living Cells in Culture.
Methods Cell Biol. Vols. 29 and 30.
Taylor, D.L., Nederlof, M., Lanni, F., and
Waggoner, A.S. 1992. The new vision of light
microscopy. Am. Sci. 80:322-335.
Zernike, F. 1942. Phase contrast: A new method
for the microscopic observation of transparent
objects. Physica 9:686-693.
Zernike, F. 1955. How I discovered phase contrast.
Science 121:345-349.
Zernike, F. 1958. The wave theory of microscope
image formation. In Concepts in Classical Optics (J. Strong, ed.) pp. 525-536. W.H. Freeman,
San Francisco.
Microscopy
2A.1.31
Current Protocols in Microbiology
UNIT 2A.2
ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy represents a method of exciting and
visualizing fluorophores present in the near-membrane region of live or fixed cells grown on
coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs
when light passes from a high-refractive medium (e.g., glass) into a low-refractive medium
(e.g., cell, water). The evanescent field produced by total internally reflected light excites the
fluorescent molecules at the cell-substrate interface and is accompanied by minimal exposure of
the remaining cell volume. This technique provides high-contrast fluorescence images, with very
low background and virtually no out-of-focus light, ideal for visualization and spectroscopy of
single-molecule fluorescence near a surface. This unit presents, in a concise manner, the principle
of operation, instrument diversity, and TIRF microscopy applications for the study of biological
C 2008 by John Wiley & Sons, Inc.
samples. Curr. Protoc. Microbiol. 10:2A.2.1-2A.2.22.
Keywords: total internal reflection fluorescence microscopy
INTRODUCTION
Over the past 25 years, researchers from
physics and biology have shown significant interest in developing optical techniques
for investigating nanostructures (e.g., single
molecules) beyond the diffraction limit of
light. Total internal reflection fluorescence
(TIRF) microscopy is such a technique. TIRF
was introduced in 1965 by Hirschfeld (1965)
as a technique for selective surface illumination at a solid/liquid interface. This microscopy technique was further developed and
refined by Axelrod (see Axelrod et al., 1983,
1984), with later theoretical contributions
from Reichert, Truskey (see Reichert, 1989;
Reichert and Truskey, 1990), and Burmeister
(see Burmeister et al., 1998).
TIRF microscopy represents a method of
exciting and visualizing fluorophores present
in the near-membrane region of live or
fixed cells grown on coverslips. If the microscope is thoroughly calibrated, the separation distance between cell membrane
and substrate can be measured (Reichert,
1989; Truskey et al., 1992). In addition,
any movement of fluorescent-tagged proteins
in focal adhesion contacts, at the membrane level or in its immediate proximity, can be monitored quantitatively in real
time.
PRINCIPLE OF OPERATION
TIRF microscopy is based on the total internal reflection phenomenon that occurs when
Microscopy
2A.2.1
Supplement 10
Reflection
Refraction
Refractive index
Diffraction
Diffraction limit
a
medium 2
n2
n1
n1
medium 1
c
Figure 2A.2.1 Light path at the interface between two media. (a) Refracted light at an angle
of incidence smaller than the critical angle; (b) light at an angle of incidence equal to the critical
angle of light, traveling along the interface between the two media (2 = 90 ); and (c) total internal
reflection occurring at incident angles higher than the critical angle. 1 , incident angle; 2 , refractive
angle; c , critical angle; n1 and n2 , refractive indices of the two media.
TIRF Microscopy
light passes from a medium with a high refractive index into a medium with a low refractive
index (n1 and n2 , respectively, in Fig. 2A.2.1).
At the interface, the light will bend and travel
along the interface if the incident angle is
equal to the critical angle. If the incident angle is higher than the critical angle, the light
will turn back into the high refractive medium
and only a short-range electromagnetic disturbance called the evanescent field will pass into
the low refractive medium. The evanescent
field intensity decreases exponentially with
distance from the interface (Fig. 2A.2.2), confining the eventual fluorescence excitation to
the near-field region (excitation decays over
/4 to /5; Axelrod, 2001a). This imaging
method provides high-contrast images of the
near-membrane area, with a useful maximum
depth of penetration of 100 to 200 nm. Due
TIRF Parameters
To better understand the characteristics and
the usefulness of this microscopy technique,
we will briefly discuss the theoretical TIRF
parameters. See Table 2A.2.1 for definitions
of terminology related to optics.
The relationship between the angles of incidence (1 ) and refraction (2 ) and the indices
of refraction of the two median1 and n2 are
the indices of refraction of the incident and
refractive medium, respectivelyis known as
Snells Law. This law will apply to the refraction of light in any situation, regardless of what
2A.2.2
Supplement 10
medium
z
cell
evanescent field
glass
incident beam
Figure 2A.2.2 Schematic representation of the TIRF effect. The evanescent field produces
selective excitation of the fluorophores at the cell-coverslip interface. , angle of incidence; z,
distance from the interface.
Figure 2A.2.3 A fixed vascular smooth muscle cell labeled with vinculin-Alexa 488 was imaged
by epifluorescence (A) and TIRF (B; pseudo-colored red). The TIRF image was overlapped with
the epifluorescence image (C) to emphasize the differences between the two imaging modes
(yellow represents good overlap between the two images). The discrete pixilation of vinculin-Alexa
488 in the TIRF image (B) is due to the fact that it was obtained by optically sectioning only the
very bottom of the cell at a maximum depth of 100 nm, compared to the epifluorescence image
which was obtained by collecting the fluorescence through the whole cell volume. Image size is
150 110 m. For the color version of this figure go to http://www.currentprotocols.com.
n2
n1
Equation 2A.2.2
NA
n1
Equation 2A.2.3
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z
I ( z ) = I o exp
d
Equation 2A.2.4
1
4 n12 sin 12 n22
Equation 2A.2.5
Intensity
1
c
0
40
50
60
70
80
90
Angle of incidence ( )
TIRF Microscopy
Figure 2A.2.4 Evanescent field intensities of the two planes of polarization parallel (gray line)
and perpendicular (black line) to the sample plane as a function of the incident angle.
2A.2.4
Supplement 10
Figure 2A.2.5 (A) A vascular smooth muscle cell transiently transfected with GFP-vinculin was
imaged in TIRF mode. (B) A three-dimensional representation of the fluorescence intensity and
the corresponding linear logarithmic transformation of the intensity that generates the separation
coverslip-cell distance map. In the distance map, blue indicates a position close to the coverslip
and red indicates a position distant from the coverslip. In the intensity map, blue indicates low
intensity and red indicates high intensity. The rainbow scale in the lower left shows the colors
associated with intermediate levels of distance or intensity. Image size is 70 110 m. For the
color version of this figure go to http://www.currentprotocols.com.
Table 2A.2.2 Commercially Available TIRF Systems
Manufacturer
Web site
Leica
http://www.leica-microsystems.com/products/am tirf mc
Nikon
http://www.nikon-instruments.jp/eng/page/products/tirf.aspx
Olympus
http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/
olympusaptirf.html
TILL Photonics
http://www.till-photonics.com/Applications/tirf.php
Zeiss
http://www.zeiss.de
configurations in an inverted microscope, using side or rear microscope ports for introducing the laser light or conventional arc lamp
illumination. Table 2A.2.2 lists the present
manufacturers of TIRF modules as turn-key
systems.
Introduction of the laser illumination
through the side port requires a special
dichroic mirror cube facing the side, which is
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Current Protocols in Microbiology
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condenser
prism
focusing
lens
sample
objective
mirror
condenser
microinjector
cell culture dish
coverslips
fixed to dish
fixed to prism
objective
oil layer
E
focusing lens
Figure 2A.2.6 Schematic drawings for prism-based TIRF in an inverted microscope (laser illumination). Configurations A to D use a TIR prism above the sample. In these configurations, the
buffer-filled sample chamber consists of a lower bare glass coverslip, a spacer ring (Teflon) and
the fluid cell coverslip inverted so the cells face down. The upper surface of the fluid cell coverslip
is put in optical contact with the prism by a layer of immersion oil or glycerol. The lateral position
of the prism is fixed but the sample can be translated while still maintaining optical contact. In
configuration D, two incident beams split from the same laser and intersect at the TIR surface,
thereby setting up a striped interference pattern on the sample which is useful in studying surface
diffusion. Configuration E places the prism below the sample and TIR effect depends on multiple internal reflections in the substrate. This configuration thereby allows complete access to the
sample from above for solution changing and/or electrophysiology studies. However, only air or
water immersion objectives may be used because oil at the substrate lower surface will thwart the
internal reflections. The vertical distances are exaggerated for clarity. Reproduced from Axelrod
(2001b) with permission from Blackwell Publishing.
TIRF Microscopy
2A.2.6
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SP
mirror or
prism
BFP
BFP
laser
beam expander
microscope/epi-illuminator/
filter cube/objective
laser optical fiber
D
SP
BFP
L1
Hg arc
L1
opaque
disk
image
SP
BFP
L2
laser
Figure 2A.2.7 Schematic drawings for prismless TIRF in an inverted microscope. (A) Laser
illumination through a side port. (B) Laser illumination introduced by an optical fiber through the
rear port normally used by the arc lamp. (C) Laser illumination in microscope systems containing
an equivalent back focal plane (BFP) in the rear path normally used by an arc lamp. (D) Arc lamp
TIR illumination. If (as in the Olympus IX-70) an aperture plane does not exist in the indicated
position, it can be created with a pair of lenses L1 and L2 (C and D). The illumination at the
BFP is a circular annulus; it is shown as a point on one side of the optical axis for pictorial
clarity only. In all these configurations. SP refers to sample plane and BFP refers to the objective
back focal plane or its equivalent planes (i.e., aperture planes). Components drawn with thick
lines need to be installed; components in thin lines are possibly preexisting in the standard
microscope. The BFP aperture plane (L1 and L2) in panels C and D is also provided by the
Nikon TE 2000-U microscope. Reproduced from Axelrod (2001b) with permission from Blackwell
Publishing.
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250
Depth of penetration (nm)
1.78
1.51
200
150
100
50
0
45
50
55
60
65
70
75
Angle of incidence ( )
Figure 2A.2.8 Depth of penetration of the evanescent field versus angle of incidence () for the
two different refractive indices (n) of the objective-coverslip combination used in TIRF.
Table 2A.2.3 TIRF Parameters
NA
critical a
max a
d (nm)a
1.45
61
72
<100
1.49
61
80
<100
1.65
48
67
<220
a Theoretical values calculated for = 488 nm, n = 1.516 (or 1.788 for the
1
Manufacturer
Objective
Numerical
aperture (NA)
Olympus
APO 60
1.45
1.45
APO 100
1.65a
U APO 150
1.45
Zeiss
1.45
Nikon
Plan APO 60
1.45
APO 60
1.49
1.45
APO 100
1.49
1.46
Leica
TIRF Microscopy
objective might be a better choice. This particular objective requires higher refractive
index coverslips and immersion oil (n = 1.78;
Cargille Laboratories). The high refractive
index oil is volatile and leaves a crystalline
residue in a short period of time. There
2A.2.8
Supplement 10
PRACTICAL GUIDELINES
Control of the Angle of Incidence
Figure 2A.2.9 presents, in detail, the optical
path for a through-the-lens TIRF configuration
in an inverted microscope. The laser beam used
for excitation is collimated, and then focused at
the back focal plane of the objective at a chosen angle of incidence with respect to the
optical axis of the microscope. The point of
focus in the back focal plane is off-axis (black
solid line). There is a one-to-one correspondence between the off-axis radial distance (),
which is dictated by the distance between the
optical axis of the microscope and the fiber
position at the entrance of the TIRF illuminator (d), and the angle of incidence . By
moving the micrometer that changes the fiber
position with respect to the optical axis, the
axial radial distance () will increase, and the
critical angle of incidence can be exceeded to
allow supercritical angles to be reached in a
reproducible manner. This adjustment reaches
its limit when the laser light is damped into the
edge of the objective (Axelrod, 2001c).
Microscope Stability
For obtaining reproducible TIRF images,
the microscope focus should be as stable as
possible. For quantitative or time-lapse measurements, the focus stability is of great importance. The microscope room should be kept at
a constant temperature, and the microscope
might require a warm-up time that allows
for thermal equilibrium to be achieved (see
UNIT 2C.5). The new autofocus lock-in systems
commercially available from Olympus, Applied Scientific Instrumentation, and Nikon,
may improve the focus stability performance.
Sample Preparation
Best TIRF images are obtained on cells cultured on good quality glass coverslips. Due to
the small penetration depth of the evanescent
field, the quality and thickness of the coverslip
are important. Usual TIRF images are obtained
by fluorescent labeling of the molecules at the
cell level. In this case, TIRF microscopy will
provide high-intensity fluorescence images of
the labeled molecules on a dark background
(Fig. 2A.2.3 and Fig. 2A.2.5). The contact
Figure 2A.2.9 Schematic representation of the TIRF optical path in a through-the-lens microscope configuration. The TIRF illuminator provides a micrometer that allows the controlled movement of the optical fiber off-axis to achieve the total internal reflection (TIR) condition at the sample
level. Ray a is obtained with the fiber optic centered on the optical axis. Ray b is obtained for an
incident angle at the sample plane smaller than the critical angle. Ray c is obtained when TIR
effect takes place. The fluorescence light reflected from the sample plane is collected through the
same microscope objective and is further sent to the camera where the image is recorded. ,
angle of incidence at the sample; d, distance between the optical axis and the fiber position; ,
off-axis distance of the laser beam in the back focal plane of the objective.
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Current Protocols in Microbiology
Supplement 10
Artifacts
The oil used between the objective and the
coverslip is another important aspect in obtaining good TIRF images. Air bubbles or contaminants in the oil will introduce a mismatch
into the refractive indices, producing scattered
light. If the oil does not form a film of uniform
thickness between the two glass elements, a
nonuniform illumination of the sample plane
could cause a random striped pattern of light
and dark bands (Temple, 1981), and the stripes
will move when the stage moves. Dust on
the microscope objective will produce similar
light and dark stripes in the field of view, but
the stripes will not follow the stage movement.
Dust on the optics inside the turret could produce a concentric interference fringe pattern.
The best way to clean these optical elements is
to blow dust away, avoiding wiping the optical
surfaces.
TIRF Applications
The unique features of TIRF make this microscopy technique an invaluable tool for biological studies that is finding increasing applications. TIRF can be further combined with
other techniques to cover a much larger range
TIRF Microscopy
Figure 2A.2.10 (at right) Direct imaging of DNA molecules and localization of multiple sequence
motifs of lambda phage DNA. (A) The predicted Nb.BbvC I map of lambda DNA. Positions of the
nicking sites are indicated by arrows. Nicking sites 2 to 4 and 5 to 6 are closely clustered and are
not resolvable due to the limits of optical diffraction. (B) In the intensity-scaled composite image of
linear lambda DNA, the Nb.BbvC I sites (labeled with Tamra-ddUTP) are shown as green spots,
and the DNA backbone (labeled with YOYO) is shown as blue lines. Due to the diffraction limits of
the microscope, only four labels can be fully resolved. In this field, two DNA fragments (a and b) are
fully labeled while one fragment (c) has three labels. Red arrows point to clustered sites; some of
them are brighter than others because of the presence of multiple labels. (C) The sequence motif
map in the bottom graph was obtained by analyzing 61 single molecular fluorescence images. The
solid line is the Gaussian curve fit, and the peaks correspond well to the predicted locations of
the sequence motifs. Reproduced from Xiao et al., (2007) with permission from Oxford University
Press. For the color version of this figure go to http://www.currentprotocols.com.
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Figure 2A.2.11 Direct imaging of DNA molecules and localization of multiple sequence motifs
of human adenovirus type 2. (A) Predicted Nb.BbvC I map of human adenovirus type 2. Nine sites
are found on the 35.5-kb viral DNA with two sets of clustered sites (1 to 2 and 7 to 8), leading to
seven resolvable labels. (B) In the intensity-scaled composite images of four fully labeled human
adenovirus type 2 DNA, the Nb.Bbvc I sites (labeled with Tamra-ddUTP) are shown as green spots,
and the DNA backbone (labeled with YOYO) is shown as blue lines. Due to the diffraction limits
of the microscope, only seven labels can be fully resolved. Labels 1 and 6 are generally brighter
than the other labels due to clustering. (C) The sequence motif map in the graph was obtained
by analyzing 63 single molecule fluorescence images. The solid line is the Gaussian curve fitting
and the peaks correspond well to the predicted locations of the sequence motif. Reproduced from
Xiao et al. (2007) with permission from Oxford University Press.
Current Protocols in Microbiology
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Figure 2A.2.12 Direct imaging of DNA molecules and localization of sequence motifs of human
rhinovirus 14 DNA. (A) The predicted Nb.Bsm I (GCATTC) map of human rhinovirus 14 DNA.
Only two sites are present in this small RNA virus. (B) In the large field of the intensity-scaled
composite image, numerous molecules are found. Some molecules have two green labels (red
arrows) and some have only one green label (yellow arrows). (C) The sequence motif map in
the graph was obtained by analyzing 56 single molecule fluorescence images. The solid line is the
Gaussian curve fitting and the peaks correspond well to the predicted locations of the sequence
motif. Reproduced from Xiao et al. (2007) with permission from Oxford University Press. For the
color version of this figure go to http://www.currentprotocols.com.
Current Protocols in Microbiology
Microscopy
2A.2.13
Supplement 10
Membrane-trafficking perturbations
induced by the dynamic interaction of
HIV-1 Nef with the clathrin-mediated
endocytic pathway
The nef gene product of HIV-1 associates
with cell membranes, being essential for maximal viral replication in vivo, and markedly
contributes to the pathogenesis of AIDS. The
positive influence of Nef on viral replication and infectivity is a multifactorial process
that affects trafficking of membrane proteins
within the endocytic pathway. Endocytosis internalizes plasma membraneassociated proteins through membrane-bound vesicles, supporting various cellular functions (Conner and
Schmid, 2003). One of the best characterized endocytosis pathways relies on the protein clathrin (Bonifacino and Traub, 2003).
Clathrin adaptors link clathrin to cytoplasmic
determinants of endocytic cargo during the
formation of plasma membrane invaginations
Nef-GFP
TIRF Microscopy
Ds Red-clathrin
Figure 2A.2.13 Distribution of HIV-1 Nef protein in HeLa cells observed by epifluorescence.
HeLa cells were transfected with plasmids encoding wild-type HIV-1 Nef fused to green-fluorescing
protein (Nef-GFP, A and C) and clathrin light chain fused to DsRed (B and D). Cells were then
fixed and observed by epifluorescence. The focus was made on a medial region (A and B)
and on the adherent surface (C and D) of the same cells. Insets show higher magnification
(2.5) of representative areas. Arrows point out colocalization of both markers. Scale bar, 10 m.
Reproduced from Burtey et al. (2007) with permission from Blackwell Publishing.
2A.2.14
Supplement 10
Figure 2A.2.14 Colocalization of Nef with clathrin at the plasma membrane revealed by TIRF
microscopy. Live HeLa cells expressing DsRed-clathrin (A) and Nef-GFP (B) were imaged by
TIRF microscopy. Insets show higher magnification (3) of a representative area. Arrows point
out colocalizing spots, and arrowheads show clathrin spots (CS) in which Nef is not concentrated.
Scale bar, 10 m. (C) Colocalization of Nef-GFP within CS was quantified in more than seven cells.
One thousand spots were quantified and GFP- and DsRed-associated fluorescence intensities
were measured and normalized to the local background. The presence of Nef within CS was
then quantified by selecting Nef spots with a normalized fluorescence intensity above 1.1, a value
corresponding to a detectable increased fluorescence relative to background (a spot) by direct
observation (data not shown). Conversely, the presence of clathrin with Nef spots was similarly
quantified. (D) Spots analyzed in C were plotted as function of their normalized fluorescence for
both clathrin and Nef. Spots with a normalized fluorescence below 1.1 appear in gray. Reproduced
from Burtey et al. (2007) with permission from Blackwell Publishing.
disappear from the surface, and 5% move laterally. In the analysis performed on 40 spots
from each CS category (Fig. 2A.2.15), Nef
was detected in the large majority of static
CS (91.5%), in approximately half of the
disappearing CS (50.6%), and in a minority
of laterally mobile CS (22.1%). Altogether,
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Current Protocols in Microbiology
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Ds Red-clathrin
Nef-GFP
Ds Red-clathrin
Nef-GFP
G
Cla
Ds Red-cla
Nef
4.2
3.6
5.4
18.9
19.2
19.5
19.8
21.3
Nef-GFP
D
120
0.595
0.297
DsRed-clathrin
100
80
60
40
20
0
2 3 4 5 6 7 8 9 10 11 12
1.190
70
60
50
40
30
20
10
2
1.784
2.082
Nef-GFP
00.000
00.297
00.595
00.892
01.190
01.487
01.784
02.082
H
0
2 3 4 5 6 7 8 9 10 11 12
100
90
80
70
60
50
40
30
20
10
0
91.5%
50.8%
22.1%
static
spots
disappearing
spots
laterally
mobile spots
Figure 2A.2.15 Distribution of Nef in dynamic clathrin spot (CS) populations. Live HeLa cells expressing DsRed-clathrin
(A and E) and Nef-GFP (B and F) were imaged using TIRF microscopy acquired at 300 msec/frame for 30 sec. (A to
D) Distribution of Nef in disappearing CS. A disappearing spot was identified on the clathrin frame, green and red images
were separated, and the corresponding regions were magnified (5). (C) The fluorescence intensity of each marker was
analyzed over time along a line drawn between two spots, a disappearing spot (arrow in C) and a static spot (D). The Nefand clathrin-associated fluorescence decreased with the same kinetics in the disappearing spot while remaining stable in
the neighboring static spot. (E to G) Distribution of Nef in laterally mobile CS. A laterally mobile spot was identified on the
clathrin frame, then green (F) and red images (E) were separated, and the corresponding region was magnified (5, G).
The upper panel in G corresponds to the clathrin images where a laterally mobile CS containing Nef-GFP (lower panel)
was identified (arrows). Scale bars 10 m. (H) The presence of Nef-GFP inside 40 CS (from more than five cells) from
each CS population (static, disappearing and laterally mobile) was determined. Results are expressed as the percentage
of CS of each population that contain Nef-GFP (normalized fluorescence >1.1). Reproduced from Burtey et al. (2007) with
permission from Blackwell Publishing.
TIRF Microscopy
Murine polynoma virus (Py) is a nonenveloped DNA tumor virus (Stehle et al.,
1994) that uses gangliosides GD1a and
GD1b as receptors (Tsai et al., 2003) and
relies on clathrin-independent, cholesterol-
2A.2.16
Supplement 10
18 0 time(s)
18
xy-displacement
( m)
0 time(s)
18 0 time(s)
18 0 time(s)
Figure 2A.2.16 Analysis of VLP trajectories at the bottom surface of live cells. All harvested
trajectories are plotted irrespective of the recorded length and whether the particles were bound
or free at the start of the recording. Every point represents one trajectory, and every trajectory is at
least 100 steps (5 sec) long. The longest trajectories are 2000 steps (100 sec) long. Representative
trajectories (numbered 1, 2, and 3) and one typical outlier (trajectory 4) represent rapid and random
movement (trajectory 1), confinement (trajectory 2), and confinement with a slow drift (trajectory
3). Trajectory 4 represents a VLP that changes its mode of motion during the acquisition time.
Below each of these trajectories are the respective analytical plots for absolute displacement
in m for x and y direction versus time. (Adapted from Ewers, H., Smith, A.E., Sbalzarini, I.F.,
Lilie, H., Koumouysakos, P., and Helenius, A. Copyright 2005. Single-particle tracking of murine
polyoma viruslike particles on live cells and artificial membranes. Proc. Natl. Acad. Sci. U.S.A.
102:15110-15115 with permission from the National Academy of Sciences, U.S.A.).
Each carrier (i.e., discrete VSVG-GFP single particle visible in TIRF in proximity to
cell membrane) was characterized by its movement, total fluorescence intensity, peak intensity, and the width of its intensity profile. They
were classified into three groups: (1) docked
carriers that stopped moving and remain stationary adjacent to the membrane, without fusing during the 1 min observation time; (2) carriers that exhibited a synchronous increase and
decrease in the total peak intensity due to their
movement in and out of the evanescent field,
without fusing to the membrane; and (3) carriers that showed a synchronous increase in
the total peak and width of the fluorescence
and, finally, fusion with the plasma membrane
(Fig. 2A.2.17).
When a carrier fuses with the plasma membrane, the total fluorescence intensity will increase as the vesicle flattens to the membrane,
whereas the peak intensity will transiently increase and then decrease as VSVG-GFP diffuses laterally into the plasma membrane. In
addition, the width will increase as a result
of this diffusion. Figure 2A.2.17B shows the
parameters of the fusion event plotted over
time. Table 2A.2.5 shows a qualitative summary of the kinetic behavior of the characteristic parameters for a fusing carrier. Thus, three
temporal phases can be distinguished along
this process: (1) stationary (movement of the
carrier towards the plasma membrane before
Microscopy
2A.2.17
Current Protocols in Microbiology
Supplement 10
flattening
diffusion
Distance
docking
Intensity of
evanescent field
0.05
3
4
Time [sec]
0.15
0.10
10
3
8
6
0.6
0.05
0.00
total intensity
peak intensity X3
width2
0.4
0.4
0.6
0.00
0.8
1.0 1.2
Time [sec]
Time: 0 msec 32 msec
spread
12
1
0
5
4
Width2 [ m2]
0.10
rise
14
stationary
16
Width2 [ m2]
Intensity [a.u.]
4
2
0.15
total intensity
peak intensity
width2
8
Intensity [a.u.]
96 msec
64 msec
1 m
4
1 m
8
128 msec
352 msec
928 msec
1632 msec
Figure 2A.2.17 Analysis of carriers. The vesicular stomatitis virus glycoprotein/green fluorescent protein (VSVG-GFP)
fluorescence was imaged for carriers close to the plasma membrane. The total intensity, peak intensity, and square of
the Gaussian width were plotted over time in panels A and B for the carriers shown in C and D, respectively. Selected
frames are shown from a sequence in panel C for a carrier that moved perpendicular to the coverslip, without fusing to
the plasma membrane and in panel D for a carrier which fused to plasma membrane. The intensity of the VSVG-GFP in
frames C and D is shown in pseudo-color. Each sequence was processed with a running average in time of 1 frame, and
thresholded separately to aid visualization. The radically symmetric Gaussian fit of the carrier fluorescence is shown below
each frame. The numbered arrows refer to the frames from sequences C and D. In panel B the three phases (stationary,
rise, and spread) are separated by dotted lines. Times are marked relative to the start of the rise phase. (Reproduced
from Schmoranzer et al., The Journal of Cell Biology, 149:23-31. Copyright 2000 The Rockefeller University Press. For the
color version of this figure go to http://www.currentprotocols.com.
TIRF Microscopy
2A.2.18
Supplement 10
Table 2A.2.5 Qualitative Summary of the Kinetic Behavior of the Characteristic Values for a
Fusing Carrier
Phase
Stationary
Rise
Spread
Total intensity
Constant
Relatively constant
Peak intensity
Constant
Width
Constant
ADVANTAGES OF TIRF
The prism-based TIRF configurations are
inexpensive and offer a variety of prism shapes
that can be coupled with the coverslip. In addition, the prism-based TIRF excitation produces less scattered light than through-thelense TIRF (Axelrod, 2001b). The drawback
of the prism setup is that it restricts sample access, and the entire TIRF path must be custom
assembled in the laboratory.
The through-the-lens TIRF configuration
represents the simplest method for TIRF exci-
2A.2.19
Current Protocols in Microbiology
Supplement 10
Golgi
A
z
y
x
in
laser
out
81
91
96
97
144
145
146
152
143
118
135
194
221
222
223
226
235
257
258
260
263
267
285
Relative fluorescence
100
300
Figure 2A.2.18 Live cell visualization of the late exocytic pathway by combined epifluorescence and TIRF. (A) Organelles
and carriers distant from the plasma membrane (>100 nm) are visible only by epifluorescence (red). Once carriers approach
the plasma membrane, they also become visible by TIRF (green). In an overlay of the two channels, originally red carriers
turn yellow and subsequently green as they approach the plasma membrane and later fuse. (B) Two live cells imaged
by epifluorescence (left) and TIRF (center) as individual channels are merged on the right, where boxes indicate areas
enlarged in subsequent images. (C) A carrier exits the Golgi complex and moves to the plasma membrane (small >),
where it docks and partially fuses (large >). A running subtraction of successive frames tracks the movement of the carrier
from the Golgi complex to the apparent docking site (last frame). (D) Two carriers that undergo successive fusion in close
proximity. Only the second one (>) undergoes complete fusion. The time is indicated in seconds. (E) Plot of the relative
fluorescence intensity of the second fusion event boxed in panel D. Note that the decrease in epifluorescence (red) is
accompanied by a large, sharp increase in TIRF signal (green) when fusion occurs (>). The small TIRF peak is due to an
earlier (222 sec) nearby fusion event. Bars: (B) 20 m; (C) 4 m; (D) 2 m. (Reproduced from Toomre et al., The Journal
of Cell Biology, 149:33-40. Copyright 2000 The Rockefeller University Press.). For the color version of this figure go to
http://www.currentprotocols.com.
TIRF Microscopy
2A.2.20
Supplement 10
LITERATURE CITED
Adams, M.C., Matov, A., Yarar, D., Gupton, S.L.,
Danuser, G., and Waterman-Storer, C.M. 2004.
Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and
wide-field epifluorescence FSM of the actin cytoskeleton and focal adhesions in living cells. J.
Microsc. 216:138-152.
Axelrod, D. 2001a. Total internal reflection fluorescence microscopy. In Methods in Cellular Imaging (A. Periasamy, ed.) pp. 362-380. American
Physiological Society, Bethesda, Md.
Axelrod, D. 2001b. Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764774.
Axelrod, D. 2001c. Selective imaging of surface
fluorescence with very high aperture microscope
objectives. J. Biomed. Opt. 6:6-13.
Axelrod, D., Thompson, N.L., and Burghardt, T.P.
1983. Total internal reflection fluorescent microscopy. J Microsc. 129:19-28.
Axelrod, D., Burghardt, T.P., and Thompson, N.L.
1984. Total internal reflection fluorescence.
Annu. Rev. Biophys. Bioeng. 13:247-268.
Axelrod, D., Fulbright, R.M., and Hellen, E.H.
1986. Adsorption kinetics on biological membranes: Measurements by total internal reflection fluorescence. In Applications of Fluorescence in Biomedical Sciences (L. Taylor, A.S.
Waggoner, R.F. Murphy, F. Lanni, and R.R.
Birge, eds.) pp. 461-476. A.R. Liss, New York.
Bonifacino, J.S. and Traub, L.M. 2003. Signals for
sorting of transmembrane proteins to endosomes
and lysosomes. Annu. Rev. Biochem. 72:395447.
Burmeister, J.S., Truskey, G.A., Yarbrough,
J.L., and Reichert, W.M. 1994. Imaging of
cell/substrate contacts on polymers by total internal reflection fluorescence microscopy.
Biotechnol. Prog. 10:26-31.
Burmeister, J.S., Olivier, L.A., Truskey, G.A., and
Reichert, W.M. 1998. Application of total internal reflection fluorescence microscopy to
study cell adhesion to biomaterials. Biomaterials 19:307-325.
Burtey, A., Rappoport, J.Z., Bouchet, J.,
Basmaciogullari, S., Guatelli, J., Simin, S.M.,
Benichou, S., and Benmerah, A. 2007. Dynamic
interaction of HIV-1 with the clathrin-mediated
endocytic pathway at the plasma membrane.
Traffic 8:61-76.
Conner, S.F. and Schmid, S.L. 2003. Regulated portals of entry into the cell. Nature 422:37-44.
Demuro, A. and Parker, I. 2004. Imaging the activity and localization of single voltage-gated Ca2+
channels by total internal reflection fluorescence
microscopy. Biophys. J. 86:3250-3259.
Ewers, H., Smith, A.E., Sbalzarini, I.F., Lilie,
H., Koumouysakos, P., and Helenius, A. 2005.
Single-particle tracking of murine polyoma
viruslike particles on live cells and artificial membranes. Proc. Natl. Acad. Sci. U.S.A.
102:15110-15115.
Microscopy
2A.2.21
Current Protocols in Microbiology
Supplement 10
KEY REFERENCES
Axelrod, D. 2003. Total internal reflection fluorescence microscopy in cell biology. Methods
Enzymol. 31:1-33.
Good review of TIRF theory, applications, and
practical protocols to set up a TIRF system.
Gingell et al., 1987. See above.
Quantitative theoretical approach of TIRF.
Oheim, M. 2001. Imaging transmitter release. II.
A practical guide to evanescent-wave imaging.
Laser Med. Sci. 16:159-170.
A how-to guide for constructing a TIRF system.
Wazawa, T. and Ueda, M. 2005. Total internal
reflection fluorescence microscopy in single
molecule nanobioscience. Adv. Biochem. Eng.
Biotechnol. 95:77-106.
Good review of TIRF applied to single molecule
studies.
INTERNET RESOURCES
http://www.micro.magnet.fsu.edu/primer/
index.html
Molecular Expressions Microscopy Primer. Excellent presentation of optical microscopy.
TIRF Microscopy
2A.2.22
Supplement 10
UNIT 2B.1
Transmission electron microscopy (TEM) has long been an important tool in the expanding field of microbiology. A major virtue of TEM is the potential resolution provided by
electron beams accelerated at high voltage with effective wavelengths that are shorter by
a factor of 105 than visible light. In principle, electron microscopes are capable of molecular resolution if only the wavelength (diffraction) limitation is considered; in practice,
however, other factors limit the achievable resolution, including the sample itself. Other
limitations of electron microscopy result from the fact that samples must be thin, dry,
and capable of scattering electrons in a vacuum. Typically, this necessitates fixation or
stabilization of the sample, and the introduction of electron-scattering heavy metal stains
to generate contrast for imaging.
This unit describes two types of TEM preparation techniques for examining particulate
samples as well as samples presenting more complex ultrastructural considerations that
require analysis in thin sections. Negative staining is a simple but valuable technique
that is useful for routine examination of particulate samples in suspension ranging from
bacteria to viruses to purified macromolecules. Strategies are presented for delivering
sample and stain sequentially onto a grid for TEM analysis (Basic Protocol 1), the direct
deposition of sample and stain together onto the grid (Alternate Protocol 1), the delivery
of spray droplets of sample and stain to the grid (Alternate Protocol 2), and the preparation
of grids for negative staining (see Support Protocol 1). Combination of negative staining
with immunolocalization of antigens using protein A and/or protein Ggold is presented
in Basic Protocol 2, along with the alternative procedure using gold-conjugated secondary
antibodies in place of protein A/Ggold (Alternative Protocol 3). In order to investigate
the relationships between microbes and the environments with which they interface,
more complex specimen preparation methods are required. Viruses and bacteria on or
in various substrates, including cells and tissues, can be identified by imaging in fixed
samples prepared for analysis in sections of 60- to 90-nm thickness. Due to the many
steps in sample preparation for ultrastructural analysis of thin-sectioned samples, the
major steps in the process are divided into Basic Protocols 3 and Alternate Protocols 4, 5,
6, and 7. In Basic Protocol 3, the fixation and initial processing of solid tissue or biopsy
samples is considered; Alternate Protocol 4 describes the variant procedure for pelleted
cells, Alternate Protocol 5 describes the procedure for bacterial suspensions, Alternate
Protocol 6 describes the procedure for cell monolayers; and Alternate Protocol 7 describes
the fixation and initial processing of samples to be subjected to immunogold labeling.
Basic Protocol 4 then addresses the process of embedding tissues and cell pellets into
plastic resin for sectioning, while Alternate Protocol 8 deals with embedding procedures
for monolayers grown on glass slides and Alternate Protocol 9 considers embedding
procedures for thin-section immunogold labeling. Once tissues are embedded, the cutting
(using an ultramicrotome) of uniform thin sections, free from defects, that adhere to each
other as straight ribbons, is an extremely challenging manual skill requiring a high level
of dexterity. Ultramicrotomy is not a technique that can be effectively communicated or
learned using a protocols manual, and therefore only an overview is presented in this unit
(Basic Protocol 5). It is assumed that the reader has access to ultramicrotomy support
service or hands-on training in this key element of specimen preparation for analysis of
thin sections. Once thin sections are obtained and collected onto a TEM grid, the sample
is usually stained with heavy metals as a final step before examination in the electron
microscope (Basic Protocol 6). This can be preceded by immunolocalization of antigens
in thin sections as discussed in Alternate Protocol 10.
Microscopy
Contributed by Robert C. Burghardt and Robert Droleskey
Current Protocols in Microbiology (2006) 2B.1.1-2B.1.39
C 2006 by John Wiley & Sons, Inc.
Copyright
2B.1.1
Supplement 3
SAFETY
Aside from the biohazards posed by handling and disposing of pathogenic microbes
(refer to UNIT 1A.1), many of the chemicals used in the processing of samples for electron
microscopy are either volatile, inflammatory, toxic, carcinogenic, and/or radioactive. It
is important to understand the nature of health and environmental hazards posed by these
chemicals and to handle and dispose of them according to appropriate safety regulations
(see UNITS 1A.1, 1A.3 & 1A.4). Expose electron microscopy grids containing negatively stained
pathogenic specimens to UV radiation or chemical fixation before examination and store
in a suitable container for safe temporary storage. Discard grids into a small wide-based
container with appropriate decontamination reagents upon completion of the analysis.
Rinse forceps used to handle grids with pathogenic samples in alcohol and flame before
and after use. Specimens processed for embedding and thin sectioning are typically
chemically stabilized in a fresh aldehyde-containing fixative, which will inactivate/kill
the majority of viruses and bacteria. Autoclave other disposable preparative materials that
come into contact with pathogens (e.g., Parafilm, filter paper) in appropriate containers
prior to disposal.
BASIC
PROTOCOL 1
Transmission
Electron
Microscopy
2B.1.2
Supplement 3
Each of the negative stains (see Reagents and Solutions) can be prepared with 1% (w/v)
carbohydrate (glucose or trehalose) to protect samples that are sensitive to air drying.
However, inclusion of carbohydrate involves additional considerations, as it reduces
the overall density of the stain, requiring higher stain concentration; also, the electronbeam instability of the carbohydrates necessitates use of lower beam intensities and/or
specimen cooling if possible. Lower concentrations of these stains can be prepared from
stock solutions if reduced specimen contrast is needed for any particular application.
Current Protocols in Microbiology
Negative staining can be at times a relatively dirty procedure with respect to microscope
vacuum, due to the generation of a corrosive sublimate, and in some cases can degrade
the sample itself. It is always best to limit contamination by using appropriate anticontamination devices such as ion-getter pumps and liquid nitrogen cold traps, if they are
available on the TEM instrument.
Materials
Samples: bacterial or viral suspensions
Negative staining solution: phosphotungstic acid staining solution (see recipe),
uranyl acetate staining solution (see recipe), or ammonium molybdate staining
solution (see recipe)
37% formaldehyde (optional)
Desiccant
Several sets of self-locking fine-point forceps
Formvar- or carbon-coated Formvar hexagonal 300- or 400- mesh copper grids (see
Support Protocol 1)
Whatman no. 4 filter paper
E-Series Germicidal Ultraviolet lamp (Spectroline) or equivalent, with UV monitor
and UV goggles (optional)
35-mm petri dish (optional)
Grid box (Electron Microscopy Sciences or Ted Pella)
1. Cut a piece of Parafilm from a roll (6 to 8 cm in length) along with its paper overlay
and place wax-side-down on a clean bench top. Prior to removal of the paper overlay,
score corners of the Parafilm or score parallel lines into the Parafilm with a blunt
object to loosely attach the Parafilm to the bench top.
This will provide the surface upon which to place individual droplets of sample, wash,
and staining solution in rows for sequential transfer of grids during the staining process.
2. Pipet in a row, with a spacing of 1.5 cm, a 15- to 25-l droplet of sample, two
or three droplets of Milli-Q-purified water (or equivalent) and one droplet of the
negative staining solution onto the wax surface of the Parafilm.
Several rows of these solutions can be prepared to process several grids at one time. The
number of water droplets needed depends on the concentration of solute to be removed
from the sample, as the presence of salt crystals will degrade and/or obscure the sample.
Optimization of the sample is largely by trial and error. If the sample is too concentrated,
it can be diluted in solvent and deposited on the Parafilm for further processing.
Microscopy
2B.1.3
Current Protocols in Microbiology
Supplement 3
Figure 2B.1.1 Aspects of the negative staining procedure. (A) Grids can be conveniently handled
using either self-closing forceps (top) or conventional ne-tipped forceps with a rubber O-ring to
keep the jaws closed (bottom). Specimen to be examined can be applied either by (B) oating
grid on a drop of the specimen or by (C) direct application to the grid. Stain can be applied as
illustrated in B or C. Washing (D) and uid removal (E) are performed in subsequent steps of the
procedure.
4. Before the sample has air dried on the grid, transfer the grid to the surface of
the water droplets sequentially, carefully removing the excess water between each
transfer using the edge of a wedge of filter paper.
This step is necessary if the sample has high concentrations of buffer salts or other solutes
which may interfere with uniform deposition of stain. If the sample is suspended in water,
the number of washing steps can be reduced or washing can be eliminated.
5. Transfer the grid to the surface of the negative stain solution for 5 to 30 sec, then
remove the grid and remove the excess staining solution by again allowing the grid
to touch the edge of a filter paper wedge.
The rate of removal of stain can be adjusted by changing the orientation of the filter
paper wedge, since the pointed edge adsorbs less liquid than the broader edges. Thereby,
it is possible to increase or decrease the amount of stain removed if stain density requires
modification.
Transmission
Electron
Microscopy
2B.1.4
Supplement 3
6. Allow the grid to air dry completely before TEM analysis to avoid damage to sample
and contamination of the microscope.
The grid with the sample can be placed in a container with a desiccant to accelerate
drying.
Several iterations of this basic procedure are usually required to optimize the concentration and distribution of the sample particles on the grid (see Critical Parameters and
Troubleshooting).
7. Optional: When dealing with pathogenic microbes, inactivate in one of the following
ways:
a. Expose the grids to UV irradiation (900 W/cm2 ) at 15 to 20 cm for at least
10 min.
b. Expose grids to 37% formaldehyde fumes in a 35-mm petri dish for at least 30 min.
The formaldehyde can be placed in the lid from a screw-top fixation vial and the grids
can be distributed on filter paper surrounding the lid and exposed to the fumes for at least
30 min.
8. Store grids in a grid box with desiccant at room temperature until ready for further
processing or TEM analysis.
ALTERNATE
PROTOCOL 1
Excessive handling of coated grids can be eliminated by the use of the direct deposition
technique whereby both the sample and stain are dropped onto the coated grid. This
reduces grid manipulation and can speed up the staining procedure once an optimal ratio
of sample to stain is determined. When examining pathogenic organisms, it is often
necessary to inactivate them with glutaraldehyde prior to staining. If this is the case, it
is possible to directly add the negative stain to the sample and drop the mixture in a
one-step procedure similar to that outlined below.
For materials, see Basic Protocol 1.
1. Using several sets of self-locking forceps, pick up grids by their edges and place them
with support film facing upward in a row on top of plastic-backed bench protectors
(diapers).
2. Deposit a 10-l sample droplet on top of the first grid with a pipet, let sample adhere
to the grid for 30 to 60 sec, then add a 10-l droplet of negative stain directly to the
grid with a pipet.
This adsorption step should distribute the sample and stain uniformly over the surface of
the grid (Figure 2B.1.1C).
When dealing with large volumes of sample, it is possible to combine samples, stain
together in a microcentrifuge tube, and apply them to the grid simultaneously. Several
dilutions of stain and sample can be prepared and run at the same time.
3. Briefly touch the edge of the grid to a wedge of filter paper to remove excess sample.
4. In the case of samples containing high salt concentration, wash twice, each time by
placing a 10- to 15-l drop of distilled water on the grid for a few seconds and then
removing it with a clean wedge of filter paper.
In some instances this extra washing step may cause inadequate sample contrast and will
require another brief application of the negative stain solution.
Microscopy
2B.1.5
Current Protocols in Microbiology
Supplement 3
5. Allow the grid to air dry. Inactivate biohazardous organisms as in Basic Protocol 1,
step 7, if necessary. Store grids in a grid box with desiccant at room temperature
until ready for further processing or TEM analysis.
ALTERNATE
PROTOCOL 2
2. Hold grid in front of the nebulizer using a set of locking forceps inserted into a
suitable stand so that the grid projects into the path of the spray.
3. Apply small droplets of sample onto the grid by atomizing the sample.
CAUTION: If potentially pathogenic organisms are being examined, appropriate safety
precautions must be taken (see UNIT 1A.1). These can include fixation of the sample
with glutaraldehyde before use of the nebulizer, spraying the sample onto the grid in
a biological safety hood, and/or spraying the sample onto a grid secured in front of a
disinfectant-coated towel.
The size of droplets can be adjusted by the pressure applied to the nebulizer bulb. The
density of sample on the grid can be regulated by varying the distance of the grid from
the nebulizer.
Transmission
Electron
Microscopy
2B.1.6
Supplement 3
and Marivac Inc. (the last two suppliers are accessible through a common Web site
at http://www.canemco.com). If commercial suppliers are used, it is recommended that
freshly prepared support films be purchased in relatively small lots, as the films lose
stability under the electron beam when stored for several months.
Support films are commonly prepared from Collodion (nitrocellulose), Formvar, and
carbon due to their relatively high transparency to electrons and strength under electron
irradiation. Collodion or Formvar can be cast on glass slides or a water surface. Collodion
is soluble in ethyl or amyl acetate and acetone, and films are prepared from solutions
ranging from 0.5% to 2% (w/v). Formvar is soluble in ethylene dichloride or chloroform
and can be prepared from solutions that range in concentration from 0.1% to 0.5% (w/v).
A film can be deposited by dipping slides into Coplin jars containing these solutions at a
depth of 10 cm. Once the slide is dipped, the edge is drained onto filter paper and the
slide dried in a dust-free area. The dried film is then floated off of the slide by dipping
it into a bowl of water at a 30 to 40 angle after the edges of the slide are scraped with
a razor blade to allow the hydrophobic film to float off. Once the rectangular film floats
on the water surface, a number of grids that are precleaned in acetone can be carefully
distributed over the surface of the floating film. A strip of Parafilm slightly larger than the
area of the floating support film is then gently laid on top of the grid-containing support
film and immediately lifted off of the water surface. The Parafilm with grids on the upper
surface is then placed in a covered petri dish or desiccator where the grids remain until
needed. Plastic films can be strengthened/stabilized by depositing a thin film of carbon
in a vacuum evaporator.
Carbon films are strong and very stable under the electron beam and are useful for
high-resolution electron imaging. Preparation of carbon films is a relatively laborious
procedure requiring a vacuum evaporation device with appropriately sharpened carbon
electrodes. Preparation of carbon support films as well as carbon deposition on plastic
films are described in detail in Hayat (2000).
BASIC
PROTOCOL 2
The following protocol describes basic steps necessary for immunolocalization of antigens in negatively stained preparations utilizing colloidal gold preparations conjugated
to either secondary antibodies or to protein Agold, protein Ggold, or protein A plus
protein G (protein A/G)gold, which are used as electron-opaque reporters of primary antibodies that bind to surface antigens on viruses or bacteria. The conjugates interact with
mammalian immunoglobulins; however, their individual affinity for immunoglobulins is
not equivalent for all species or for all antibody subclasses. There are many alternatives
and variations on immunoelectron microscopy approaches, only two of which are outlined below. In these examples, it is assumed that the sample is stable without fixation
and that optimal conditions for sample concentration and delivery onto a grid, as well
as for negative staining of the sample, have already been determined. If it is suspected
that the stability of the sample will be jeopardized by the number of steps and duration
of the protocol, the samples can be fixed in appropriately buffered, freshly prepared, 1%
paraformaldehyde. This is followed by washing in a glycine-containing buffer to remove
residual free aldehydes resulting from fixation prior to immunolocalization of antigens
and negative staining.
Materials
Samples: bacterial or viral suspensions
Primary antibody against antigen of interest and irrelevant primary antibody of
same species/class as control
Immunogold antibody dilution buffer (see recipe)
Microscopy
2B.1.7
Current Protocols in Microbiology
Supplement 3
Protein Agold or protein A plus protein Ggold solution: e.g., Structure Probe/SPI
Supplies or Nanoprobes (see SUPPLIERS APPENDIX) or Aurion
(http://www.aurion.nl)
96-well microtiter plate
Humidified chamber: e.g., Tupperware or Rubbermaid plastic container with
sealable lid, containing water-saturated paper towels
Several sets of self-locking fine-point forceps
Formvar-coated 300- or 400-mesh nickel grids.
Additional reagents and equipment for negative staining (see Basic Protocol 1)
1. In a well of a microtiter plate, mix 25 l sample suspension with an equal volume
of a suitable dilution of primary antibody in immunogold antibody dilution buffer.
Incubate 1 hr at 25 to 37 C in a humidified chamber.
The antibody dilution needs to be empirically determined. Initial tests should include
a serial dilution of antibody that increases by factors of 10. This will help determine
optimum conditions for labeling with minimum background. A 1/100 and 1/1000 dilution
provide a reasonable starting point.
To streamline the process, controls can be processed at the same time and should include
an irrelevant primary antibody of the same species and/or class, and another preparation
without primary antibody.
3. Using several sets of self-locking forceps, pick up grids by their edges and place them
with support film facing upward in a row on top of plastic-backed bench protectors
(diapers).
4. Deposit a 10- to 15-l droplet of the mixture from step 2 directly onto the grid with
a pipet and allow to stand 5 min.
5. Cut a piece of Parafilm from a roll (6 to 8 cm in length) along with its paper overlay
and place wax-side-down on a clean bench top. Prior to removal of the paper overlay,
score corners of the Parafilm or score parallel lines into the Parafilm with a blunt
object to loosely attach the Parafilm to the bench top.
6. Pipet, in a row, with a spacing of 1.5 cm, two drops of immunogold antibody
dilution buffer and one drop of distilled water onto the wax surface of the Parafilm.
Before the sample dries on the grid, wash by sequential immersion in the two drops
buffer and the drop of distilled water. Carefully remove the excess liquid between
transfers by allowing the grid to touch the edge of a wedge of Whatman no. 4 filter
paper.
7. Perform negative staining as in Basic Protocol 1, using 2% phosphotungstic acid and
bringing the sample-containing grid into contact with staining solution for 30 sec.
Transmission
Electron
Microscopy
2B.1.8
Supplement 3
ALTERNATE
PROTOCOL 3
6. Rinse grids by sequentially transferring them through four successive drops of immunogold antibody dilution buffer followed by one drop of distilled water using the
technique described in Basic Protocol 1. Use a clean wedge of filter paper at the edge
of the grid to wick off the water.
7. Perform negative staining as described in Basic Protocol 1.
Microscopy
2B.1.9
Current Protocols in Microbiology
Supplement 3
of these preparations may involve a variety of different strategies. The following protocols
describe the basic steps necessary for the chemical fixation and initial processing of tissue
pieces, cell pellets, bacterial suspensions, cells in monolayer culture, and samples that
are to be immunolabeled in thin sections, each outlined separately.
CAUTION: Due to the volatility and toxicity of the fixatives used in the following
protocols (i.e., glutaraldehyde, paraformaldehyde, and osmium tetroxide), these items
should be handled in a fume hood using latex or nitrile gloves. Protective eyeware should
also be worn.
BASIC
PROTOCOL 3
Materials
Tissue sample
TEM primary fixative 1 (see recipe)
Phosphate/sucrose rinse buffer (see recipe), 4 C
Osmium tetroxide post-fixative (see recipe)
Uranyl acetate staining solution (see recipe)
Graded ethanol series: 50% and 75% ethanol (4 C) and 95% and 100% ethanol
(room temperature)
Acetone or propylene oxide
Polypropylene cutting board
Single-edged razor blades
Fixation vials: dram vials with caps
Platform rocker or orbital shaker (optional)
Additional reagents and equipment for embedment (Basic Protocol 4)
Fix tissue
1. Obtain a fresh piece of tissue and, as quickly as possible, slice tissue on a polypropylene cutting board with a razor blade into pieces <1 mm in thickness (or smaller
than 1 mm3 ).
It is preferable to cover the tissue with a small amount of TEM primary fixative 1 during
slicing.
2. Place several small pieces of tissue in a fixation vial containing freshly prepared
TEM primary fixative 1 at a volume at least 20 times greater than that of the tissue
pieces.
3. Incubate tissue at room temperature for 60 min, them move to a 4 C ice bath for an
additional 60 min. Alternatively, fix tissues overnight at 4 C.
Transmission
Electron
Microscopy
4. Decant fixative from vials and replace with phosphate/sucrose rinse buffer at 4 C.
Replace the buffer with fresh phosphate/sucrose rinse buffer several times over a
30-min interval to wash the tissue.
2B.1.10
Supplement 3
8. Using the technique described in step 4, wash tissue three times in cold distilled
water to remove uranyl acetate.
ALTERNATE
PROTOCOL 4
Microscopy
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Obtain pellet
1. Harvest bacteria or cultured cells in buffered saline or serum-free culture medium
and centrifuge in either hard- or soft-walled microcentrifuge tubes.
A variety of microcentrifuge tubes can be used for pelleting samples. When using hardwalled tubes such as polypropylene microcentrifuge tubes, pellets can be overlaid with
a glutaraldehyde-based fixative for stabilization of cells. Polyethylene microcentrifuge
tubes have soft walls and the tips of these tubes containing the pellet can be cut off with
a razor blade and dropped directly into fixative for initial tissue processing.
Fix cells
2a. If the sample is collected in a hard-walled microcentrifuge tube: Carefully remove
supernatant from pellet. In a fume hood, slowly add freshly prepared TEM primary
fixative 1 using a volume at least 20 times greater than that of the pellet. Fix for 1 hr
at 4 C.
2b. If the sample is collected in a soft-walled microcentrifuge tube: Cut off the tip of the
microcentrifuge tube containing the sample pellet and drop the pellet along with the
end of the tube into a fixation vial containing freshly prepared TEM primary fixative
1 at a volume at least 20 times greater than that of the pellet. Fix for 1 hr at 4 C.
3. Replace fixative with phosphate/sucrose rinse buffer and carefully remove the pellet
from the microcentrifuge tube or cut tip of tube.
Transmission
Electron
Microscopy
10. Stain tissue en bloc in 1% aqueous uranyl acetate for 90 to 120 min at 4 C with
periodic agitation.
Alternatively stain in 0.5% aqueous uranyl acetate overnight at 4 C.
2B.1.12
Supplement 3
11. Using the technique described in step 7, rinse pellets three times in cold distilled
water to remove uranyl acetate.
ALTERNATE
PROTOCOL 5
2. Immediately invert mixture and place on a horizontal mixer 60 min at room temperature.
3. After initial room temperature fixation, transfer cell suspension to a 4 C ice bath for
an additional 1 hr of fixation.
4. Centrifuge cell suspension 10 min at 1000 g, 4 C, and discard supernatant.
5. Resuspend cells in phosphate/sucrose rinse buffer and incubate at 4 C for 10 min.
6. Centrifuge cells 10 min at 1000 g, 4 C, and discard supernatant.
2B.1.13
Current Protocols in Microbiology
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8. Centrifuge the agar and cell suspension 5 min at 5000 g, room temperature, to
pellet cells.
9. Chill tubes briefly on ice to solidify agar.
10. Using a single-edged razor blade, cut through the microcentrifuge tube just above
the cell pellet in the agar to remove the pellet. Slice the removed agar encased
pellet into thin slices, 0.5 mm wide, and place in fixation vials containing chilled
phosphate/sucrose rinse buffer.
15. Wash three times in cold distilled water to remove uranyl acetate.
2B.1.14
Supplement 3
1. In a fume hood, slowly remove medium from the cultured cells and add TEM primary
fixative 2 to the chamber so as to cover the monolayer with 3 to 5 mm of fixative
(6 ml fixative per slide). Cover, and incubate cells in fixative for 30 min at room
temperature followed by an additional 30 min at 4 C.
The plastic chamber should not removed until after the fixation and uranyl acetate staining
are completed.
2. Remove fixative and replace with an equal volume of cold phosphate/sucrose rinse
buffer. Cover and incubate 10 min.
3. Remove phosphate/sucrose rinse buffer and replace with an equal volume of osmium
tetroxide post-fixative. Incubate at 4 C for 60 min.
4. Remove post-fixative solution and rinse once in phosphate/sucrose rinse buffer.
5. Rinse chambers with cold distilled water six times over the course of 1 hr.
6. Add 1 ml uranyl acetate staining solution to the chamber and incubate 1 hr at 4 C.
7. Rinse chambers twice with cold distilled water.
8. Dehydrate and embed cells on the chamber slide (see Alternate Protocol 8).
ALTERNATE
PROTOCOL 7
The fixation and processing of samples that will permit labeling of antigens in thinsectioned materials pose substantial challenges. Methods commonly used for optimal
ultrastructural morphology must usually be modified to ensure that antigens are not
only retained but available for labeling. This often involves optimization, with compromises between the fixation, processing, and embedding steps; however, careful selection
of preparation methods can yield high-quality combinations of structural detail and
immunochemical labeling.
10 min
10 min
10 min
30 min
30 min.
Microscopy
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Materials
Fixed and processed tissue (Basic Protocol 3), cell pellet (Alternate Protocol 4), or
bacterial sample (Alternate Protocol 5)
Lufts epoxy mixture (see recipe)
Acetone or propylene oxide
Orbital shaker
Transfer forceps or applicator stick
Embedding molds or capsules (available from Ted Pella; BEEM flat embedding
molds, cat. no. 111-2, PTFE flat embedding molds, cat. no. 10509, or BEEM
embedding capsules, size 00, cat. no. 13)
60 C oven
1. Pipet out the last rinse of acetone or propylene oxide from the fixation vial and replace
with a mixture of 50% (v/v) Lufts epoxy mixture and 50% acetone or propylene
oxide. Place the vial on an orbital shaker and rotate sufficiently to suspend the tissue
pieces or pellet in the resin.
Upon initial introduction of the resin, the tissue pieces or pellets will float to the top. With
time, they will sink as they become infiltrated.
2. Incubate at room temperature for 30 min with rotation on the orbital shaker to aid
infiltration.
Transmission
Electron
Microscopy
2B.1.16
Supplement 3
3. Remove all of the 50-50 resin mixture and replace with a mixture of 75% (v/v) Lufts
epoxy mixture and 25% acetone or propylene oxide. Incubate at room temperature
for 30 min with rotation to aid infiltration.
The pellets will again float to the top of the suspension and will slowly sink as the resin
infiltrates the pellets.
Current Protocols in Microbiology
4. Remove the resin from step 3 and replace it with 100% Lufts epoxy mixture. Rotate
on an orbital shaker for 1 hr at room temperature, then remove the epoxy mixture
and replace with freshly prepared 100% Lufts epoxy mixture.
5. Remove the resin and, using transfer forceps or an applicator stick, carefully transfer
pellets to suitable embedding molds containing freshly prepared 100% Lufts epoxy
mixture. Transfer molds to a vented 60 C oven and incubate overnight.
A variety of embedding molds and capsules are commercially available that allow tissue to
be optimally positioned for sectioning once released from the mold. For pelleted material,
polyethylene caps for snap-cap sample vials (Electron Microscopy Sciences #64257-30)
work well for this purpose. They come in a variety of sizes, allow for easy placement of
the sample, have plenty of surface area for outgassing of residual solvent if necessary,
and are reusable; in addition, the polymerized resin is easily removed. Pellets or tissue
can be excised from the resin using a jewelers saw or a single-edged razor blade if
the resin is slightly heated and mounted on a blank BEEM capsule or Plexiglas pegs
(Ladd Research #21830) using superglue. Small pieces of tissue can be directly placed in
resin-filled BEEM capsules for polymerization. However, when BEEM capsules are used
for direct embedding of tissue, it is often advantageous to employ flat embedding molds
for embedding extra pieces of tissue if the need arises.
ALTERNATE
PROTOCOL 8
Materials
Fixed and processed monolayer cells in chamber slides (Alternate Protocol 6)
50%, 75%, 95%, and 100% ethanol
Mollenhauers no. 2 resin mixture (see recipe)
Single-edged razor blade
1. Dehydrate the cell monolayer in the chamber slide by sequentially adding the following solutions at room temperature to the chamber, leaving for the indicated length
of time, and then draining and filling with the next solution:
50% ethanol
50% ethanol
75% ethanol
75% ethanol
95% ethanol
95% ethanol
100% ethanol
100% ethanol
100% ethanol
5 min
5 min
5 min
5 min
5 min
5 min
5 min
5 min
5 min.
Each dehydration step is performed multiple times to insure adequate exchange of fluid
around the corners of the chambers.
2. Remove the last change of ethanol and then quickly invert chamber onto an absorbent
paper towel to remove excess ethanol, being careful not to allow cells to dry out
or touch the paper towel. Quickly refill chambers with a mixture of 50% (v/v)
Mollenhauers no. 2 resin mixture/50% ethanol.
3. Rotate the chamber slide gently to mix contents and decant mixture. Invert over a
fresh paper towel and then refill the chamber with fresh resin mixture of the same
composition that was used in step 2. Cover and incubate for 30 min, rotating the
slide occasionally.
Microscopy
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4. Repeat the procedure used in steps 2 and 3 with a mixture of 75% Mollenhauers
no. 2 resin mixture/25% ethanol, then again with 100% Mollenhauers no. 2 resin
mixture. Replace the 100% resin with fresh resin after an initial 30 min incubation,
for a total incubation time of 60 min in 100% resin.
5. Using a pair of forceps, carefully pull up the plastic chamber from the slide, leaving
the silicone gasket intact on the glass. Carefully refill each gasket-outlined chamber
with fresh 100% resin to just below the top of the gasket surface. Place in a level
60 C oven to polymerize.
6. After resin polymerization, remove the gasket from the slide and then remove the
embedded cells from the slide using a single-edged razor blade.
The application of heat to the slide may be necessary for the removal of the embedded
chambers. If that is the case, set the slide on an 80o C hot plate for 30 to 45 sec.
Pieces of the embedded monolayer can be cut out and mounted on plastic pegs so that they
are sectioned either en face or in cross-section. When sectioning across the monolayer,
the use of grids with Formvar support will provide stability to the section.
ALTERNATE
PROTOCOL 9
Materials
Fixed and processed tissue or cell sample (Alternate Protocol 7)
LR White resin (Polysciences)
LR White accelerator (Polysciences)
Embedding molds (available from Ted Pella; BEEM flat embedding molds, cat. no.
111-2, PTFE flat embedding molds, cat. no. 10509, or BEEM embedding
capsules, size 00, cat. no. 13)
Cooled water bath or cooling block (optional)
Jewelers saw
Plexiglas pegs (Ladd Research, cat. no. 21830; http://www.laddresearch.com/ )
Cyanoacrylate glue (e.g., Superglue, Krazy Glue)
1. Following the last two washes of tissue in 100% ethanol described Alternate
Protocol 7, infiltrate the sample with LR White resin as follows. Remove the ethanol
and replace with 100% LR White resin, incubate 1 hr at room temperature, then
replace with fresh 100% LR White resin and incubate an additional 1 hr at room
temperature. Finally, replace again with fresh 100% LR White resin and incubate an
additional 1 hr to overnight at room temperature.
See technical data sheet for LR White resins at http://www.polysciences.com/shop/
assets/datasheets/305A.pdf.
Transmission
Electron
Microscopy
2B.1.18
Supplement 3
or a wooden stick with a fine tip. Deposit the tissue pieces into the resin-containing
embedding mold(s). Allow molds to polymerize for 48 hr at room temperature.
If flat embedding molds are used, it may be necessary to exclude air from the surface of
the resin and overlay the mold with a piece of ACLAR film (Ted Pella, cat. no. 10501-10)
to exclude as much air as possible from the surface of the mold. Embedding in BEEM or
gelatin capsules is preferred over the use of flat embedding molds, as the surface area
exposed to air is minimized and the capsules do not require the film overlay.
OVERVIEW OF ULTRAMICROTOMY
As noted previously, the goal of ultramicrotomy is to prepare thin sections between
50 and 90 nm thick. The generation of sections that adhere to one another as straight
ribbons using an ultramicrotome is an extremely challenging manual skill that requires a
high level of dexterity. Ultramicrotomy is not a technique that can be effectively learned
using a protocols manual and is therefore given as an overview rather than a method in
this unit.
BASIC
PROTOCOL 5
While it is assumed that the reader has access to ultramicrotomy support service or handson training in this key element of specimen preparation for analysis of thin sections, a
brief description of ultramicrotomy follows. In order to thin-section embedded tissue that
has been placed in an embedding mold, it must be mounted in a holder for trimming. Some
ultramicrotomes have different types of holders that can grip flat embedded tissue or hold
a cylinder of plastic with a tapered end (pyramid shape) containing the tissue. The tapered
end must be trimmed further to form a small trapezoid of 200 m2 . This is usually
done by hand with a single-edged razor blade under a stereo microscope and requires
considerable dexterity and practice. Some microtomes have specimen holders, which
permit rapid trimming of the block to form the trapezoid with a highly polished face.
Thin sections are cut with either a glass or diamond knife. Glass knives are normally
made freshly with a knife-maker instrument, which breaks 1-in. (2.5-cm) squares
of glass from high-quality glass strips. The squares of glass are scored diagonally and
carefully broken to form triangular pieces, one edge of which contains the knife edge.
A commercially made plastic trough is sealed with dental wax. Alternatively, a trough
or boat is fashioned around the knife edge with Mylar or other tape that will provide
a distilled waterfilled reservoir that reaches the edge of the knife and that will allow
sections to float away from the edge of the knife from the block during the cutting
stroke of the microtome arm. The wide side of the trapezoid-shaped block face makes
first contact with the knife edge during the cutting stroke of the microtome. Prior to
collecting sections, the block face must be polished in preparation for thin sectioning
by cutting initial sections until the block face appears to have a mirrored surface. Thin
sections are then cut, and individual trapezoid-shaped sections adhere to the knife edge
until the subsequent section displaces the previous section during the cutting stroke of
the microtome arm. With each consecutive section, a ribbon resembling the shape of
a tapeworm floats into the boat (Fig. 2B.1.2). These ribbons are typically manipulated
into parallel rows with a fine eyelash attached to the end of a small applicator stick just
prior to picking up the sections on a 3-mm-diameter electron microscope grid. Sections
are adhered to the grid by lowering it with a pair of forceps above the sections, gently
touching the grid to the surface of the water, and then lifting the grid off the water surface
in a smooth motion. Residual water is removed from the edge of the grid with a wedge
of filter paper as is done for negative staining.
Microscopy
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Figure 2B.1.2 Thin sections (70 to 80 nm) prepared on a diamond knife as seen through a
stereo microscope. Trapezoid-shaped sections are cut sequentially, adhering to the knife edge
and forming ribbons during sectioning (black arrow). They are visible as the result of reected
light from above, which generates an interference pattern that can be used to estimate section
thickness. As sections accumulate, they can be separated into smaller ribbons, which oat on the
water surface and can be aligned for collection onto a grid. The width of the trough of the diamond
knife shown is 10 mm.
Copper grids are routinely used for most biological samples; nickel grids are usually
used with samples prepared for immunogold electron microscopy. The 50- to 90-nm
thickness of each section can be estimated by adjusting the microtome stereomicroscope
viewing and illumination system to detect interference colors reflecting from the sections.
Generally, gray sections indicate a thickness of less than 60 nm, while silver sections
range from 60 to 90 nm (the typical section thickness for most biological samples), and
gold sections range from about 90 to 150 nm. The diamond knife, which contains a highquality diamond with a cleaved knife edge mounted in an aluminum holder fashioned
into a trough for floating sections, is a considerably more expensive and longer-lasting
alternative to disposable, freshly made glass knives.
BASIC
PROTOCOL 6
Materials
Transmission
Electron
Microscopy
2B.1.20
Supplement 3
ALTERNATE
PROTOCOL 10
Materials
Thin sections on 300-mesh Formvar-coated nickel grids (Basic Protocol 5)
Immunogold blocking buffer (see recipe)
Primary antibody against antigen of interest and control (irrelevant) primary
antibody of same Ig class
TBS-Tween (see recipe)
Reagents for colloidal gold labelingone of the following:
Colloidal gold-labeled secondary antibody against species from which primary
antibody was obtained
Biotinylated secondary antibody against species from which primary antibody
was raised, and streptavidin-conjugated colloidal gold
Colloidal gold-labeled protein A and/or protein G
TEM primary fixative 2 (optional; see recipe)
Whatman no. 4 filter paper
Spray bottle
Grid box
Microscopy
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Current Protocols in Microbiology
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Additional reagents and equipment for etching of epoxy embedding resin (optional;
see Support Protocol 2) and uranyl acetate/lead citrate staining of thin sections
(see Basic Protocol 6)
1. If the sample is not embedded in LR White resin, perform Support Protocol 2.
Block sample
2. Place an 50-l drop of immunogold blocking buffer onto Parafilm and float samplecontaining grid on top of it for 30 min as a blocking step.
Treat with primary antibody
3. Prepare appropriate dilution of primary antibody in TBS-Tween, place 50-l droplets
of the diluted antibody onto Parafilm, then place grids on droplets and incubate at
room temperature for 2 hr.
Controls can be processed at the same time and should include both an irrelevant primary
antibody of the same class and another preparation without primary antibody.
4. Remove grid and draw off excess solution with a filter paper wedge (see Basic
Protocol 1) and gently rinse with a stream of TBS-Tween from a spray bottle.
Transmission
Electron
Microscopy
2B.1.22
Supplement 3
SUPPORT
PROTOCOL 2
The following steps may precede Alternate Protocol 10 if the tissue is not embedded
in LR White resin. Tissues subjected to optimal fixation for immunolocalization with
colloidal gold reagents embedded in an epoxy resin may be etched to increase access of
primary antibody to antigenic sites in the sectioned tissue.
Materials
Plastic etching solution: 5% (w/v) sodium metaperiodate (prepare fresh)
Thin sections on 300-mesh Formvar-coated nickel grids (Basic Protocol 5)
TBS-Tween (see recipe)
Spray bottle
1. Place 50-l drops of plastic etching solution onto clean Parafilm.
2. Place grids section-side-down on etching solution for 10 min at room temperature.
3. Gently run a stream of TBS-Tween from a spray bottle down the tips of the forceps
onto the grid to wash off etching solution.
Microscopy
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Current Protocols in Microbiology
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Transmission
Electron
Microscopy
2B.1.24
Supplement 3
TBS-Tween
To 1 liter of distilled water add:
6.1 g Trizma base
9 g NaCl
Mix to dissolve
Adjust pH to 7.6 using 1 N HCl
Add 0.5 ml Tween 20
Store up to 6 months at 4 C
Final concentrations: 0.5 M Tris-buffered saline, 0.05% Tween 20.
COMMENTARY
Background Information
Negative staining TEM
Negative staining is one of the simplest yet
most useful methods for imaging small particles in suspension. Introduced in the late 1950s
Microscopy
2B.1.25
Current Protocols in Microbiology
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Transmission
Electron
Microscopy
2B.1.26
Supplement 3
Critical Parameters
and Troubleshooting
Negative staining TEM
Several iterations of this basic procedure
are usually required to optimize the concentration and distribution of the sample particles on
the grid. The sample dilution is usually determined empirically. Stain density can be varied
by adjusting the amount of excess stain during removal with the filter paper wedge, by
Microscopy
2B.1.27
Current Protocols in Microbiology
Supplement 3
Problem
Possible cause
Solution
Grids with support films tend Grids with support films can be glow
to be hydrophobic
discharged shortly before staining.
Wetting agents can be used make
grids more hydrophobic. Incubate
grids for 30-60 sec on a drop of
bacitracin (50 g/ml), poly-L-lysine
(1 g/ml), or BSA (0.05% to 0.005%
w/v).
Alternatively, wetting agents can be
added directly to the sample to aid
spreading.
Grids with support films tend Can add wetting agents at lower
to be hydrophobic
concentrations than those mentioned
above directly to the stain
If using phosphotungstic acid, switch
to a more hydrophilic stain such as
ammonium molybdate
Structural details of
sample surface are not
resolved
Stain precipitate
2B.1.28
Supplement 3
Problem
Possible cause
Solution
No gold labeling
The primary antibody may be bad, e.g., due If available, run a positive control to check
to poor titer, age, improper storage, improper
dilution, or excessive freezing and thawing
The pH of solutions may be excessively
acidic or alkaline
Adjust the pH
The antigen may be destroyed by preparative Use a different procedure. Consider brief
procedures
fixation in 1% paraformaldehyde.
Excessive
background gold
particles
Free aldehyde groups in fixed tissue may be Reduce by exposing sections to 0.5 M
a source of background
ammonium chloride for 1 hr before incubations.
The primary antibody concentration may be Dilute by orders of magnitude
too high
Clustering of gold
particles
Dilute further
Nonspecific binding
Microscopy
2B.1.29
Current Protocols in Microbiology
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Transmission
Electron
Microscopy
2B.1.30
Supplement 3
Preparation of tissues and cells for immunogold labeling requires a balance between
preservation of tissue/cell ultrastructure and
the preservation of antigens. The goal is to
optimize these compromises in order to combine adequate structural detail and adequate
immunochemical labeling. Concentrations of
glutaraldehyde that are normally used for ultrastructure most often destroy or cross-link
antigens. Therefore paraformaldehyde alone
or in combination with dilute glutaraldehyde
(0.1% to 1%) is a useful starting point for fixation. Tissues processed for immunolabeling in
thin sections require special embedding procedures, as discussed below.
The protocols presented in this section
can be classified as representing classical approaches to the preservation of tissue for examination by thin-section electron microscopy.
Other techniques are available that offer the
advantages of potentially better preservation
of specimen ultrastructure and possibly shorter
processing times at the expense of having
specialized processing equipment, additional
safety concerns, and special modifications of
the electron microscope itself. Freeze substitution, accomplished by the formation of vitreous ice within the sample by either plunging the sample into cooled liquid propane or
ethane or exposing samples to a jet of liquid
propane or to high-pressure freezing, offers
the advantage of better preservation of ultrastructure, and usually better antigen preservation as well. Microwave processing techniques
offer the advantage of rapid specimen fixation and embedding times. All of these techniques require specialized preparation protocols adapted to suit the specific equipment on
hand, and are beyond the scope of this chapter.
Embedding techniques for TEM
By their nature, epoxy resins and their
components are viscous, and this viscosity
tends to increase with time after the individual
components of the resin are mixed. Because of
this, it is advisable to prepare resins in small
batches just prior to use. Dilutions of complete
resin mixtures in dehydration agents are prepared by dispensing the resin components into
new disposable beakers. Preparations of resin
and dehydration agent are kept capped to prevent evaporation of the agent over time, an
especially important consideration when
working in a fume hood. In the case of epoxy
resin, accurate weighing of all components
and dilution of resins is of critical importance
in their preparation to ensure a resin mixture that will give the proper characteristics.
It is also important that the individual components of the resin be completely mixed. Inadequate mixing of resin components can result in
poorly infiltrated samples that section poorly,
that have poor specimen contrast, and that do
not hold up in the electron beam. Shaking or
vigorous stirring results in well mixed resin.
Any resulting entrapped air is liberated upon
standing or can be removed by use of a vacuum
chamber for 5 to 10 min or until the generation
of gas bubbles stops (the vacuum provided by
a one-stage rotary pump is sufficient). Caution
must be used when degassing the resin mixture, as it can boil out of the disposable beaker;
careful monitoring of the mixture in the vacuum chamber is necessary. Furthermore, the
ability to introduce air into the chamber to
minimize initial boiling of the resin during degassing is also necessary.
The decision to process cultured cells as
pellets or as intact monolayers warrants additional comment. While scraping and pelleting monolayers provides a useful approach for
collecting and concentrating a sufficient number of cells for processing, the disadvantage of
this approach is that scraping of monolayers
can damage some cells and disrupt the cellsubstrate interactions, and often cell-cell interactions as well. Figure 2B.1.3 shows an example of virus-infected cultured chicken spleen
cells prepared as a pellet.
When there is a need to evaluate the polarity
of cells and/or cell-cell and cell-substrate interactions, processing of monolayers in chambered slides is a useful, albeit more complicated alternative that poses some special
challenges. Figure 2B.1.4 shows an example of
HeLa cells prepared as a monolayer in chamber slides along with the appearance of the
sample at different stages of the processing
steps, ending with the embedment of the entire monolayer for thin sectioning.
The individual epoxy resinfilled chambers with embedded cells, outlined by the silicone gasket, should come free cleanly from
the glass microscope slide surface. Each individual chamber can be further subdivided
and mounted for sectioning purposes. It is important when processing monolayers to ensure
that there is an adequate exchange of fluids
during dehydration and resin infiltration. Areas of the monolayers near the edges of the
gasket on chambered slides tend to retain fluid
during exchanges, which can inhibit uniform
dehydration and infiltration of the monolayer.
If the monolayer being prepared is not confluent, identification of the areas of embedded
chambers where cells are located can be aided
Microscopy
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Current Protocols in Microbiology
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Figure 2B.1.3 Transmission electron micrograph of cultured chicken spleen cells infected with
avian reticuloendotheliosis virus, embedded using Basic Protocol 4. (A) Clusters of virus particles
(arrows) are seen adjacent to the plasma membrane of an individual spleen cell. (B) Higher
magnication of an area of plasma membrane with shedding virus (arrows) similar to that shown
in A. Bars = 1.0 m (A) and 0.1 m (B).
Transmission
Electron
Microscopy
visualized. En face sectioning of the embedded monolayers allows for the observation of
cells from the attachment surface of the cells
up through the cells to the apical surface of
the cell. While positioning the attachment surface of the monolayer parallel to the knife edge
while sectioning will allow one to serially section through the cells, sectioning on an axis
slightly off parallel will enable both the apical and basal surfaces of the monolayer to be
visualized simultaneously as the block is sectioned. If cross-sections of the monolayer are
2B.1.32
Supplement 3
Figure 2B.1.4 Monolayer processing in chambered slides. (A) The 4-well chambered slide
consists of plastic wells mounted onto a glass slide (1) with a silicone gasket. When the plastic
chamber is removed (2), a thin layer of unpolymerized epoxy resin is added (3) at the thickness
of the silicone gasket (4). Embedded wells containing cells are removed from the glass slide (5)
and pieces of the resin embedded wells are mounted onto plastic pegs for sectioning (6). (B)
Transmission electron micrograph of a HeLa cell monolayer grown and processed for electron
microscopy on a 4-well chambered slide. In this preparation, a cross-section of the monolayer was
prepared following removal of the embedded monolayer from the glass slide, and an additional
layer of fresh resin (5 in panel A) was added to the substrate side. A piece of the monolayer
was then cut with a jewelers saw and mounded on plastic pegs (6 in panel A) for sectioning.
Bar = 0.5 m.
Microscopy
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Current Protocols in Microbiology
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Transmission
Electron
Microscopy
2B.1.34
Supplement 3
Troubleshooting suggestions for immunogold staining of negatively stained preparations are summarized in Table 2B.1.2 and are
generally relevant to immunogold staining of
thin sections. It is important that a suitable
nonspecific binding control be included for all
samples. This should at least consist of a section blocked according to the standard procedure described above and then incubated on
the colloidal gold suspension, eliminating the
primary antibody incubation step. The addition of a grid incubated with a purified IgG
from the same species as that in which the
specific primary antibody was raised and used
at the same concentration as the primary antibody is also appropriate. Before any meaningful assessment of labeling is performed, these
controls must be evaluated for labeling. In addition, the degree of labeling in the portions
of the grid containing only support film and,
if present, plastic without sample, provides an
index of background labeling. While it is not
absolutely necessary to use Formvar-coated
grids in this procedure, they greatly aid the
retention of sections on the grid, as the numerous wash steps in this procedure increase the
chances of losing sections from uncoated grid
surfaces.
Anticipated Results
Negative staining TEM
A successful negatively stained preparation
has sample distributed on all Formvar-coated
grid squares; aggregates of sample and stain
can be easily detected at low magnification
in the electron microscope. Figures 2B.1.5A
and B show low and higher magnification view
of a grid in which a relatively uniform distribution of sample and stain. Figures 2B.1.6A
and B provide representative examples of negatively stained bacteria and bacteriophage.
Immunoelectron microscopy
When successfully labeled and stained,
the negatively stained organisms should be
dispersed onto the grid with colloidal gold
Microscopy
2B.1.35
Current Protocols in Microbiology
Supplement 3
Figure 2B.1.7 Immunogold-labeled and negatively stained preparation of Salmonella typhimurium prepared using Basic Protocol 2. Bacteria were deposited on the grid and incubated
with an anti-mbrial primary antibody; this was followed by incubation with protein Ggold and
negative staining with 1% ammonium molybdate. The 10-nm gold particles (arrows) decorate the
mbrial structures as seen in Figure 2B.1.5 A.
2B.1.36
Supplement 3
Time Considerations
Negative staining TEM
The protocol for negative staining is relatively simple, and, when dealing with an ideal
sample (i.e., high concentration of particulates
in an easily diluted solvent, with a relatively
stable structure in the negative staining solution), grids can be ready for viewing in a TEM
within 30 min to 1 hr. Similarly, when viewing grids with the TEM, recording of sample
images can begin relatively soon after initial
evaluation of the preparation. It is usually necessary to survey the grid at low magnification
to assess the quality of the preparation and
to select areas for higher-magnification examination. Typically, areas with moderate stain
that outlines aggregated sample and/or sometimes debris should be carefully screened.
Suitable specimens for image acquisition can
usually be found within minutes at higher
magnification. Prior to image acquisition, it is
sometimes necessary to stabilize the area of
the grid to be photographed with the electron
beam to reduce movement of the Formvarcoated film. Sample dilution and stain density
can be adjusted empirically; technical problems (e.g., poor spreading of the negative stain,
structural damage to sensitive samples caused
by low pH in samples stained with aqueous
uranyl acetate, or unstable support films) require thoughtful adjustment with subsequent
preparations, which can significantly extend
the duration of the specimen preparation and
examination process.
TEM of thin sections
Successful heavy metal staining of thin sections results in sections that are relatively easy
to visualize on the phosphor screen of the electron microscope. The deposition of sufficient
stain is required to record images, as identification of optimal focus in a TEM is another procedure that requires considerable care
and practice. Figure 2B.1.3 and Figure 2B.1.4
show examples of samples suitably stained
with uranyl acetate and lead. Figure 2B.1.8
provides an example of the use of immunogold
Figure 2B.1.8 Propionibacterium xed and initially processed for immunogold labeling and embedded in LR White resin. In panels (A) and (B) shown at the same magnication, the LR White
embedded sections were labeled rst with a primary monoclonal antibody specic for this particular
isolate of Propionibacterium; this was followed by secondary labeling with 10-nm protein Ggold
(white arrows). Panel A shows inherent specimen contrast without staining uranyl acetate and lead
staining of the section, whereas panel B shows the added contrast obtained by employing those
stains. Note that while the ultrastructural detail of cells is improved with heavy metal staining, the
visualization of gold particles can be more difcult and may require examination of sections at
higher magnication.
Microscopy
2B.1.37
Current Protocols in Microbiology
Supplement 3
Literature Cited
Immunoelectron microscopy
These procedures require extensive manipulation of grids and several incubations, which
will require several hours to complete. It is
therefore desirable to process several girds simultaneously, not only to speed up the process of evaluating samples, but to ensure that
a sufficient number of samples are observed
along with appropriate controls. It is also possible to run several dilutions of antibody and
gold conjugate simultaneously to expedite the
procedure.
Transmission
Electron
Microscopy
Key References
Doane, F.W. and Anderson, N. 1987. Electron
Microscopy in Diagnostic Virology. Cambridge
University Press, New York.
Includes a number of protocols dealing with the use
of electron microscopy in diagnostic virology.
2B.1.38
Supplement 3
Internet Resources
http://www.ncbi.nlm.nih.gov/ICTVdb/#ICTVdB
The Universal Virus Database, ICTVdB, is a resource that provides an Index of Viruses and includes searchable of virus isolates, species, genera,
families, orders, and images of many viruses, as
well as links to genomic and protein databanks.
http://www.polysciences.com/shop/assets/
datasheets/305A.pdf
Polysciences, Inc. Technical Data Sheet no. 305A
for LR White resin.
Microscopy
2B.1.39
Current Protocols in Microbiology
Supplement 3
UNIT 2C.1
2C.1.1
Figure 2C.1.1 Applications of laser scanning microscopy. (A, B). Imaging in thick specimens.
Neurons in a Drosophila embryo were immunolabeled with antibodies against three different
transcription factors (images provided by Dr. Ward Odenwald of the National Institutes of Health,
Bethesda, Md.; reproduced from Kamabadur et al., 1998). (A) A single optical section (2.5-m)
captured with 25, 0.8-NA objective. Labeled neurons in the plane of focus appear sharply dened,
while those outside it are not visualized. (B) A maximum projection of 65 optical sections collected
at 2-m intervals in the z axis. (C) Imaging intracellular structures. Dissociated rat broblasts
were immunolabeled with anti-tubulin antibodies to visualize microtubules (green) and stained
with uorescent probes for mitochondria (Mitotracker, red) and DNA (DAPI, blue). The image is
a projection of 20 optical sections (0.3-m intervals) captured with a 100, 1.4-NA objective. (D)
Measuring molecular mobility in living cells. In a living broblast expressing a Golgi membrane
protein (galactosyltransferase) fused to GFP (S65T), GFP uorescence (green) is localized in
the Golgi complex, shown superimposed on a DIC image of the cell. After the rst image was
collected, the boxed region (yellow) was scanned with full laser power to photobleach the GFP in
the boxed area. The second image was collected 2 sec later. Subsequent images (not illustrated)
showed that the GFP-galactosyltransferase rapidly diffused back into the photobleached area.
Images were captured with a LSM410 (Carl Zeiss, Inc.). For the color version of this gure go to
http://www.currentprotocols.com.
section (Practical Guidelines) provides guidelines for preparing specimens and configuring the critical parameters for confocal imaging. The Commentary provides references to
sources of additional information.
2C.1.2
Current Protocols in Microbiology
Figure 2C.1.2 3-D Imaging in living specimens. Comparison of water- and oil-immersion objectives. (A,B,E,F) Living yeast cells expressing a GFP construct that targets to the mitochondrial
matrix were visualized with C-APO 63 1.2 NA (water) objective (A,B) or a Planapochromat
100 1.4 NA (oil) objective (E,F). The images show xy and yz projections of stacks of 40 images
collected at 0.2-m intervals along the optical axis. The xy projections appear sharper than the
yz projections because the resolution is higher in the focal plane of the objective than along the
optical axis. The yeast were embedded in an aqueous solution with 0.2% agarose. Panels C and
G are yz projections of images of 0.19-m uorescent beads captured with a 63 (water; C) or
100 (oil; G) objective. The beads were embedded in an aqueous solution with 2% agarose. D
and H are intensity proles along the horizontal and vertical axes of the beads. A 63 1.2 NA
(water) objective provides better axial resolution than an 100 1.4 NA objective (oil) in specimens
in an aqueous solution. Scale bars = 5 m (A,B,E,F); 0.5 m (C,G). Images were captured with
a LSM510 (Carl Zeiss, Inc.).
emit fluorescence in all directions. The objective captures a portion of the emitted light. The
objective projects light from the focal spot in
the specimen to a conjugate spot in an image plane. The pinhole aperture is positioned
in the image plane so as to be centered on this
spot. The light that passes through the aperture
is detected by a photomultiplier tube (PMT).
Light from out-of-focus areas of the specimen
is spread out at the image plane and is largely
blocked by the pinhole aperture.
The diameter of the pinhole determines how
much of the fluorescence emitted by the illuminated cone in the specimen is detected, as
well as the thickness of the optical section.
From wave optics, it is known that a point light
source in the plane of focus of an objective produces a three-dimensional diffraction pattern
in the image plane. The cross-section at the image plane is an Airy disk, a circular diffraction
pattern with a bright central region. The radius
of the bright central region of the Airy disk in
the reference frame of the specimen is given by
RAiry = 0.61/NA where is the emission
wavelength and NA is the numerical aperture
of the objective (Inoue and Spring, 2002). At
the image plane, the radius of the central region is RAiry multiplied by the magnification
at that plane (Wilson, 1995).
Adjustment of the pinhole to a diameter
slightly less than the diameter of the central region of the Airy disk allows most of
the light from the focal point to reach the
detector and reduces the background from
out-of-focus areas by 1000-fold relative to
wide-field microscopy (Sandison et al., 1995).
The separation of the in-focus signal from the
out-of-focus background achieved by a properly adjusted pinhole is the principal advantage of confocal microscopy for examination
of thick specimens.
Point illumination and the presence of a
pinhole in the detection light path also produce
improved lateral and axial resolution relative
to conventional microscopy (Table 2C.1.1).
The actual extent of improvement depends on
the size of the pinhole. Near-maximal axial
resolution is obtained with a pinhole radius of
0.7 RAiry , whereas optimal lateral resolution is obtained with a pinhole smaller than
0.3 RAiry (Wilson, 1995). However, a pinhole smaller than 0.7 RAiry significantly
Microscopy
2C.1.3
Current Protocols in Microbiology
Figure 2C.1.3 Applications of confocal microscopy in microbial research. (A, B). Human red
blood cells (RBC) infected with malaria parasites (Plasmodium falciparum; 3D7 strain). Biotinylated
human RBC were labeled with streptavidin-conjugated Quantum Dots 525 (CA; green color;
http://www.qdots.com) and 0.5- to 4-M FM-64 (Molecular Probes, red color). The cells were
injected into chambers (HybriWell HBW20 from Grace Bio-Labs) and were examined with an LSM
510 confocal microscope (Carl Zeiss, Inc.) with a 100 1.4-NA oil objective. Panel A shows two
RBC, one containing a parasite at the trophozoite stage; panel B shows a parasite at the schizont
stage. The schizont was extruded from the RBC for better imaging of individual parasites (red).
The green signal in the center of the schizont represents autouorescence from hemazoin in
the digestive vacuole. Images were provided by Dr. Svetlana Glushakova (National Institutes of
Health, Bethesda, Md.; methods described in Glushakova et al., 2005). (C, D) Biolm composed
of microcolonies of nitrifying bacteria (ammonia oxidizers; Nitrosomonas sp.) and nitrite oxidizers
(Nitrospira sp.). Both populations were labeled by uorescence in situ hybridization (FISH) with
16S rRNA-targeted oligonucleotide probes. Nitrosomonas colonies are green; Nitrospira colonies
are red. Images are 3-D reconstructions created using the Daime imaging program (see Daims
et al., 2006, and Internet Resources) from stacks of optical sections collected with an LSM 510
(Carl Zeiss, Inc). Panel C shows an overview of the projected image of the biolm, while panel D
shows a smaller region at higher zoom. Images were provided by Dr. Holger Daims (Universitat
Wien, Vienna, Austria). For the color version of this gure go to http://www.currentprotocols.com.
Basic Confocal
Microscopy
2C.1.4
Current Protocols in Microbiology
Figure 2C.1.4 The basis of optical sectioning. Illumination from a point light source is reected
by a dichroic mirror into the back aperture of a microscope objective. The objective lens focuses
the light to a diffraction-limited spot within the specimen. Fluorophores at the focal spot and within
the cones of illumination above and below it are excited, emitting uorescence in all directions.
The uorescence captured by the objective passes through the dichroic mirror because the uorescence is at a longer wavelength than the excitation. The confocal pinhole allows uorescence
from the focal spot to reach the photodetector and blocks uorescence from out-of-focus areas.
Redrawn from Shotton (1993).
Table 2C.1.1 Theoretical Resolutions of Confocal and Conventional Microscopesa, b
Objective
x /em
Ax. res.
Lat. res.
Ax. res.
Lat. res.
Ax. res.
0.55
4.50
0.26
0.99
0.16
0.56
568/590
0.64
5.17
0.30
1.09
0.18
0.64
647/677
0.72
5.88
0.34
1.28
0.21
0.72
0.79
6.48
0.37
1.43
0.24
0.93
590
0.90
7.38
0.42
1.63
0.28
1.06
680
1.04
8.50
0.49
1.88
0.32
1.22
ex
and em , excitation and emission wavelengths; lat. res. and ax. res., lateral and axial resolutions.
Microscopy
2C.1.5
Current Protocols in Microbiology
Figure 2C.1.5 The light path of a laser-scanning confocal microscope. The diagram illustrates
the light path of a LSCM set up for simultaneous imaging of FITC and lissamine rhodamine. The
488- and 568-nm lines of a krypton-argon laser are reected by dichroic beam splitter 1 into the
optical axis of the microscope. The beam is reected by a mirror into the microscope objective,
which focuses the beam to a diffraction-limited spot in the specimen. The scanner consists of a pair
of galvanometer mirrors that deect the laser beams so as to scan the spot across the specimen
in a raster pattern. Fluorescence emitted as each point is illuminated travels the reverse path
through the scanning system. The FITC uorescence (peak at 520 nm) and lissamine rhodamine
uorescence (peak at 590 nm) pass through dichroic beam splitter 1 to dichroic beam splitter
2, which transmits the lissamine rhodamine uorescence to photomultiplier tube 1 and reects
the FITC uorescence to photomultiplier tube 2. A variable pinhole in front of each photodetector
blocks light from out-of-focus areas of the specimen while allowing light from the focal plane to
reach the detector.
CONFIGURATION OF AN LSCM
Basic Confocal
Microscopy
Confocal microscopes use lasers for illumination because they provide intense excitation within a narrow range of wavelengths.
Mixed krypton-argon gas lasers are popular
for multicolor confocal microscopy because
they emit at three wavelengths (488, 568, and
647 nm) that excite many commonly-used
fluorophorese.g., FITC, rhodamine, Cy3,
Cy5, Alexa 488/555/568/647, green fluorescent protein (GFP), and red fluorescent protein (mRFP or DsRed). The disadvantage of
krypton-argon lasers is that their life spans are
short (2000 hr). Another way to achieve multiwavelength excitation is to combine the outputs of multiple lasers. Many of the confocal
microscopes currently on the market combine
an argon laser (488 nm) with a green heliumneon (HeNe) laser (543 or 594 nm) and a red
HeNe laser (633 nm). The argon laser also
may provide 458 and 514 nm lines, which can
be used to excite the cyan and yellow variants
of GFP (CFP and YFP). Some confocal microscopes can accommodate a 405-nm diode
2C.1.6
Current Protocols in Microbiology
front of the detectors. Incorporation of a separate pinhole for each detector allows the user
to optimize the pinhole settings for different
wavelengths.
The photodetectors in LSCMs are photomultiplier tubes (PMTs), which generate electrons at a rate proportional to the intensity of
the incoming fluorescence signal (Art, 1995).
The PMT output is converted to a digital image
that can be displayed on a computer monitor
and stored as a digital file for later analysis.
Digitization may be at 8-bit (256 gray levels), 10-bit (1024 gray levels) or 12-bit resolution (4096 gray levels). Confocal microscopes
typically have two to four PMTs for reflected
light/epifluorescence imaging and may have,
in addition, a photodetector for transmitted
light.
In spinning disk confocal microscopes, the
illumination from a laser or white light source
passes through pinholes in the Nipkow disk so
as to excite fluorescence at multiple (1000)
sites within the specimen. The disk revolves
rapidly (1000 to 5000 rpm) causing the illuminating spots to sweep across the specimen as uniformly-spaced scan lines (Inoue and
Inoue, 2002). Fluorescence emitted by the
specimen that is collected by the objective returns through the same pinholes in the Nipkow
disk that provided the excitation light before
it is detected by a full-field CCD camera. In
this way, point light sources and detector pinholes to block out-of-focus fluorescence are
provided, with the advantage of higher collection speeds than a spot scanner. Drawbacks
of this approach include decreased illumination to the specimen from light loss through
the pinholes and the inability to change the
pinhole diameter. This means that, unlike the
case with a spot scanner, optimal confocality is achieved only for one objective magnification, and the thickness of the optical section cannot be changed. Although the reduced
specimen illumination generates a smaller fluorescent signal, scientific-grade CCD cameras
have significantly higher quantum efficiencies
than the PMTs used for fluorescence detection in LSCMs and are able to more than adequately detect these levels of fluorescence.
Fluorescent specimens have been reported to
undergo less photobleaching during examination with a spinning disk confocal microscope
than with a LSCM. The lower rate of photobleaching is thought to be due to the lower
illumination levels (Inoue and Inoue, 2002).
A new type of slit-scanning confocal microscope (LSM 5 Live; Carl Zeiss, Inc.) that
allows images to be acquired at rates as fast
Microscopy
2C.1.7
Current Protocols in Microbiology
PRACTICAL GUIDELINES
Sample Preparation
The preeminent goal in preparing samples
for imaging with a confocal microscope is to
maximize the fluorescence signals while preserving the three-dimensional structure of the
specimen. Ideally, the sample should be less
than 50 m in thickness, although thicker
samples can be visualized. Guidelines for
preparing fixed and living samples are described below.
Fixation
Basic Confocal
Microscopy
Choices of fluorophores
The choice of fluorophores should take into
account the available laser lines and the detector channels of the confocal microscope. Excitation is most efficient at wavelengths near
the peak of the excitation spectrum of the
fluorophore, but a precise match is not required. For experiments that require imaging
multiple fluorophores with standard photodetectors (PMTs), it is best to select fluorophores
that are excited by different laser lines, in
order to minimize spectral crossover (bleedthrough) between the channels (Fig. 2C.1.6).
Excitation and emission spectra for many fluorophores are available via the Internet (see
Internet Resources). A recommended combination of fluorophores for excitation at 405
nm, 488 nm, 543/561 nm, and 633 nm would
comprise Marina Blue, Alexa 488, Alexa 555,
and Alexa 647 (all available from Molecular Probes/Invitrogen). The nucleic acid stain
DAPI can be excited by illumination at 405
nm, although ultraviolet excitation (350 nm)
is more optimal. The cyanine dyes Cy2,
Cy3, and Cy5 (available from Jackson ImmunoResearch Laboratories) are also suitable
for confocal microscopy. For multiwavelength
imaging with a spectral detector and spectral
unmixing, it is important to select fluorophores
that have distinct emission spectra, but there
is no advantage in using fluorophores that
have differing excitation spectra. Indeed, it
is best to use fluorophores that have similar excitation maxima, so that they can be
excited with a single laser line reflected
into the microscope with a single-wavelength
dichroic mirror. Other important criteria to
consider in selecting fluorophores for confocal
microscopy are the quantum efficiencies and
rates of photobleaching. In addition, the
staining protocol should be designed so as
to produce similar signal intensities in each
channel. More information about selecting fluorophores for confocal imaging is available at
the Molecular Expressions Web site (see Internet Resources).
2C.1.8
Current Protocols in Microbiology
Control samples
Confocal microscopes rely on electronic
image enhancement techniques that can make
even dim autofluorescence signals or nonspecific background staining look bright. In
order to distinguish a real signal from background, it is essential to prepare and examine
appropriate control samples. For immunofluorescence experiments with one primary antibody, the appropriate control samples are
unstained specimens and specimens treated
with the secondary antibody but no primary
antibody. Other control experiments may be
required to verify the specificity of labeling.
Experiments with two primary and secondary
Microscopy
2C.1.9
Current Protocols in Microbiology
Medium
RI
Immersion media
Air
1.00
Water
1.338
Glycerol
1.47
Immersion oil
1.518
Mounting media
50% glycerol/PBS/DABCO
1.416a
1.474a
1.473a
1.458a
1.415b
1.3865b,c
c Refractive index (RI) for liquid medium. (RI for solidified medium will be higher.)
Basic Confocal
Microscopy
Living specimens
Microscopy on living specimens grown in
vitro is most conveniently performed with an
inverted microscope, because the specimens
can be viewed through the bottom of the
culture chamber and the top can be opened
for access. To allow imaging with an oil- or
water-immersion objective, the culture chamber substrate should be a coverglass. The glass
coverslip can be coated with poly-L-lysine
(using a 1 mg/ml solution; Sigma) to promote adhesion of the specimens (either
eukaryotic cells or microorganisms). Nonadherent specimens can be immobilized by embedding them in a thin layer of low-meltingpoint agar (0.2% for eukaryotic cells, up to
2% for smaller organisms). Motile specimens
2C.1.10
Current Protocols in Microbiology
Microscopy
2C.1.11
Current Protocols in Microbiology
Pinhole size
As explained in the section on the Basis of
Optical Sectioning, the size of the detector pinhole has a critical influence on image quality.
A pinhole with a diameter slightly less than
or equal to the diameter of the bright central
region of the Airy disk will let most of the
light from the plane of focus reach the detector, while blocking most of the out-of-focus
flare. The lateral resolution will be 10% better than that obtainable by conventional microscopy with the same optics (Centonze and
Pawley, 1995), although not as good as can be
achieved with a smaller pinhole. Lateral resolution continues to improve as pinhole radius
is decreased down to a pinhole size of 0.2
Airy disk radius, but a pinhole this small excludes 95% of the signal (Wilson, 1995).
Axial resolution improves as pinhole size decreases, down to 0.7 Airy disk radius, then
levels off. The best trade-off between signal
intensity and resolution will depend on the
characteristics of the sample and the required
resolution.
Scan zoom
Basic Confocal
Microscopy
Illumination intensity
Fluorescence emission increases linearly
with illumination intensity up to a level at
which emission saturates. Optimal signalto-background and signal-to-noise ratios are
obtained with illumination levels well below
saturation (Tsien and Waggoner, 1995). The
illumination intensity on a laser-scanning microscope can be adjusted by operating the
laser at submaximal power and by inserting
neutral-density filters into the light path or
varying the transmission through the AOTF.
In general, the best images are obtained with
illumination levels that are as high as possible without producing unacceptable rates of
photobleaching.
2C.1.12
Current Protocols in Microbiology
Averaging
Confocal images of dimly fluorescent specimens captured at the fastest scan rate on a
typical LSCM (0.5 sec/frame) appear noisy
because of the small numbers of photons collected from each spot. Improved signal-tonoise can be attained by scanning the specimen
at a slower rate or by scanning multiple times
and averaging the signals. Current LSCMs allow individual lines in the image to be repeatedly scanned and averaged. Line averaging generally produces sharper images than
frame averaging (which averages full frames)
because there is less risk of blurring due to
movements or changes in the specimen.
Image display
Confocal images are typically displayed as
8-bit grayscale or 24-bit RGB (red/green/blue)
color images. Each channel of an RGB image can represent a different fluorophore (Fig.
2C.1.1A to C). Color mixtures indicate colocalization of fluorophores within a pixel. A
RGB fluorescence image can be merged with
a grayscale transmitted light image by adding
the transmitted light image to each channel of
the RGB image (Fig. 2C.1.1D; fluorescence in
green channel merged with DIC image).
The three-dimensional dataset obtained by
capturing a series of optical sections through
the specimen can be used to compute views
of the specimen from different viewing angles. Commercial confocal microscopes typically include the capability to generate orthogonal views of the specimen (xy, xz, and
yz) and may permit views from arbitrary angles. An xy projection or z-series projection
is a two-dimensional display formed by merging multiple image planes (Fig. 2C.1.1B,C,
Fig. 2C.1.2A,E). The most common type
of projection is a maximum projection in
which each pixel represents the intensity of
the brightest pixel in the z-axis. Another type
of projection, referred to as surface render,
displays the most superficial pixels with intensities above a defined threshold. Projections also can be created for different viewing angles (Fig. 2C.1.2B, F). Projections of
the specimen from different viewing angles
can be combined to create an animation in
which the specimen appears to rotate in space.
Such animations give the viewer a striking
impression of the three-dimensional geometry of the specimen. Generating two projections at azimuths differing by 4 to 10 creates stereo pairs that can be visualized with
a stereo viewer or color-coded and merged
to form a stereo anaglyph. Volume-rendering
Microscopy
2C.1.13
Current Protocols in Microbiology
imaging software is available that provides additional options for three-dimensional visualization and measurements (see Internet Resources).
COMMENTARY
Basic Confocal
Microscopy
2C.1.14
Troubleshooting
Test samples are useful for monitoring the
performance of a confocal microscope. A micrometer slide should be used to check the spatial calibration of each objective. Fluorescent
microspheres with mixtures of fluorophores
(FluoSpheres; Molecular Probes) are useful
for checking the x,y and axial alignment of
images acquired at different excitation wavelengths. Misalignment of the images in the xy
plane may indicate that the pinholes are not
centered or that the lasers need to be aligned;
misalignment in the z axis may be due to incorrect setting of a collimating lens, misalignment
of pinholes, or chromatic aberration in the objective. The optical resolution of the microscope can be measured by capturing images of
submicroscopic (<0.2 nm) fluorescent microspheres (Fig. 2C.1.2C,G). The images of the
microspheres should be radially symmetrical
in the xy plane and elliptical in the z-axis (Fig.
2C.1.2C). Horizontal and axial resolutions are
defined by the full width at half maximal intensity (FWHM) of intensity profiles along the
horizontal and vertical axes of the beads (Fig.
2C.1.2D,H).
Anticipated Results
Confocal microscopy provides sharp images of fluorescent structures in thick specimens (Fig. 2C.1.1, Fig. 2C.1.2, Fig. 2C.1.3C,
D). The maximum depth at which adequate
images can be obtained depends on the objective and the optical properties of the specimen.
With a high-NA immersion objective, it may
be possible to capture images at depths of >100
m in a specimen that is transparent and not
heavily stained (Centoze and Pawley, 1995).
However, if the specimen scatters light, both
the illumination intensity and the proportion
Current Protocols in Microbiology
Microscopy
2C.1.15
Current Protocols in Microbiology
Acknowledgements
The author wishes to thank Dr. Albert
Neuztner (National Institutes of Health,
Bethesda, Md.) for providing the cells illustrated in Fig. 2C.1.3 and Dr. James Galbraith
(also of the National Institutes of Health) for
helpful comments on the manuscript. This research was supported by the Intramural Research Program of the National Institute of
Neurological Diseases and Stroke at the National Institutes of Health, Bethesda, Md.
LITERATURE CITED
Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno,
H., and Miyawaki, A. 2002. An optical marker
based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl.
Acad. Sci. U.S.A. 99:12651-12656.
Basic Confocal
Microscopy
Art, J. 1995. Photon detectors for confocal microscopy. In Handbook of Biological Confocal
Microscopy, 2nd ed. (J. Pawley, ed.) pp. 183196. Plenum, New York.
2C.1.16
Current Protocols in Microbiology
Microscopy
2C.1.17
Current Protocols in Microbiology
http://www.uhnres.utoronto.ca/facilities/
wcif/imagej/
A manual written by Tony Collins that describes
the use of ImageJ to visualize and analyze confocal
images.
http://www.molecularexpressions.com
The Molecular Expressions Web site is a rich source
of information about all aspects of light microscopy,
including confocal microscopy. It includes sections
on the basic principles of confocal imaging, instrumentation, sample preparation, and choices of fluorophores. An interactive tutorial Choosing fluorophore combinations for confocal microscopy
allows the user to determine the extent of spectral cross-over that will occur when imaging different combinations of fluorophores with specific laser
lines and filter sets.
KEY REFERENCES
http://www.nikonusa.com
Nikon, Inc.
Covers the basics of light microscopy, video microscopy, and much more.
http://www.microscopyu.com
http://www.olympusconfocal.com/
Olympus, Inc.
http://www.perkinelmer.com/
http://www.solameretech.com/
PerkinElmer, Inc.
Solamere Technology.
INTERNET RESOURCES
http://www.microbial-ecology.net/daime
Daime Web site, from which the Daime software application can be downloaded. Daime (digital image
analysis in microbial ecology) is an open-source
software program for 2-D and 3-D image analysis
developed by Holger Daims, Sebastian Lucker, and
Michael Wagner (Universitat Wien, Vienna, Austria). The features of Daime and its application to
analysis of biofilms are described in Daims et al.
(2006).
http://rsb.info.nih.gov/ij
Basic Confocal
Microscopy
ImageJ is a public domain image analysis program developed by W. Rasband (Research Services Branch, National Institute of Mental Health,
NIH) for operating systems running Java (including
Windows/PC and OSX/Macintosh). ImageJ has
many useful tools for analysis of confocal images.
Spectra of uorophores
http://fluorescence.nexus-solutions.net/
frames6.htm
Biorad Microsciences fluorochrome database and
charting application.
http://home.earthlink.net/fluorescentdyes/
George McNamara Multiprobe Microscopy.
http://www.probes.com/
Molecular Probes.
http://www.molecularexpressions.com
Molecular Expressions.
http://www.olympusfluoview.com/resources/
specimenchambers.html
Sources of chambers for maintaining living specimens during observation by microscopy.
2C.1.18
Current Protocols in Microbiology
http://listserv.buffalo.edu/user/sub.html
Many topics of interest to confocal microscopists
are discussed on the confocal listserver operated
by the listserver at the University at Buffalo. To
subscribe to the lis,t go to the URL and type confocal in the box that asks which list one wishes to
join.
Microscopy
2C.1.19
Current Protocols in Microbiology
UNIT 2C.2
ABSTRACT
The atomic force microscope (AFM) is an important tool for studying biological samples due to its ability to image surfaces under liquids. The AFM operates by physical
interaction of a cantilever tip with the molecules on the cell surface. Adhesion forces
between the tip and cell surface molecules are detected as cantilever deflections. Thus,
the cantilever tip can be used to image live cells with atomic resolution and to probe
single molecular events in living cells under physiological conditions. Currently, this is
the only technique available that directly provides structural, mechanical, and functional
information at high resolution. This unit presents the basic AFM components, modes of
operation, useful tips for sample preparation, and a short review of AFM applications
C 2008 by John Wiley &
in microbiology. Curr. Protoc. Microbiol. 8:2C.2.1-2C.2.17.
Sons, Inc.
Keywords: AFM r imaging r force curves
INTRODUCTION
Understanding the function of microbial cell surfaces requires knowledge of their structural and physical properties. Most of the classical methods used to investigate these
structures (e.g., electron microscopy, X rays) require cell drying, which may compromise the accuracy of the analysis by denaturing the surfaces (Dufrene, 2002, 2003).
Because of microorganisms small size, the information is generally obtained from a
large number of cells and not at the individual cell level. Thus, there is a need for new,
nondestructive technologies capable of probing single cell surfaces at high resolution.
The atomic force microscope (AFM) has emerged as a nondestructive technique, offering resolution comparable to electron microscopy but allowing the study of biological
material in its native environment. AFM was invented in 1986 by Binnig et al. (1986) as
a novel technique that allows imaging of surfaces and force measurements at the atomic
scale, whether the sample is in air or under fluid. This last property makes it very attractive as a tool for biological applications for live cell imaging (Radmacher et al., 1992;
Lal and John, 1994) and single molecule force spectroscopy (Willemsen et al., 2000;
Wojcikiewicz et al., 2004; Trache et al., 2005), providing structural, mechanical, and
functional information with high resolution under physiological conditions. Biologists
are using the AFM in an increasing number of creative ways, and the referenced literature
is vast (see Key References). This unit presents the basic AFM components and modes of
operation, gives useful tips for sample preparation, and provides a short review of AFM
applications in microbiology.
PRINCIPLE OF OPERATION
The principle of operation of the AFM is very similar to that of a stylus profilometer: a
sharp cantilever tip interacts with the sample surface, sensing the local forces between
the molecules of the tip and sample surface (Fig. 2C.2.1). This instrument is not a
conventional microscope that collects and focuses light. The word microscope has been
Microscopy
Current Protocols in Microbiology 2C.2.1-2C.2.17, February 2008
Published online February 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc02c02s8
C 2008 John Wiley & Sons, Inc.
Copyright
2C.2.1
Supplement 8
Figure 2C.2.1 Schematic illustration of the AFM system. A exible cantilever with a tip at the
end is rigidly connected to an xyz piezoelectric element. The optical lever consists of a laser diode
beam that is focused on the back of the cantilever and bounces off, reaching the quadrant photodetector. In contact mode the xyz position of the cantilever is given by recording the corresponding
position of the piezoelectric element, and the detector reading is proportional to the cantilever
deection. In tapping mode the AFM cantilever is vibrated at its resonant frequency under an
external electrical excitation (dashed line).
associated with this instrument because it is able to measure microscopic features of the
sample. The most characteristic property of the AFM is that the images are acquired
by feeling the sample surface without using light. A common analogy is to imagine
a blindfolded person who perceives the characteristics of a surface just by touching it.
In this way, not only the sample topography can be recorded with atomic resolution,
but also material characteristics and the eventual strength of the interaction between the
sample surface and the AFM tip. Due to the fact that no light is involved in acquiring the
sample properties, the resolution achieved by the AFM is not limited by the wavelength
of the radiation that investigates the sample, as in classical light microscopy. Thus, the
AFM reaches a resolution far below the diffraction limit offered by light microscopy,
being limited only by the tip radius and the spring constant of the cantilever.
The main components of an AFM (Gad and Ikai, 2001) consist of the AFM probe (a
sharp tip mounted on a soft cantilever), the optical lever that allows for measuring the
cantilever deflections, the feedback loop that allows for monitoring the interaction forces
between the molecules on the tip with the ones on the cell surface, the piezoelectric
scanner that moves the tip in respect with the sample in a 3D pattern, and a conversion
system from raw data acquired by the instrument into an image or other useful display.
A short overview of the system components is presented below.
Atomic Force
Microscopy
The most sensitive part of an AFM is the tip that interacts directly with the sample
surface. The most common probes are constructed from silicon or silicon nitride using
microfabrication techniques (Binnig et al., 1987). Each probe can have multiple integrated
cantilevers (Fig. 2C.2.2). The properties and dimensions of the cantilever and tip play a
major role in determining the sensitivity and resolution of the AFM (Morris et al., 1999).
For special applications the pyramidal tip can be replaced with glass or polystyrene beads
with a diameter of 1 to 5 m. The AFM tip must be chosen carefully depending on the
specific application. In general, the cantilever should be soft enough to be deflected at
2C.2.2
Supplement 8
Figure 2C.2.2 Scanning electron microscope images of AFM cantilevers. (A) Typical AFM probe
for force measurements (magnication 45). At the very end of the cantilever is a pyramid-shaped
tip with a 4-m base, 35 half angle of the tip, and a radius of 50 nm (inset, magnication 16,000).
(B) Typical AFM probe for mechanical stimulation measurements (magnication 10,000). The
cantilever is V-shaped, with a spring constant of 0.06 N/m and a 2-m glass bead attached at
its end (inset, magnication 10,000). Images were taken at the Microscopy & Imaging Center of
TAMU, College Station, Texas. Reproduced from Trache and Meininger (2005) with permission of
the International Society for Optical Engineering.
Table 2C.2.1 Most Common Cantilever Parameters
Cantilever material
Silicon nitridea
Silicona
Resonant frequency
20-100 N/m
200-400 kHz
Cantilever
configuration
V-shaped
Reflective coating
Single beam
gold
Uncoated, aluminum
b
Tip shape
Pyramid
Sphere
glass/polystyrene
Pyramid
Tip radius of
curvature/diameter
20-60 nm
0.6-45 mb
5-10 nm
Sidewall angles
a Adapted from SPM Training Notebook (2003) with permission of Veeco Instruments.
b Parameters from Novascan Technologies.
very small forces, and the tip radius should be comparable with the features of interest.
Table 2C.2.1 presents the properties of the most common cantilevers.
Use of the optical lever is the most common technique for detecting the cantilever
deflection. A laser beam coming from a laser diode is reflected off the back of the
cantilever onto a photodetector. The photodetector consists of a split photodiode that
senses the change in light intensity from the reflected beam due to changes in the
deflection of the cantilever following tip-sample interaction. The sensitivity of such a
system is in the range of 0.1 nm (Meyer and Amer, 1990).
Microscopy
2C.2.3
Current Protocols in Microbiology
Supplement 8
The AFM scanners are made from piezoelectric material, which expands or contracts
proportionally with an applied voltage. The piezoelectric scanner can move very precisely
with very good reproducibility for small displacements, but for displacements >70% of
the full-scale displacement, the piezoelectric response is nonlinear. The commercially
available AFM featuring closed-loop feedback systems independently monitors the scanner movement and corrects its motion for nonlinearity. There are two types of scanner
configurations: scanned tip AFM (where the piezoelectric scanner is rigidly attached
to the probe and is moved over the sample surface, which stands still) and scanned
sample AFM (where the scanner is attached to the sample and is moved under the tip).
Both designs could be integrated with optical microscopes or used as stand-alone AFM
systems.
A dual monitor computer interface allows for display of data (images or force curves) on
the display monitor and the parameters of the system on the control monitor.
Atomic Force
Microscopy
2C.2.4
Supplement 8
Figure 2C.2.3 Diagram of generic force curves. Approach (black thin line) and retraction force
curves with (black thick line) and without (middle line) adhesions are presented. The x axis
represents the piezoelectric element displacement, and the y axis represents the force (see text
for details).
measurements of the relative stiffness of the cell surface (approach curve) and adhesion
force measurements between the biologically functionalized AFM tip and the cell surface
(retraction curve) are acquired (Fig. 2C.2.3). When the probe is extended towards the
cell surface (a), a contact point is established with the cell (b), and thereafter the cell
surface is indented. Due to cell stiffness, further probe extension causes an opposing
force of increasing magnitude to be generated along with increasing indentation in the
cell membrane (b to c). The upward deflection of the cantilever as it bends in response
to this force, results in an increasing deflection signal. When the probe retracts from
the sample (c to d), the force between probe and sample gradually decreases until the
cantilever returns to the original position, at which time the deflection signal returns to
the original value (a). However, if adhesions occurred between the probe and sample
surface, the force causes the cantilever to bend downward, and the deflection signal will
be lower than the original value (e to g). When all adhesions are broken, the cantilever
returns to the original position (a), and the deflections (unbinding of adhesion events) are
recorded on the retraction force curve. The adhesion force is calculated as the product of
the deflection height associated with the unbinding event and the spring constant of the
cantilever.
On soft samples such as cells, the force application results in an indentation of the
cell that is a measure of the local elastic properties of the cell membrane. Due to this
elasticity of the cell membrane, only a portion of the piezoelectric element movement
causes deflection of the probe; the remainder results in indentation of the cell membrane.
The membrane indentation part of the approach curve could be analyzed using different
models (Sneddon, 1965; Costa and Yin, 1999) in order to determine the local values of
the Young modulus of elasticity as a measure of the local apparent elasticity of the cell
membrane.
PRACTICAL GUIDELINES
Although AFM has great potential in microbiological studies, it is important to understand
that accurate data collection is sometimes difficult due to the technical limitations associated with the technique itself. Images produced by AFM always represent a convolution
of the tip shape with the sample features. It is important to correlate the tip geometrical
Microscopy
2C.2.5
Current Protocols in Microbiology
Supplement 8
Figure 2C.2.4 Schematics of tip artifacts. (A) The smaller the tip radius of curvature, the smaller
the sample feature that can be resolved. A dull or dirty (B) tip will affect the image prole. Adapted
from SPM Training Notebook (2003) with permission of Veeco Instruments.
features with the expected features of the sample to obtain the best images. A tip that is
too large will not be able to record features in the sample smaller than the tip parameters
(Fig. 2.C2.4A). Also, a contaminated, broken, or blunt tip will introduce distortions into
an image, affecting its sharpness or even introducing noise, with the geometry of the tip
dominating the geometry of the sample (Figure 2.C2.4B). A detailed description of AFM
artifacts is presented at http://www.pacificnanotech.com/afm-artifacts single.html.
Atomic Force
Microscopy
Sample Immobilization
One of the most important aspects of imaging live cells under physiological conditions
using the AFM is sample immobilization. If the sample is not firmly attached to the
bottom of cell culture dish, the AFM tip can easy lift the sample. In this case the tip
will break or get contaminated in the process, and no measurements can be performed.
Most live cells (e.g., smooth muscle cells, endothelial cells, fibroblasts) attach well
to classical substrates like polystyrene or glass. Some cells (e.g., myocytes) can be
problematic for AFM measurements due to their intrinsic movement or poor attachment
to the substrate. Coating the cell culture dish with gelatin or an extracellular matrix
protein (e.g., fibronectin, laminin) can considerably enhance the cell attachment. Live cell
imaging can be performed at room temperature or 37 C depending on the experimental
requirements. For imaging single bacteria, yeast, or fungal cells under physiological
conditions, a concentrated cell suspension is absorbed through an isopore polycarbonate
membrane with a pore size comparable to the cell size (Kasas and Ikai, 1995; Dufrene
et al., 1999). Imaging of reconstituted microbial layers or DNA is performed by absorbing
the layers of interest on a flat substrate, i.e., fresh cleaved mica or glass (Muller et al.,
1997; Colton et al., 1998). Other immobilization methods involve air-drying the sample
(a droplet of a concentrated cell suspension is placed on a mica or glass substrate and
2C.2.6
Supplement 8
allowed to dry in air; Amro et al., 2000) or covalently bonding bacteria to silanized glass
slides (Camesano et al., 2000).
2C.2.7
Current Protocols in Microbiology
Supplement 8
Organism
Sample
Bacillus coagulans
S-layer
Covalent linkage to
glass/mica
Oblique lattice; r 10 nm
(study of antibody binding)
Ohnesorge et al.
(1992)
Recrystallization on
silanized silicon
Covalent linkage to
glass/mica
Square lattice; r 12 nm
(agreement with electron
microscopy)
Ohnesorge et al.
(1992)
Recrystallization on
silanized silicon
Recrystallization on
supported lipid bilayers
Recrystallization on
various silicon surfaces
Ring-shaped hexamers; r = 1
nm (correlation with electron
microscopy)
Adsorption on mica
Conformational change of
central pores
Adsorption on HOPG
Bacillus
stearothermophilus
S-layer
Deinococcus
radiodurans
Reference
Halobacterium
laobium
Purple
membrane
Adsorption on mica,
silanized glass, and
supported lipid bilayers
Hexagonal symmetry; r =
1.1 nm
Halobacterium
salinarum
Purple
membrane
Adsorption on mica
Escherichia coli
Escherichia coli
Porin trimers; r = 1 nm
(comparison with X-ray
structure; detection of two
conformations)
a Reproduced from Dufrene (2002) with permission of the American Society for Microbiology.
b r-values are lateral resolutions determined directly from the images or after image processing.
Atomic Force
Microscopy
2C.2.8
Supplement 8
Organism
Observations
Reference
Saccharomyces cerevisiae
Dufrene (2002)
Phanerochaete chrysosporium
Aspergillus oryzae
Lactococcus lactis
Streptococcus salivarius
Pseudomonas aeruginoas
Pseudomonas putida
a Reproduced from Dufrene (2002) with permission of the American Society for Microbiology.
Microscopy
2C.2.9
Current Protocols in Microbiology
Supplement 8
Table 2C.2.4 Probing Cell Surface Properties, Molecular Interactions, and Elasticitya
Organism
Method
Results
References
Escherichia coli
Force-distance curves
with cell-coated probes
Shewanella oneidensis
Force-distance curves
with cell-coated probes
Burkholderia cepacia,
Pseudomonas putida
Force-distance curves
Electrostatic and steric interactions
with silicon nitride probes
Phanerochaete
chrysosporium
Force-distance curves
with chemically
functionalized probes
Dufrene (2000)
Magnetospirillum
gryphiswaldense
Streptococcus salivarius
Methanospirillum hungatei
Depression technique
Xu et al. (1996)
Escherichia coli,
Pseudomonas aeruginosa
Depression technique
Deinococcus radiodurans
Aspergillus oryzae
a Reproduced from Dufrene (2002) with permission of the American Society for Microbiology.
Figure 2C.2.7 shows high-magnification images of individual virions that exhibit a surface appearing to be an open network with large spaces of almost hexagonal shape.
Atomic Force
Microscopy
2C.2.10
Supplement 8
Figure 2C.2.5 Height and deection images of A. naeslundii genospecies 1 (ATCC #12104) adherent to the
mica substrate after a 5-day period of anaerobic growth. The shape of the outer bacterial surface and the typical
diptheroidal arrangements (V, Y, or T forms and palisades) of Actinomyces species are seen in these images.
Reprinted from Archives of Oral Biology, vol. 49. Tang, G., Yip, H.K., Samaranayake, L.P., Chan, K.Y., Luo, G., and
Fang, H.H.P. Direct detection of cell surface interactive forces of sessile, mbriated, and nonmbriated Actinomyces
spp. using atomic force microscopy. pp. 727-738. Copyright 2004, with permission from Elsevier.
Figure 2C.2.6 When virus particles are clustered into two-dimensional arrays on the mica substrate, they are more rmly
immobilized and, therefore, yield better AFM images. The progressively magnied hexagonal arrangement seen here is
of the insect tipula iridescent virus (TIV). The little capsid structure is visible, and the polygonal structure of the capsid is
evident. Reproduced from Kuznetsov et al. (2001) with permission of the Society for General Microbiology.
Microscopy
2C.2.11
Current Protocols in Microbiology
Supplement 8
Figure 2C.2.7 At higher magnication, the honeycomb appearance of the surface lattice of Paramecium bursaria chlorella virus type 1 (PBCV-1) is evident. Scars and defects are common on the virion
faces, and the unique pentagonal clusters at the vertices can be seen in some cases. In panel D a large
fragment of a trisymmetronis attened on the mica substrate, thereby allowing high-resolution imaging.
In panels E and F are two higher-magnication images of the network of trimeric proteins making up
the honeycomb arrangement. Reprinted from Journal of Structural Biology, vol. 149, Kuznetsov, Y.G.,
Gurnon, J.R., Van Etten, J.L., and McPherson, A. Atomic force microscopy investigation of a chlorella
virus, PBCV-1. pp. 256-263. Copyright 2005, with permission from Elsevier.
Figure 2C.2.8 AFM height image (6 6 m, z range 1.5 m), in aqueous solution, of a single
Saccharomyces cerevisiae cell trapped in a porous membrane. The cell (c) is located at the center
of the image, while the surrounding at area represents the polymer membrane (m). The 1 m
circular protrusion is attributed to a bud scar. Note that the cell is surrounded by an artifactual
structure (a) resulting from the contact between the AFM probe and the pore edges Reproduced
from Dufrene
(2002) with permission from the American Society for Microbiology.
Atomic Force
Microscopy
2C.2.12
Supplement 8
Figure 2C.2.9 Mapping cell surface charges using chemically functionalized probes. (A) Force-distance curves
recorded in solutions of varying pH between the surface of Saccharomyces cerevisiae and an AFM probe functionalized with carboxyl groups. (B) Adhesion force maps recorded at pH 7 and pH 4. The differences in adhesion forces
observed with pH were related to a change of ionization state of the cell surface. Abbreviation: nN, nanoNewton.
Y.F. Recent progress in the application of atomic
Reprinted from Current Opinions in Microbiology, vol. 6. Dufrene,
force microscopy imaging and force spectroscopy to microbiology. pp. 317-323. Copyright 2003, with permission
from Elsevier.
Microscopy
2C.2.13
Current Protocols in Microbiology
Supplement 8
Table 2C.2.5 Characteristics of Force-Distance Curves Measured by AFM between Two Strains of Mutants Streptococci
and Enamel Particles with and without a Salivary Pellicle in the Absence and Presence of a Biosurfactant Coatinga,b
Enamel coating
S. sobrinus
HG 1025
S. mutans
ATCC 25175
F0 (nN)
(nm)
Dmax (nm)f
Fmax (nN)g
80 78
0.9 0.9
3.9 1.7
53 45
77
Biosurfactant
14.3 9.5
253 110
Saliva
4.6 1.4
83 14
10
107 44
0.3 0.2
Saliva +
biosurfactant
4.3 1.4
315 90
178 89
2.0 0.4
0.9 0.7
30 11
78
39 19
0.8 0.7
Biosurfactant
0.8 0.4
59 20
10
106 110
0.2 0.1
Saliva
5.5 1.2
186 62
Saliva +
biosurfactant
1.3 0.8
333 58
a Results are means of 50 force-distance curves, taken over five different organisms on ten randomly selected locations per organism, and including
max ,
gF
max ,
average maximum distance at which a local maximum in adhesion forces occurred upon retraction.
average local maximum in adhesion forces upon retraction.
resonance energy transfer (FRET; Ebenstein et al., 2004), surface plasmon resonance
(SPR) measurements (Chen et al., 1996), total internal reflection fluorescence (TIRF)
microscopy and/or internal reflection microscopy (IRM; Hugel et al., 2002; Trache and
Meininger, 2005), or confocal microscopy (Horton et al., 2000; Noy and Huser, 2003;
UNIT 2C.1).
The future should bring advances to the challenging aspects of the present technology,
most importantly the development of instrumentation for increasing acquisition speed to
allow for real time imaging of fast biological processes, an improved sample preparation
and immobilization technology, and better technology for preparing AFM functionalized
bioprobes.
LITERATURE CITED
Ahimou, F., Denis, F.A., Touhami, A., and Dufrene,
Y.F. 2002. Probing microbial cell surface
charges by atomic force microscopy. Langmuir
18:9937-9942.
Amro, N.A., Kotra, L.P., Wadu-Mesthrige, K.,
Bulychev, A., Mobashery, S., and Liu, G.Y.
2000. High-resolution atomic force microscopy
studies of the Escherichia coli outer membrane: Structural basis for permeability. Langmuir 16:2789-2796.
Arnoldi, M., Kacher, C.M., Bauerlein, E.,
Radmacher, M., and Fritz, M. 1998. Elastic
properties of the cell wall of Magnetospirillum
gryphiswaldense investigated by atomic force
microscopy Appl. Phys. A. 66:S613-S617.
Atomic Force
Microscopy
2C.2.14
Supplement 8
Boulbitch, A., Quinn, B., and Pink, D. 2000. Elasticity of the rod-shaped Gram-negative eubacteria. Phys. Rev. Lett. 85:5246-5249.
Butt, H.J., Downing, K.H., and Hansma, P.K.
1990. Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.
Biophys. J. 58:1473-1480.
Camesano, T.A. and Logan, B.E. 2000. Probing bacterial electrosteric interactions using
atomic force microscopy. Environ. Sci. Technol.
34:3354-3362.
Camesano, T.A., Natan, M.J., and Logan, B.E.
2000. Observation of changes in bacterial cell
morphology using tapping mode atomic force
microscopy. Langnuir 16:4563-4572.
Chen, X., Davies, M.C., Roberts, C.J., Shakesheff,
K.M., Tendler, S.J. B., and Williams, P.M. 1996.
Dynamics surface events measured by simultaneous probe microscopy and surface plasmon
detection. Anal. Chem. 68:1451-1455.
Colton, R.J., Engel, A., Frommer, J.E., Gaub,
H.E., Gewirth, A.A., Guckenberger, R., Rabe,
J., Heckel, W.M., and Parkinson, B. (eds.) 1998.
Procedures in Scanning Probe Microscopies.
John Wiley & Sons Ltd., Chichester, U.K.
Costa, K.D. and Yin, F.C.P. 1999. Analysis of indentation: Implications for measuring mechanical properties with atomic force microscopy. J.
Biomechan. Eng. 121:462-472.
Lehenkari, P.P. and Horton, M.A. 1999. Single integrin adhesion forces in intact cells measured
by atomic force microscopy. Biochem. Biophys.
Res. Commun. 259:645-650.
Dufrene, Y.F. 2003. Recent progress in the application of atomic force microscopy imaging and
force spectroscopy to microbiology. Curr. Opin.
Microbiol. 6:317-323.
Dufrene, Y.F., Boonaert C.J.P., Gerin, P.A., Asther,
M., and Rouxhet, P.G. 1999. Direct probing
of the surface ultrastructure and molecular interactions of dormant and germinating spores
of Phanerochaete chrysosporium. J. Bacter.
181:5350-5355.
Ebenstein, Y., Mokari, T., and Banin, U.
2004. Quantum-dot-functionalized scanning
probes for fluorescence-energy-transfer-based
microscopy. J. Phys. Chem. B 108:93-99.
Gad, M. and Ikai, A. 1995. Method for immobilizing microbial cells on gel surface for dynamic
AFM studies. Biophys. J. 69:2226-2233.
Gad, M. and Ikai, A. 2001. Imaging living cells and
mapping their surface molecules with the atomic
force microscope. In Methods in Cellular Imaging (A. Periasamy, ed.) pp. 409-423, University
Press, Oxford.
Hinterdorfer, P., Kienberger, F., Raab, A., Gruber,
H.J., Baumgartner, G.W., Kada, G., Riener, C.,
Wielert-Badt, S., Borken, C., and Schlinder,
H. 2000. Poly(ethylene glycol) an ideal
spacer for molecular recognition force mi-
Microscopy
2C.2.15
Current Protocols in Microbiology
Supplement 8
Pum, D. and Sleytr, U.B. 1996. Molecular nanotechnology and bioimetics with S-layers. In
Crystalline Bacterial Cell Surface Proteins (U.B.
Sleytr, P. Messner, D. Pum, and M. Sara, eds.)
pp. 175-209. R.G. Landes, Austin, Texas.
Atomic Force
Microscopy
Wetzer, B., Pum, D., and Sleytr, U.B. 1997. Slayer stabilized solid supported lipid bilayers.
J. Struct. Biol. 119:123-128.
Williams, T. 1998. Invertebrate iridescent viruses.
In The Insect Viruses (L.K. Miller and L.A. Ball,
eds.) pp. 31-68. Plenum Press, New York.
Willemsen, O.H., Snel, M.M., Cambi, A., Greve,
J., De Grooth, B.G., and Figdor, C.G. 2000.
Biomolecular interactions measured by atomic
force microscopy. Biophys. J. 79:3267-3281.
Wojcikiewicz, E.P., Zhang, X., and Moy, V.T. 2004.
Force and compliance measurements on living
2C.2.16
Supplement 8
Key References
Morris et al., 1999. See above.
Covers AFM imaging on biological samples.
Braga, P.C. and Ricci, D. (eds.) 2004. Atomic
force microscopy: Biomedical methods and
applications. In Methods in Molecular Biology,
Vol.242, Humana Press, Totowa, N.J.
Covers AFM imaging on biological samples and
some functional studies.
Colton, R.J., Engel, A., Frommer, J.E., Gaub,
H.E., Gewirth, A.A., Guckenberger, R., Rabe,
J., Heckl, W.M., and Parkinson, B. 1998. Procedures in Scanning Probe Microscopies. Wiley
and Sons, West Sussex, England, U.K..
Good source of practical information on AFM
imaging.
Internet Resource
http://www.pacificnanotech.com/afmartifacts single.html
Good presentation of imaging artifacts.
Microscopy
2C.2.17
Current Protocols in Microbiology
Supplement 8
UNIT 3A.1
ABSTRACT
Tick-borne illnesses are emerging as a major concern for human health in recent years.
These include the human monocytic ehrlichiosis caused by the Amblyomma americanum
tick-transmitted bacterium, Ehrlichia chaffeensis; human ewingii ehrlichiosis caused by
Ehrlichia ewingii (also transmitted by A. americanum ticks); and human granulocytic
anaplasmosis caused by the Ixodes scapularis tick-transmitted pathogen, Anaplasma
phagocytophilum. Likewise, tick-borne rickettsial pathogens are also a major concern
to the health of various vertebrates including dogs, cattle, and several wild animals. In
vitrocultured pathogens grown in a vertebrate host cell and a tick cell culture system
will be useful in studies to understand the pathogenic differences as well as to perform
experimental infection studies and to generate large quantities of purified antigens. In
this unit, methods for culturing E. chaffeensis and Ehrlichia canis (a canine monocytic
ehrlichiosis pathogen) in cell lines to represent vertebrate and tick hosts are described.
The unit also includes methods useful in purifying bacteria from the host cells and to evaluate proteins by 2-D gel electrophoresis and western blotting. Curr. Protoc. Microbiol.
C 2008 by John Wiley & Sons, Inc.
9:3A.1.1-3A.1.21.
Keywords: Ehrlichia r culture r macrophage r tick cell r 2-D gel electrophoresis r
IFA r polychromatic staining r cell-free Ehrlichia
INTRODUCTION
Several emerging tick-borne rickettsial infections have been reported in recent years
and have been shown to significantly impact the health of humans and other vertebrates
(Paddock et al., 1993; De Silva et al., 1996). These include infections by Ehrlichia
chaffeensis, a human monocytic agent; E. ewingii, a granulocytic ehrlichiosis agent;
and Anaplasma phagocytophilum, a human granulocytic anaplasmosis agent (UNIT 3A.2;
Dawson et al., 1991; Chen et al., 1994; De Silva et al., 1996; Caspersen et al., 2002).
Ehrlichia canis has been identified as the major cause of tick-borne infections in dogs in
nearly all parts of the world for several decades (Haas and Meyer, 1986). E. chaffeensis
and E. canis may have evolved common strategies in host-pathogen adaptation based
on the fact that they share extensive genetic similarity, contain several immunogenic
antigens with shared epitopes, and are macrophage-tropic pathogens (Dawson et al.,
1991; Palmer et al., 2000). Understanding the biology of parasitism and host-pathogen
interactions requires the growth of these organisms under in vitro conditions.
This unit presents protocols for culturing the Ehrlichia species E. chaffeensis and E. canis.
In addition, it includes methods for identifying Ehrlichia organisms in culture or tissue
samples through the use of polychromatic staining and IFA methods. Protocols are also
included for purifying bacteria from host cells from in vitro cultures in support of isolating
Alpha
Proteobacteria
Current Protocols in Microbiology 3A.1.1-3A.1.21, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc03a01s9
C 2008 John Wiley & Sons, Inc.
Copyright
3A.1.1
Supplement 9
Ehrlichia genomic DNA, RNA, and total protein. Proteome analysis protocols, such as
2-D gel electrophoresis and western blotting, are also discussed.
CAUTION: E. chaffeensis and E. canis are Biosafety Level 2 (BSL-2) pathogens. Follow
all appropriate guidelines and regulations for the use and handling of these pathogenic
microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more
information.
CAUTION: The protocols described in this unit involve the use of human and/or other
animal blood or tissue samples that may also be infected with Ehrlichia species or
other potential pathogens. Follow all appropriate guidelines and regulations for the use
and handling of human- and animal-derived materials. See UNIT 1A.1 and other pertinent
resources (APPENDIX 1B) for more information.
NOTE: When preparing solutions, use deionized or double distilled water and all of
the highest quality reagents that are available from a vendor. Sterilization by filtration
through a 0.22-m filter or by autoclaving is recommended for most solutions and is essential for cell culture media. Discard any reagent that shows evidence of contamination,
precipitation, or discoloration.
BASIC
PROTOCOL 1
Materials
E. chaffeensis and E. canis strains of interest (ATCC)
Complete MEM medium (see recipe)
Canine macrophage cell line, DH82 (ATCC # CRL-10389), grown to 60%
confluence in 25-cm2 tissue culture flask (see Support Protocol 1)
Minimum essential medium with Earles salt (MEM; Mediatech cat.
no. 15-010-CV)
25-cm2 sterile tissue culture flasks
37 C, 5% CO2 humidified incubator
1. Obtain frozen stocks of E. chaffeensis or E. canis from ATCC or from a laboratory
stock stored in a liquid nitrogen tank.
2. Thaw a vial of frozen culture by warming to 37 C in a water bath until culture is
thawed. Transfer the contents of the vial into a 15-ml centrifuge tube containing
10 ml complete MEM medium using a sterile 1-ml pipet. Centrifuge 10 min at
200 g, 4 C.
3. Discard the medium and resuspend the culture pellet in 1 ml complete MEM medium.
Transfer into a 25-cm2 tissue culture flask containing at least 60% confluent monolayer DH82 cells (prepared as in Support Protocol 1).
4. Transfer the flask to a 37 C, 5% CO2 humidified incubator.
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
5. Check the infectivity two times a week by making a Cytospin slide and evaluating
under a light microscope as described in Basic Protocol 5.
6. If the infectivity is less than 50% (as estimated by calculating the ratio of infected
cells to total cells counted under a microscope), change half of the medium (see Fig.
3A.1.1 for identification of Ehrlichia).
3A.1.2
Supplement 9
7. When the infectivity reaches 80% to 90%, subculture by inoculating 10% to 20%
of the infected culture into a new 25-cm2 tissue culture flask having at least 60%
confluence of DH82 cultures. Alternatively, harvest the cultures for protein preparations (as described in Basic Protocol 7) or for nucleic acid isolation as described in
UNIT 3A.3.
This culture procedure can also be used to inoculate a specimen (such as a blood or a
tissue sample) suspected to be positive for Ehrlichia in an effort to recover an isolate. For
recovering Ehrlichia species from a specimen, refer to UNIT 3A.3.
SUPPORT
PROTOCOL 1
Materials
70% (v/v) ethanol
Complete MEM medium (see recipe)
Fetal bovine serum (FBS; Atlanta Biologicals cat. no. S11550)
L-Glutamine (Mediatech cat. no. 25005CI)
Canine macrophage cell line, DH82 (ATCC # CRL-10389 or a previously stored
frozen stock)
Biosafety Class II (Type B2) cabinet
37 C water bath
15-ml centrifuge tubes (Falcon)
25- and 75-cm2 tissue culture flasks
37 C, 5% CO2 humidified incubator
Inverted microscope
4-mm glass beads (Fisher Scientific cat. no. 11-312 A), sterile
1. Sterilize the biosafety cabinet by turning on the UV light for at least 30 min prior to
initiating the culture work.
2. Disinfect the work surface area inside the biological safety cabinet with 70% ethanol.
3. Prepare complete MEM medium inside the biosafety cabinet by mixing 500 ml
MEM, 6 ml L-glutamine, and 35 ml heat-inactivated FBS (heat inactivate 30 min at
56 C).
4. Thaw a vial of frozen DH82 culture by warming to 37 C in a water bath until cultures
are thawed, transfer the contents of the vial into a 15-ml centrifuge tube containing
10 ml complete MEM medium, and centrifuge 10 min at 200 g, 4 C.
5. Discard the medium and resuspend the culture pellet in 5 ml of complete MEM
medium.
6. Transfer the contents into a 25-cm2 tissue culture flask and incubate in a 37 C, 5%
CO2 humidified incubator.
7. Change 50% to 100% of the medium every 3 to 4 days. Replace 50% of the medium
if the cell confluence is 50% (as assessed by viewing under inverted microscope at
40 magnification) and replace the entire medium if the confluence is 50%.
8. When the cells reach 90% to 100% confluence, replace the entire volume of medium
and detach the cultures by gently rocking the culture flask by hand after adding 15
sterile 4-mm glass beads (typically takes 15 to 30 sec).
Current Protocols in Microbiology
Alpha
Proteobacteria
3A.1.3
Supplement 9
9. Dilute the culture in complete MEM medium two- to ten-fold depending on the need
to expand the cultures, then transfer 5 or 15 ml each to new 25- or 75-cm2 tissue
culture flasks, respectively, and continue growing the cultures in a 37 C, 5% CO2
humidified incubator.
BASIC
PROTOCOL 2
Materials
70% ethanol
E. chaffeensis- or E. canis-infected DH82 cultures (see Basic Protocol 1) or healthy
DH82 cultures (from a 25-cm2 tissue culture flask; see Support Protocol 1)
DH82 culture freezing medium (see recipe)
Liquid nitrogen tank
Biosafety Class II (Type B2) cabinet
15-ml sterile polypropylene centrifuge tubes (Falcon)
Refrigerated tabletop centrifuge
Alcohol-resistant marker pen
2-ml screw-cap cryovials
Nalgene Cryo 1 C freezing container (Fisher Scientific cat. no. 5100-0001)
1. Sterilize a biosafety cabinet by turning on UV light for at least 30 min prior to
initiating the culture work.
2. Disinfect the work surface area of the biological safety cabinet with 70% ethanol.
3. Transfer E. chaffeensis- or E. canis-infected DH82 cultures (see Basic Protocol 1)
or 5 ml (1 107 cells) healthy DH82 cultures (from a 25-cm2 tissue culture flask;
see Support Protocol 1) to a 15-ml sterile centrifuge tube.
CAUTION: This step and subsequent handling of cultures must be performed inside the
biosafety cabinet.
NOTE: It is important to avoid defrosting the frozen vials during the transfer from a
80 C freezer to a liquid nitrogen tank. The transfer must be performed very rapidly.
3A.1.4
Supplement 9
BASIC
PROTOCOL 3
Materials
E. chaffeensis and E. canis (ATCC or from laboratory stock)
Complete Ehrlichia-infected tick cell culture medium (see recipe)
Glassware (see Support Protocol 3)
25-cm2 tissue culture flasks
34 C humidified incubator
1. Prepare glassware for tick cell culture (see Support Protocol 3).
2. Obtain frozen stocks of E. chaffeensis and E. canis.
3. Thaw a vial of frozen tick cell stock by following Support Protocol 2, steps 2 to 9,
but using complete Ehrlichia-infected tick cell culture medium.
4. Inoculate the culture pellet from the frozen stock into a 25-cm2 tissue culture flask
containing tick cells having at least 80% confluent monolayer (prepared as described in Support Protocol 2).
5. Transfer the flask to a 34 C humidified incubator.
6. Check the infectivity two times a week by making a Cytospin slide and evaluating
under a light microscope as described in Basic Protocol 5.
7. If the infectivity is <50%, change half of the medium.
8a. When the infectivity reaches 80% to 90%, subculture by inoculating 10% to 20% of
the infected culture into a new 25-cm2 culture flask having at least 80% confluence
of tick cells.
8b. Alternatively, harvest the cultures for nucleic acid or protein preparations (UNIT 3A.3).
SUPPORT
PROTOCOL 2
Alpha
Proteobacteria
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Current Protocols in Microbiology
Supplement 9
and Kurtti (1989), and Munderloh et al. (1994). All three tick cell lines are cultured using
exactly the same procedures.
Materials
70% ethanol
Complete tick cell culture medium (see recipe)
Frozen uninfected tick cell culture
Glassware for tick cell culture (see Support Protocol 3)
Biosafety Class II (Type B2) cabinet
37 C water bath
15-ml centrifuge tubes
1- and 5-ml sterile serological pipets
25- and 75-cm2 tissue culture flask
34 C humidified incubator
1. Prepare glassware for tick cell culture (see Support Protocol 3).
2. Sterilize the biosafety cabinet by turning on UV light for at least 30 min prior to
initiating the culture work.
3. Disinfect the work surface area of the biological safety cabinet with 70% ethanol.
4. Prepare complete tick cell culture medium (see Reagents and Solutions) inside the
biosafety cabinet using the base medium, L15B 300.
5. Obtain a vial of uninfected tick cell culture from a frozen stock and defrost rapidly
by incubating for few minutes in a 37 C water bath.
6. When the cell suspension is defrosted completely, wipe the culture vial with a 70%
ethanol-soaked paper towel.
7. Carefully transfer the entire contents of culture into a sterile 15-ml centrifuge tube
and centrifuge 10 min at 400 g, 4 C.
8. Discard the supernatant and add 1 ml of complete tick cell culture medium to the
pellet and gently mix using a 1 ml pipet.
9. Transfer the contents to a 25-cm2 tissue culture flask containing 5 ml of complete
tick cell culture medium and incubate the flask in a 34 C humidified incubator.
10. Replace the entire medium the next day and from then once every week until the
desired growth of culture is achieved.
11. Monitor the growth of the culture by following Basic Protocol 5.
12. When the cells reach 90% to 100% confluence, replace the entire medium and detach
the cultures by pipetting the medium up and down using a 5-ml sterile pipet to create
a stream of medium at the cell layer.
13. Dilute the culture in complete tick cell culture medium (two- to ten-fold depending
on the need to expand the cultures) and transfer 5 or 15 ml of the diluted cultures to
a new 25- or 75-cm2 tissue culture flask, respectively.
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
Splitting tick cell cultures too soon may result in the complete loss of cultures. Enzymatic treatment, such as trypsin, or the glass bead method to separate the cells are not
recommended, as these procedures can damage the tick cells.
3A.1.6
Supplement 9
SUPPORT
PROTOCOL 3
Materials
Glassware detergent
Glassware for cell culture
Autoclave
Aluminum foil
180 C oven
1. Wash glassware once with a detergent designed for laboratory glassware washing.
2. Rinse several times with tap water and then several times with deionized water.
3. Fill the glassware completely with deionized water and autoclave 15 min at 121 C.
4. Discard water, cover with two layers of aluminum foil, and bake glassware 2 hr in a
180 C oven.
BASIC
PROTOCOL 4
The procedure of making tick cell stocks is slightly different from that described for
preparing DH82 culture stocks. Medium for freezing tick cells (both infected and uninfected) is composed of the complete medium (infected or uninfected), but with an FBS
concentration raised to 20% and also by including 10% dimethyl sulfoxide (DMSO).
Materials
Ehrlichia-infected tick cells (Basic Protocol 3)
Freezing medium for uninfected tick cell culture (see recipe)
Freezing medium for infected tick cell culture (see recipe)
Liquid nitrogen tank
2-ml cryovials
Nalgene Cryo 1 C freezing container
1. Check the appearance of healthy cells and infectivity of Ehrlichia-infected tick
cells by evaluating cultures using the polychromatic staining method (see Basic
Protocol 5).
For preparing infected tick cell culture stocks, select cultures having infectivity ranging
from 50% to 70%.
Alpha
Proteobacteria
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Current Protocols in Microbiology
Supplement 9
BASIC
PROTOCOL 5
Materials
Uninfected or Ehrlichia-infected host cells
Hema staining kit (Fisher Scientific cat. no. 23-122929, 23-122937, 23-122952)
containing:
Hema-3 fixative solution
Hema-3 solution I
Hema-3 solution II
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
3A.1.8
Supplement 9
ALTERNATE
PROTOCOL
Following this procedure, uninfected culture cells will be visible under a microscope
with no prominent fluorescence detected, while Ehrlichia in morulae will appear as
bright green dots (Fig. 3A.1.2).
Materials
Cytospin slides (see Basic Protocol 5)
Acetone
Phosphate-buffered saline (PBS), pH 7.4 (see recipe) containing 1% (v/v) BSA
Polyclonal E. chaffeensis or E. canis antiserum (obtained from a commercial
vendor, e.g., VMRD, cat. no. 211-P-EC, or from infected vertebrate host blood)
1 FA rinse buffer (see recipe)
Fluorescein isothiocyanate (FITC)-conjugated host-specific secondary antibody
Antifade gel mounting medium
Glass staining jars
37 C incubator
Slide box
Paper towels
Aluminum foil
Coverslips
Fluorescence microscope with filters for detecting FITC
1. Prepare Cytospin slides as described for polychromatic staining (see Basic Protocol 5,
steps 1 to 3).
Alpha
Proteobacteria
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Current Protocols in Microbiology
Supplement 9
Figure 3A.1.2 Indirect uorescent antibody (IFA) staining of in vitro cultured E. chaffeensisinfected macrophage cell line, DH82. Uninfected DH82 cells are used as a control to demonstrate
specic staining only in E. chaffeensisinfected cells. Ehrlichia organisms in the phagosomes of
cytoplasmic compartment of infected macrophages retain strong green uorescence (identied
with arrows). For a color version of this gure, see http://www.currentprotocols.com.
6. Soak the slides for 10 min with 1 FA rinse buffer in a glass staining jar.
7. Place slides in a moist chamber and add 10 l of FITC-conjugated host-specific
secondary antibodies.
8. Incubate slides 30 min in a dark moist chamber at 37 C (cover slide box with a piece
of aluminum foil).
9. Soak slides 10 min with 1 FA rinse buffer in a glass staining jar protected from
exposure to light.
10. Add one drop of antifade gel mounting medium and place a coverslip over a slide to
allow the medium to spread along the surface of the slide viewing area.
11. Gently press the surface of the coverslip to remove any air bubbles.
12. Examine the slides under a fluorescence microscope initially using a 10 or 40
objective and then using a 100 oil immersion objective.
BASIC
PROTOCOL 6
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
EHRLICHIA PURIFICATION
Recently, methods for purifying host cell-free Ehrlichia for use in proteome analysis
have been described (Singu et al., 2005, 2006). The protocols have been proven to be
useful for isolating different E. chaffeensis isolates and E. canis cultured organisms. This
method can also be used to recover cell-free Ehrlichia for use in antigen analysis or
for infection studies using cell-free Ehrlichia, or possibly any other molecular biology
experiments.
3A.1.10
Supplement 9
Materials
Uninfected and Ehrlichia-infected (Basic Protocol 1) canine macrophage cell line
(DH82) or tick cells in a 75-cm2 confluent flask
SPK buffer (see recipe)
Sonic Dismembrator (Fisher Scientific or equivalent)
3- and 5-m sterile isopore membrane filters (Millipore)
1. Harvest 15 ml of macrophage or tick cell culture from a 75-cm2 confluent flask
having 80% to 100% Ehrlichia infection and centrifuge 15 min at 15,560 g, 4 C,
to recover infected host cells and cell-free Ehrlichia.
This protocol can be scaled up or down depending on the volume of purified Ehrlichia
needed.
2. Resuspend the resulting culture pellet in 10 ml of SPK buffer and sonicate two times
for 30 sec, each time at a setting of 6.5 in a Sonic Dismembrator to release Ehrlichia
organisms from host cells.
The sonication setting may vary from instrument to instrument and minor standardization
may be required.
3. Centrifuge lysates 5 min at 100 g, 4 C, to pellet host nucleus and other host cell
debris.
4. Filter the supernatant sequentially by passing it through 5- and 3-m sterile isopore
membrane filters.
5. Centrifuge Ehrlichia organisms from filtrates 15 min at 15,560 g, 4 C and wash
pellets two times with 10 ml SPK buffer.
Avoid disrupting the pellets during this wash protocol. Alternatively, centrifuge cells 5 min
at 15,560 g, 4 C, after each wash.
6. Check the purity of cell-free Ehrlichia by preparing a smear on a glass slide stained
with a polychromatic stain and examined under a light microscope (see Basic
Protocol 5).
BASIC
PROTOCOL 7
Materials
Ehrlichia organisms (Basic Protocol 6)
Protein lysis buffer (see recipe)
4:1 (v/v) acetone/trichloroacetic acid, ice cold
Protein sample buffer (see recipe)
RC DC protein assay kit (BioRad)
1. Lyse Ehrlichia by adding 0.5 ml of protein lysis buffer to the pelleted organisms
obtained in Basic Protocol 6, and incubating 1 hr at room temperature.
2. Precipitate Ehrlichia proteins by adding 2 vol of ice-cold 4:1 acetone/trichloroacetic
acid and store overnight at 20 C.
3. Centrifuge precipitated protein 15 min at 15,000 g, 4 C, dissolve the final protein
pellet in 100 l sample buffer.
4. Estimate the protein concentration using the RC DC protein assay kit and following
the manufacturers step-by-step protocol.
Alpha
Proteobacteria
3A.1.11
Current Protocols in Microbiology
Supplement 9
BASIC
PROTOCOL 8
Materials
Ehrlichia proteins (Basic Protocol 7)
Equilibration buffer I (see recipe)
Equilibration buffer II (see recipe)
4% to 20% gradient polyacrylamide gels (BioRad cat. no. 3450104)
Tris-glycine electrophoresis buffer (see recipe)
Silver-staining kit (BioRad)
Blocking solution: 5% nonfat dairy milk in PBS
Primary antibodies (polyclonal sera against E. chaffeensis or E. canis raised in
mouse, dog, or human)
PBS containing 0.1% Tween 20 (PBS-T; see recipe for PBS)
Anti-mouse and anti-dog horseradish peroxidase (HRPO)conjugated secondary
antibodies
ECL western blotting detection reagents (Amersham)
Multiphor II electrophoresis system (Amersham)
Immobiline dry strips (11-cm, pH 3 to 10; Amersham)
Criterion cell apparatus (BioRad cat. no. 165-6001) or equivalent
Hybond-N nitrocellulose membrane (Amersham cat. no. RPN303D)
Protean Trans-blot cell (BioRad) or equivalent
X-ray film (Amersham)
1. Resolve 100 g Ehrlichia proteins in a Multiphor II electrophoresis system using
Immobiline 11-cm dry strips, pH 3 to 10.
The IEF parameters are 300 V, 2 mA, and 5 W for 1 min, followed by 3500 V, 2 mA, and
5 W for 245 min.
3A.1.12
Supplement 9
Figure 3A.1.3 Proteome maps of E. chaffeensis grown in (A) macrophage and (B) tick cells
(Singu et al., 2005). Scanned images of silver-stained 2-D gels of E. chaffeensis proteomes are
presented. The approximate location of the pI values of the protein migration and the protein
molecular weight standards are shown on the top and right side of the gel images, respectively.
(Ganta et al., 2002, 2004). More details about infection are provided in Ganta et al.
(2002, 2004, 2007).
8. Wash the membrane three times, 10 min each time, at room temperature in saturating
volumes of PBS-T.
9. Incubate the membrane with appropriately diluted HRPO-conjugated anti-mouse or
anti-dog secondary antibody for 1 hr at room temperature.
10. Wash the membrane with PBS-T three times as described in step 8.
11. Detect the antigen-antibody complexes by exposing an X-ray film for 2 to 5 min
using the ECL western blotting detection reagents.
Silver-stained gels and X-ray film having western blot data may be digitalized using a
scanner and the images may be analyzed with the aid of image analysis software to
perform comparisons of 2-D gels.
Alpha
Proteobacteria
3A.1.13
Current Protocols in Microbiology
Supplement 9
3A.1.14
Supplement 9
To prepare 100 ml, mix 37.5 ml of 1 M TrisCl, pH 8.8, 36.04 g urea, 2 g SDS,
and 2 g DTT. Bring volume up to 100 ml with deionized water, dispense into 2-ml
aliquots, and store up to 1 year at 20 C.
This buffer is commercially available from BioRad.
Current Protocols in Microbiology
Equilibration buffer II
375 mM TrisCl, pH 8.8 (APPENDIX 2A)
6 M urea
2% SDS
2.72 mg iodoacetamide/ml
0.01% bromophenol blue
To prepare 100 ml, mix 37.5 ml of 1 M TrisCl, pH 8.8, 36.04 g urea, 2 g SDS,
272 mg iodoacetamide, and 10 mg bromophenol blue. Bring volume up to 100 ml
with deionized water, dispense into 2-ml aliquots, and store up to 1 year at 20 C.
This buffer is commercially available from BioRad.
FA rinse buffer, 1
2.85 g Na2 CO3
8.4 g NaHCO3
2.125 g NaCl
Add distilled deionized water to 1000 ml
Adjust pH to 9 to 9.5 with 1 N NaOH
Store up to 1 year at room temperature
Freezing medium for uninfected tick cell culture
7 ml complete tick cell culture medium (see recipe)
2 ml FBS (Invitrogen cat. no. 26140079)
1 ml DMSO
Prepare fresh
Freezing medium for infected tick cell culture
7 ml complete Ehrlichia-infected tick cell culture medium (see recipe)
2 ml FBS (Invitrogen cat. no. 26140079)
1 ml DMSO
Prepare fresh
HEPES, 1 M
23.83 g HEPES (Sigma-Aldrich cat. no. H-3375)
Add cell culturegrade water to 100 ml, filter sterilize using a 0.22-m filter unit,
and store up to 6 months at 4 C.
L15B medium
1 packet of L15B medium powder (Invitrogen cat. no. 41300-039)
0.299 g L-aspartic acid (Sigma-Aldrich cat. no. A-4534)
0.299 g -ketoglutaric acid (Sigma-Aldrich cat. no. K-1128)
0.292 g L-glutamine (Sigma-Aldrich cat. no. G-6392)
0.3 g proline (Sigma-Aldrich cat. no. P-5607)
0.5 g L-glutamic acid (Sigma-Aldrich cat. no. G-8415)
14.41 g D-glucose (Sigma-Aldrich cat. no. G-7021)
1 ml mineral stock D (see recipe)
1 ml vitamin stock (see recipe)
Add the above ingredients to a 1000-ml volumetric flask, make up the volume to
1000 ml with cell culturegrade water, adjust pH to 6.4 to 6.6 with 10 N NaOH,
filter sterilize using a 0.22-m filter unit, and store up to 6 months at 4 C, covering
with aluminum foil to protect from light damage.
Alpha
Proteobacteria
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Supplement 9
L15B300 medium
1000 ml L15B medium (see recipe)
333 ml cell culturegrade water
Store up to 2 months at 4 C
Mineral stock solution A
20 mg CoCl2 6H2 O (Sigma-Aldrich cat. no. C-8661)
20 mg CuSO4 5H2 O (Sigma-Aldrich cat. no. C8027)
160 mg MnSO4 H2 O (Sigma-Aldrich cat. no. M1144)
200 mg ZnSO4 7H2 O (Sigma-Aldrich cat. no. Z0251)
Prepare the mineral stock solution by weighing the desired weights in 100 ml cell
culturegrade water (Invitrogen cat. no. 15230-162), filter sterilize using a 0.22-m
filter unit, dispense into 1-ml aliquots into sterile microcentrifuge tubes, and store
up to 2 years at 20 C for future use.
NaHCO3, 5%
Add 5 g of NaHCO3 in 100 ml of cell culturegrade water, filter sterilize using a
0.22-m filter unit, and store up to 2 months at 4 C.
NaOH, 1 N
Add 4 g of NaOH in 100 ml culture gradewater, filter sterilize using a 0.22-m
filter unit, and store up to 6 months at 4 C.
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
8 g NaCl
0.2 g KH2 PO4
0.2 g KCl
Plus one of the following:
2.17 g Na2 HPO4 7H2 O
continued
3A.1.16
Supplement 9
TPB medium
Add 29.59 g tryptose phosphate broth (TPB; Difco cat. no. 260300) to culture
gradewater and make up volume to 1000 ml, dispense into 100-ml aliquots, and
autoclave with loose caps for 15 min at 121 C. Store up to 1 year at 4 C.
The color should be pale yellow; if it darkens, the TPB should be discarded as it
will be toxic to the tick cells.
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Proteobacteria
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Vitamin stock
100 mg 4-aminobenzoic acid (PABA; Sigma-Aldrich cat. no. A-9878)
50 mg cyanocobalamine (B12; Sigma-Aldrich cat. no. V2876)
10 mg D-biotin (Sigma-Aldrich cat. no. B-4639)
Prepare the vitamin stock by weighing these ingredients and transferring to 100 ml
cell culturegrade water, filter sterilize using a 0.22-m filter unit, and store as 1-ml
aliquots up to 1 year at 20 C for use.
COMMENTARY
Background Information
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
3A.1.18
Supplement 9
Critical Parameters
Culturing Ehrlichia in canine macrophage
cell line, DH82, and tick cell lines
Sterile culture techniques are a very critical step for Ehrlichia culture as all the cultures must be maintained in the absence of
any antibiotics to insure bacterial growth. Always keep infected cultures separate from
uninfected cultures. Also, maintaining duplicate sets of infected and uninfected cultures by
independently prepared media and reagents is
preferred.
Tick cells seem to be more sensitive to minor changes of pH and cell density. Cell density of tick cell culture should be between 20%
and 80% for optimal growth. Tick cell culture can also be affected due to impurities that
may be introduced from glassware and other
culture supplies. Thus, it is recommended to
use the culture supplies from the same vendor
when one finds a product(s) useful in growing
Ehrlichia in tick cell culture.
Preparing cell culture stocks
One must prepare liquid nitrogen culture
stocks on a regular basis to prevent the loss
of cell lines due to accidental contamination
Current Protocols in Microbiology
Troubleshooting
Table 3A.1.1 presents some of the common
problems associated with the protocols in this
unit.
Anticipated Results
Growth of Ehrlichia in DH82 and tick cell
culture
DH82 cell culture is easier to maintain as
compared to tick cell culture. When recovering a frozen healthy DH82 culture stock, the
culture growth should be visible within 2 to 3
days. By 5 to 7 days, a 25-cm2 tissue culture
flask should be confluent and ready to be split.
The doubling time for tick cells is longer, and
it will take 7 to 10 days for a flask to reach
confluency.
When an Ehrlichia culture has 70% to 90%
infectivity, it is subcultured by inoculating 5%
to 10% into a flask containing a confluent
monolayer. Infections should reach 80% to
90% within 3 to 5 days after inoculation.
Detection of Ehrlichia-infected cells by
polychromatic staining and IFA methods
When examining a Cytospin slide stained
with a polychromatic stain, the morulae (cytoplasmic inclusions of Ehrlichia) stain purple, while the host cell nucleus stains pink.
Morulae in tick cells are more loosely packed
compared to those in DH82 cells.
Problem
Possible cause
Solution
Bacterial or fungal
contamination, or pH may be
too low
If contamination was
confirmed, discard the medium.
If pH too low, adjust pH with
NaOH.
Overgrowth of Ehrlichia
Alpha
Proteobacteria
3A.1.19
Current Protocols in Microbiology
Supplement 9
Acknowledgements
This study was supported by the National
Institutes of Health grants AI055052 and
AI070908. The manuscript is published as
Kansas Agricultural Experiment Station contribution no. 08-225-B.
Literature Cited
Bell-Sakyi, L., Paxton, E.A., Munderloh, U.G., and
Sumption, K.J. 2000. Growth of Cowdria ruminantium, the causative agent of heartwater, in a
tick cell line. J. Clin. Microbiol. 38:1238-1240.
Caspersen, K., Park, J.H., Patil, S., and Dumler,
J.S. 2002. Genetic variability and stability of
Anaplasma phagocytophila msp2 (p44). Infect.
Immun. 70:1230-1234.
Chen, S.M., Dumler, J.S., Bakken, J.S., and Walker,
D.H. 1994. Identification of a granulocytotropic
Ehrlichia species as the etiologic agent of human
disease. J. Clin. Microbiol. 32:589-595.
Dawson, J.E., Anderson, B.E., Fishbein, D.B.,
Sanchez, C.Y., Goldsmith, C.Y., Wilson, K.H.,
and Duntley, C.W. 1991. Isolation and characterization of an Ehrlichia sp. from a patient
diagnosed with human ehrlichiosis. J. Clin.
Microbiol. 29:2741-2745.
De Silva, A.M., Telford, S.R., Brunet, L.R.,
Barthold, S.W., and Fikrig. E. 1996. Borrelia burgdorferi OspA is an arthropod-specific
transmission-blocking Lyme disease vaccine.
J. Exp. Med. 183:271-275.
Ganta, R.R., Wilkerson, M.J., Cheng, C., Rokey,
A.M., and Chapes, S.K. 2002. Persistent
Ehrlichia chaffeensis infection occurs in the
absence of functional major histocompatibility
complex class II genes. Infect. Immun. 70:380388.
Ganta, R.R., Cheng, C., Wilkerson, M.J., and
Chapes, S.K. 2004. Delayed clearance of
Ehrlichia chaffeensis infection in CD4+ T-cell
knockout mice. Infect. Immun. 72:159-167.
Ganta, R.R., Cheng, C., Miller, E.C., McGuire,
B.L., Peddireddi, L., Sirigireddy, K.R., and
Chapes, S.K. 2007. Differential clearance and
immune responses to tick cellderived versus macrophage culturederived Ehrlichia chaffeensis in mice. Infect. Immun. 75:135-145.
Laboratory
Maintenance and
Recovery of
Ehrlichia
chaffeensis and
canis
p44 epitope by the human granulocytic ehrlichiosis agent. J. Infect. Dis. 184:1445-1450.
Kurtti, T.J., Munderloh, U.G., and Ahlstrand, G.G.
1988. Tick tissue and cell culture in vector research. In Advances in Disease Vector Research
(K.F. Harris, ed.), pp. 87-109. Springer-Verlag,
New York.
Lohr, C.V., Brayton, K.A., Shkap, V., Molad,
T., Barbet, A.F., Brown, W.C., and Palmer,
G.H. 2002. Expression of Anaplasma marginale
major surface protein 2 operon-associated
proteins during mammalian and arthropod
infection. Infect. Immun. 70:6005-6012.
Munderloh, U.G. and Kurtti, T.J. 1989. Formulation of medium for tick cell culture. Exp. Appl.
Acarol. 7:219-229.
Munderloh, U.G., Liu, Y., Wang, M., Chen, C., and
Kurtti, T.J. 1994. Establishment, maintenance
and description of cell lines from the tick Ixodes
scapularis. J. Parasitol. 80:533-543.
Munderloh, U.G., Madigan, J.E., Dumler, J.S.,
Goodman, J.L., Hayes, S.F., Barlough, J.E.,
Nelson, C.M., and Kurtti, T.J. 1996. Isolation
of the equine granulocytic ehrlichiosis agent,
Ehrlichia equi, in tick cell culture. J. Clin.
Microbiol. 34:664-670.
Munderloh, U.G., Jauron, S.D., Fingerle, V.,
Leitritz, L., Hayes, S.F., Hautman, J.M., Nelson,
C.M., Huberty, B.W., Kurtti, T.J., Ahlstrand,
G.G., Greig, B., Mellencamp, M.A., and
Goodman, J.L. 1999. Invasion and intracellular development of the human granulocytic
ehrlichiosis agent in tick cell culture. J. Clin.
Microbiol. 37:2518-2524.
Munderloh, U.G., Tate, C.M., Lynch, M.J.,
Howerth, E.W., Kurtti, T.J., and Davidson, W.R.
2003. Isolation of an Anaplasma sp. organism
from white-tailed deer by tick cell culture. J.
Clin. Microbiol. 41:4328-4335.
Paddock, C.D., Suchard, D.P., Grumbach, K.L.,
Hadley, W.K., Kerschmann, R.L., Abbey, N.W.,
Dawson, J.E., Anderson, B.E., Sims, K.G.,
Dumler, J.S., and Herndier, B.G. 1993. Brief report: Fatal seronegative Ehrlichiosis in a patient
with HIV infection. N. Engl. J. Med. 329:11641167.
Palmer, G.H., Brown, W.C., and Rurangirwam,
F.R. 2000. Antigenic variation in the persistence
and transmission of the Ehrlichia, Anaplasma
marginale. Microbes. Infect. 2:167-176.
Ristic, M., Huxsoll, D.L., Weisiger, R.M.,
Hildebrandt, P.K., and Nyindo, M.B. 1972.
Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect
Immun. 6:226-231.
Singu, V., Liu, H., Cheng, C., and Ganta, R.R. 2005.
Ehrlichia chaffeensis expresses macrophageand tick cellspecific 28-kilodalton outer membrane proteins. Infect. Immun. 73:79-87.
3A.1.20
Supplement 9
Alpha
Proteobacteria
3A.1.21
Current Protocols in Microbiology
Supplement 9
Laboratory Maintenance of
Anaplasma phagocytophilum
UNIT 3A.2
Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA; formerly human granulocytic ehrlichiosis). This unique bacterium resides
within salivary glands of its Ixodes tick vector or within the intracytoplasmic connes of
neutrophils in its mammalian hosts. Because of its obligate intracellular nature, A. phagocytophilum is maintained in the laboratory in eukaryotic cell culture or within murine
hosts. The human-derived promyelocytic leukemia cell line HL-60 is routinely used for
in vitro cultivation of A. phagocytophilum. The susceptibility of these cells to A. phagocytophilum infection, like that of human neutrophils, is attributable to the expression of
P-selectin glycoprotein ligand-1 (PSGL-1) on the cell surface. The rapid rate at which
HL-60 cells grow and their permissiveness with respect to A. phagocytophilum infection
allows one to obtain high yields of in vitrocultured bacteria. However, the ability of
A. phagocytophilum to successfully establish in vivo infection declines considerably
when continually maintained in cell culture. It is therefore important that the bacterium
also be maintained in vivo. Laboratory mice are excellent in vivo models, as infection in
these mice partially mimics many of the hematological parameters and histopathological lesions associated with HGA. Peripheral blood infection of immunocompetent mice
resolves by the third or fourth week post-inoculation. Moreover, A. phagocytophilum
pathogenicity wanes with repeated passage through immunocompetent mice. SCID mice
are unable to clear A. phagocytophilum infection, and are therefore used for continual
maintenance of the pathogen in vivo. This unit describes the inoculation and maintenance
of A. phagocytophilum in vivo using a murine model of infection (see Basic Protocol 1)
and in vitro using HL-60 cells (see Basic Protocol 2). Methods for identifying A.
phagocytophiluminfected cells in a peripheral blood sample (see Support Protocol 1)
or infected HL-60 cells (see Support Protocol 2) using either cytological staining (see
Support Protocol 3) or immunouorescent microscopy (see Support Protocol 4) are also
discussed. Additionally, cryopreservation of A. phagocytophiluminfected HL-60 cell
lines (see Support Protocol 3) is described. Furthermore, this unit describes the recovery
of semipure preparations of viable A. phagocytophilum from infected HL-60 cells (see
Basic Protocol 2). Techniques for further purifying these bacterial preparations by density gradient centrifugation (see Support Protocol 6) or for uorescently labeling them to
track infection (see Support Protocol 7) are also provided. Figure 3A.2.1 demonstrates
how these protocols are interrelated.
CAUTION: Anaplasma phagocytophilum is a Biosafety Level 2 (BSL-2) pathogen. Follow
all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: The protocols described in this unit involve the use of human and/or murine
blood that is infected with A. phagocytophilum. Syringe lysis of A. phagocytophilum
infected HL-60 cells is also involved. Follow all appropriate guidelines and regulations
for the use and handling of human-derived materials and blood-borne pathogens, as
well as storage and disposal of sharps. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
CAUTION: These experiments require Animal Biosafety Level 2 (ABSL-2) conditions.
Follow all appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1
and other pertinent resources (APPENDIX 1B) for more information. Protocols using live
animals must rst be reviewed and approved by an Institutional Animal Care and Use
Committee (IACUC) or must conform to governmental regulations regarding the care
and use of laboratory animals.
Alpha
Proteobacteria
3A.2.1
Figure 3A.2.1
BASIC
PROTOCOL 1
Laboratory
Maintenance of
Anaplasma species
3A.2.2
Current Protocols in Microbiology
While the protocol below calls for C3H/Smn.CicrHsd/scid or C3H/HeN mice, C3H/HeJ
and their congenic C3H-scid counterparts may also be used. There is no observable
difference in bacterial loads between the C3H/ HeN and C3H/ HeJ strains.
Materials
5% (v/v) Lysol
70% (v/v) ethanol
Mouse: C3H/Smn.CicrHsd/scid (SCID; 3 to 5 weeks old; Harlan Sprague-Dawley)
or C3H/HeN (3 to 5 weeks old; National Cancer Institute Animal Production
Program, Frederick Cancer Research Center)
Ketamine/xylazine anesthetic cocktail (see recipe) or other anesthetic agent as
outlined in approved institutional protocol
Anticoagulant-treated, A. phagocytophiluminfected peripheral blood (either from
an A. phagocytophiluminfected mouse or a HGA patient that has not yet
received antibiotic therapy)
Class II, Type B2, vertical ow biological safety cabinet
1-cc syringes with 28-G, 12 -in. (12.7-mm) needles
1-cc tuberculin syringes with 26-G, 12 -in. (12.7-mm) needles
Gauze
Additional reagents and equipment for anesthesia (Donovan and Brown, 1998) and
intraperitoneal injection (Donovan and Brown, 1995a) of mice
NOTE: The total blood volume of a mouse is 5.5 ml per 100 g body weight. Do not
collect more than 20% of total volume at one time. Do not collect more than this volume
weekly. There is no limit to the number of times a mouse may be bled, provided that
the aforementioned parameters are observed. For repeated blood collection via the retroorbital route, alternate eyes should be used.
Anesthetize mice
1. Disinfect the work surfaces of a Class II, Type B2, vertical ow biological safety
cabinet with 5% Lysol followed by 70% ethanol. Place a paper towel on the work
surface of the biological safety cabinet.
2. Anesthetize the mouse (Donovan and Brown, 1998) by injecting 0.1 ml/20 g body
weight of ketamine/xylazine anesthetic cocktail intramuscularly using a 1-cc tuberculin syringe with a 28-G, 12 -in. needle.
This dosage should provide effective anesthesia for 30 to 40 min.
Once the mouse becomes unresponsive to a toe pinch, the procedure may be continued.
If the mouse inches or otherwise responds to the pinch, wait 5 min and check again. If
the mouse still responds, administer an additional dose of 0.1 ml anesthetic cocktail/20 g
body weight.
3. Transfer the anesthetized mouse to the biological safety cabinet, placing it on the
paper towel covering the at work surface.
Inoculate mice
4. In the biological safety cabinet, ll a 1-cc tuberculin syringe tted with a 26-G, 12 -in.
needle with 100 l of A. phagocytophiluminfected blood.
5. While holding a mouse against the at surface, slowly inject the 100 l blood sample
into the peritoneal cavity (Donovan and Brown, 1995a).
6. Carefully withdraw the needle and use gauze to apply digital pressure at the injection
site to maintain hemostasis.
Alpha
Proteobacteria
3A.2.3
Current Protocols in Microbiology
7. Place the injected mouse in a spare cage and continue injections with additional mice.
When injections are nished, check the animals for normal behavior and return them
to their normal housing location.
8. Remove unused materials from the biological safety cabinet. Disinfect the interior
of the biosafety cabinet with 5% Lysol followed by 70% ethanol.
SUPPORT
PROTOCOL 1
Materials
A. phagocytophiluminfected mice (see Basic Protocol 1)
Glass tubes coated with anticoagulant (e.g., EDTA, heparin)
Glass microscope slides with frosted ends
Additional reagents and equipment for blood collection from mice (Donovan and
Brown, 1995b)
NOTE: A blood sample from a human patient suspected of having HGA who has yet to
receive antibiotic therapy is processed in a manner identical to that described in steps 2
through 6.
1. Collect blood from A. phagocytophiluminfected mice (Donovan and Brown, 1995b)
via retro-orbital bleed, saphenous vein puncture, or other approved technique and
transfer to anticoagulant-treated glass tubes.
2. Place 1 to 2 l of the anticoagulant-treated blood sample onto one end of a glass
slide with frosted end.
3. Holding the slide steady with one hand, touch the end of another slide held at a 45
angle to the drop of blood.
The blood will spread along the interface of the two slides.
4. Drag the angled slide along the surface of the held slide in a single sweeping motion.
Repeat this process to generate at least 3 slides per blood sample.
Laboratory
Maintenance of
Anaplasma species
The desired result is to achieve a tongue-shaped smear of blood along the slides surface.
The vast majority of neutrophils and other leukocytes are present in the arch-shaped
periphery (tongue) of the smear.
3A.2.4
Current Protocols in Microbiology
BASIC
PROTOCOL 2
The human-derived promyelocytic leukemia cell line HL-60 is the predominant choice
for in vitro cultivation of A. phagocytophilum. Blood from either an A. phagocytophilum
infected mouse or a human patient suspected of having HGA that has yet to be treated
with antibiotics is inoculated into HL-60 cells to generate a primary in vitro culture of
A. phagocytophilum. The infected HL-60 cells are subsequently passaged to maintain
proper cell density and supplemented with uninfected HL-60 cells to prevent lysis of the
entire host cell population.
Materials
5% (v/v) Lysol
70% (v/v) ethanol
HL-60 cells (ATCC #CCL240) growing in culture
IMDM-10: Iscoves modied Dulbeccos medium (Invitrogen 12440-053)
supplemented with 10% (v/v) heat-inactivated FBS
A. phagocytophiluminfected blood sample(s) collected in glass tubes coated with
anticoagulant (e.g., EDTA, heparin)
Class II, Type B2, vertical ow biological safety cabinet
15-ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
25-cm2 tissue culture asks with vented caps (BD Falcon)
Additional reagents and equipment for counting cells using a hemacytometer
(Strober, 1997a) and determining cell viability by trypan blue exclusion
(Strober, 1997b)
NOTE: All solutions and equipment coming into contact with living cells must be sterile
and aseptic technique should be used accordingly.
NOTE: All incubations are performed in a humidied 37 C, 5% CO2 incubator unless
otherwise specied.
Alpha
Proteobacteria
3A.2.5
Current Protocols in Microbiology
c. Monitor cultures for infection load to make sure that the bacteria do not outgrow and completely lyse the HL-60 cells, by cytological or immunouorescence
staining as described in Support Protocols 2, 3, and/or 4.
Morulae, or intracellular inclusions of A. phagocytophilum, should be apparent by
5 days post-inoculation. The percentage of infected HL-60 cells will depend on the
bacterial load of the inoculum.
Laboratory
Maintenance of
Anaplasma species
Peripheral blood, either from an A. phagocytophiluminfected mouse or a patient suspected of having HGA that has yet to receive antibiotic therapy may be used in place of
A. phagocytophiluminfected HL-60 cells in the protocol below. However, examination
of peripheral blood using the smear method (Support Protocol 1) is preferable because
of the rapidity and ease with which it can be performed.
3A.2.6
Current Protocols in Microbiology
Materials
5 105 cell/ml suspension of A. phagocytophiluminfected HL-60 cells in
IMDM-10 (see Basic Protocol 2)
5 105 cell/ml suspension of uninfected HL-60 cells in IMDM-10 (control)
0.5% (v/v) sodium hypochlorite
Glass microscope slides with frosted ends
Cytocentrifuge setup (Thermo Electron Corp.) including:
Shandon Stainless Steel Cytoclip
Shandon TPX Filter Cards
Shandon TPX Sample Chamber
Shandon Cytospin 4 Cytocentrifuge
Absorbent bench paper with plastic backing
Prepare slides
1. Label the frosted portion of each slide with the appropriate sample designation. Use
a Shandon Stainless Steel Cytoclip to secure a TPX Filter Card and TPX Sample
Chamber to each slide, situating the lter card between the sample chamber and slide
with the holes of the sample chamber and lter card aligned. Place the assemblies in
the cytocentrifuge and balance the rotor properly.
The use of a cytocentrifuge is recommended because the nuclei of HL-60 cells are quite
large and can obscure detection of morulae. Centrifugation of HL-60 cells attens them,
which greatly facilitates viewing of their cytoplasmic contents.
3. Centrifuge the suspensions onto the slides for 2 min at 70 g, using a Shandon
Cytospin 4 Centrifuge.
4. Unclip the assembly, remove the sample chambers, and soak them in 0.5% sodium
hypochlorite for 30 min. Afterwards, rinse the sample chambers in deionized water.
Dispose of the lter cards into a biohazard-labeled trash receptacle.
Disposable cytospin funnels are also available from Thermo Electron.
5. Place the slides at a 45 angle on a level surface covered with a sheet of absorbent
bench paper with plastic backing. Allow to air dry at room temperature for at least
20 to 30 min, until completely dry.
SUPPORT
PROTOCOL 3
Alpha
Proteobacteria
3A.2.7
Current Protocols in Microbiology
Materials
Protocol Hema-3 staining solutions (Fisher):
Protocol Hema-3 xative
Protocol Hema-3 solution I
Protocol Hema-3 solution II
Microscope slides with blood smear (see Support Protocol 1 or 2) or HL-60 cells
(see Support Protocol 2)
Four glass staining dishes with removable racks (Wheaton model 900200)
Absorbent bench paper with plastic backing
Light microscope (UNIT 2A.1)
Stain slides
1. Fill three staining dishes, respectively, with Protocol Hema-3 xative, staining solution I, and staining solution II, to a depth that will completely immerse the
slides when placed in the dishes. Similarly ll a fourth staining dish with deionized
water.
2. Place the slides in the staining rack. Immerse the staining rack in the xative for 60
sec. Remove and allow for drainage of residual xative.
3. Immerse the staining rack in solution I for 45 sec. Remove and allow for drainage of
residual stain. Immerse briey in deionized water, remove, and allow for drainage of
residual water.
4. Immerse the staining rack in Solution II for 45 sec. Remove and allow for drainage
of residual stain. Immerse briey in deionized water, remove, and allow for drainage
of residual water.
Laboratory
Maintenance of
Anaplasma species
5. Place the slides on a sheet of absorbent bench paper placed on a level surface at a
45 angle to facilitate drainage of residual water. Allow the slides to air dry for 2 hr
to overnight.
3A.2.8
Current Protocols in Microbiology
Examine slides
6. Observe the slides by light microscopy under oil immersion using the 100 objective.
Look for neutrophils with the presence of morulae.
Morulae are stippled in appearance and stain more darkly than the host cells nucleus
(Fig. 3A.2.2).
SUPPORT
PROTOCOL 4
This method is useful if ones laboratory does not possess a Cytospin cytocentrifuge, and
therefore serves as an alternative to Support Protocol 3. Also, the increased sensitivity of
immunouorescence microscopy allows one to visualize individual organisms. This is an
advantage over cytological staining (see Support Protocol 3), which is better suited for
detecting clusters of A. phagocytophilum. Slides of A. phagocytophiluminfected cells are
prepared by aliquotting the cells into wells of Teon-coated immunouorescence slides.
Alternatively, if one so chooses, slides prepared using the Cytospin procedure (see Support
Protocol 2) may be used. The slides are incubated with polyclonal antiserum against
A. phagocytophilum, followed by incubation with uorescein isothiocyanate (FITC)
conjugated secondary antibody. The slides are subsequently observed using a uorescence
microscope. Prepare antibodies during the blocking step.
Materials
5% (v/v) Lysol
70% (v/v) ethanol
A. phagocytophiluminfected HL-60 cells (see Basic Protocol 2)
Uninfected HL-60 cells (control)
Phosphate-buffered saline, pH 7.4 (PBS; APPENDIX 2A)
Acetone
PBS/1% and 5% BSA: phosphate-buffered saline, pH 7.4 (PBS; APPENDIX 2A)
containing 1% or 5% (v/v) bovine serum albumin (BSA)
Polyclonal A. phagocytophilum antiserum (rabbit or murine; see Cooper and
Patterson, 1995)
Fluorescein isothiocyanate (FITC)conjugated secondary antibody: either goat
anti-rabbit IgG (Sigma cat. no. F-6005) or goat anti-mouse IgG (Sigma cat. no.
F-8264)
PBS-T: phosphate-buffered saline, pH 7.4 (PBS; APPENDIX 2A) containing 0.05%
(v/v) Tween 20
20 g/ml propidium iodide (light-sensitive; store up to 6 months at 4 C; optional)
Antifade gel-mounting medium (Biomeda Gelmount BMM-01)
Clear nail polish
12-well (5-mm diameter) Teon-coated immunouorescence slides (Carlson
Scientic, cat. no. 101205) with coverslips
Class II, Type B2, vertical ow biological safety cabinet
Plastic, light-impermeable container large enough to accommodate slides
1-ml serological pipets
Forceps
Fluorescence microscope with lters for detecting FITC and rhodamine emission
Additional reagents and equipment for counting cells using a hemacytometer
(Strober, 1997a)
NOTE: Propidium iodide staining (steps 30 to 31) is optional. Propidium iodide binds
DNA, which results in the host cell nuclei staining red. This can facilitate easier identication of host cells though it is not necessary.
Alpha
Proteobacteria
3A.2.9
Current Protocols in Microbiology
7. Pipet 5 l of PBS to the two wells adjacent to well no. 1 (i.e., wells no. 2 and 3).
8. Pipet 5 l of infected HL-60 cells into well no. 2. Mix by pipetting to yield a two-fold
dilution (5 103 cells).
9. Transfer 5 l of the two-fold dilution from well no. 2 to well no. 3 containing PBS.
Mix by pipetting to yield a four-fold dilution (2.5 103 cells).
10. Repeat steps 7 to 9 for wells nos. 4 to 6 and nos. 7 to 9 (see Fig. 3A.2.3).
11. Repeat steps 6 to 10 to prepare a control slide using uninfected HL-60 cells.
12. Allow the slides to air dry at room temperature for at least 30 min.
If cytospin preparations are used, these must also be xed with acetone.
3A.2.10
Current Protocols in Microbiology
18. Using forceps, remove the slides and allow excess acetone to drain into the staining
jar. Allow residual acetone to evaporate from the slides.
This will take only a few seconds.
19a. If slides are not to be screened immediately: Place pairs of slides back-to-back and
wrap in a Kimwipe. Wrap once more using aluminum foil. Label the foil with the
sample designations and store up to 6 months (or possibly longer) at 20 C.
19b. If slides are to be screened immediately: Proceed to step 20.
Alpha
Proteobacteria
3A.2.11
Current Protocols in Microbiology
22. Remove PBS by aspiration. Add 5 l PBS/5% BSA to each sample-containing well.
Close the humidied chamber and incubate 1 hr at room temperature.
This blocking step will reduce nonspecic binding of antibodies.
24. Dilute the FITC-conjugated secondary antibody to 1:1000 in PBS/1% BSA. Store at
4 C protected from light until use.
33. Allow the slide to dry at room temperature in the dark for 2 hr to overnight. Once
the mounting medium has dried, seal the edges with clear nail polish.
Laboratory
Maintenance of
Anaplasma species
3A.2.12
Current Protocols in Microbiology
SUPPORT
PROTOCOL 5
It is good practice to maintain frozen stocks of the cell lines. To protect against complete
loss of cell lines due to accidental contamination or other incidents, stocks of uninfected
and A. phagocytophiluminfected HL-60 cells should be stored. The cells are pelleted,
resuspended in medium containing DMSO, frozen in an isopropanol bath at 80 C, and
transferred to liquid nitrogen for long-term storage.
Materials
5% (v/v) Lysol
70% (v/v) ethanol
A. phagocytophiluminfected HL-60 cells growing in culture (see Basic Protocol 2)
100% isopropanol
IMDM-10 with and without 12% (v/v) dimethylsulfoxide (DMSO)
0.5% (v/v) sodium hypochlorite
Class II, Type B2, vertical ow biological safety cabinet
10- and 1-ml serological pipets
Alpha
Proteobacteria
3A.2.13
Current Protocols in Microbiology
15-ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
Refrigerated tabletop centrifuge
Alcohol-resistant pen (VWR)
250-ml beaker for waste
2-ml screw-cap cryovials
Nalgene Cryo 1 C freezing container
80 C freezer
Cryobiological storage vessel containing liquid nitrogen
25-cm2 tissue culture asks with vented caps (BD Falcon)
NOTE: All solutions and equipment coming into contact with living cells must be sterile
and aseptic technique should be used accordingly.
NOTE: All incubations are performed in a humidied 37 C, 5% CO2 incubator unless
otherwise specied.
Pellet cells
1. Disinfect the work surfaces of a Class II, Type B2, vertical ow biological safety
cabinet with 5% Lysol followed by 70% ethanol.
2. Using a serological pipet, transfer 1 107 of A. phagocytophiluminfected HL-60
cells to a 15-ml conical centrifuge tube.
Uninfected HL-60 cells are processed in an identical manner.
5. Add 100% isopropanol to the ll line of the container. Replace the foam insert and
vial holder.
This will require a volume of 250 ml. Do not overll.
Freeze cells
6. Using a 1-ml serological pipet, gently resuspend the cells from step 3 in 1 ml IMDM10 containing 12% DMSO. Immediately transfer the suspension to the corresponding
labeled, 2-ml screw-cap cryovial. Close the cap.
DMSO serves as a cryopreservative, which enables the HL-60 cells to survive the freezing
process.
7. Immediately transfer the tube to a hole in the vial holder of a Nalgene the Cryo 1 C
freezing container. Place the Cryo 1 C freezing container in a 80 C freezer. Leave
undisturbed for a minimum of 4 hr.
This achieves a controlled cooling rate of 1 C per minute, which is recommended for
eukaryotic cell lines.
Laboratory
Maintenance of
Anaplasma species
Cells should be stored for no more than 1 week at 80 C before transferring to liquid
nitrogen. Longer storage at the lower temperature will compromise the viability of the
culture upon thawing. A temperature of 130 C or below is necessary for long-term
storage.
3A.2.14
Current Protocols in Microbiology
8. After a period of at least 4 hr, remove the frozen vial from the freezing container
and place in a cryobiological storage vessel containing liquid nitrogen for long-term
storage.
The long-term storage vessel should have a temperature of 130 C or below. Cells may
be stored indenitely.
Thaw cells
9. Remove the frozen sample from storage and place on ice to thaw slowly.
10. Using a 1-ml serological pipet, gently resuspend and transfer the tubes contents to
a 15-ml conical centrifuge tube.
Wash cells
11. Using a 10-ml serological pipet, immediately add 10 ml of IMDM-10.
DMSO will cause terminal differentiation of HL-60 cells. It is therefore important to
thoroughly wash the cells immediately upon thawing to remove all traces of DMSO.
12. Centrifuge the tube as in step 3, then remove and discard the supernatant in the waste
beaker.
13. Repeat steps 11 to 12 two additional times.
14. Resuspend cells in 10 ml IMDM-10 and transfer to a 25-cm2 tissue culture ask with
a vented cap.
15. Place newly resuspended culture in incubator and continue culturing as described in
Basic Protocol 2.
BASIC
PROTOCOL 3
Preparations of A. phagocytophilum that are relatively free from contaminating HL-60 cell
components are used for a variety of in vitro applications. A. phagocytophiluminfected
HL-60 cells are lysed via syringe passage, and a semipure preparation of viable bacteria
is recovered by differential centrifugation. These preparations can be used in subsequent
in vitro infection assays, inoculated into mice (see Basic Protocol 1), subjected to further
purication by density gradient centrifugation (see Support Protocol 6), or uorescently
labeled for tracking infection (Support Protocol 7).
Materials
5% (v/v) Lysol
70% (v/v) ethanol
A. phagocytophiluminfected HL-60 cells growing in culture (see Basic Protocol 2)
Uninfected HL-60 cells (control)
Phosphate-buffered saline, pH 7.4 (PBS; APPENDIX 2A), prechilled
0.5% (v/v) sodium hypochlorite
Class II, Type B2, vertical ow biological safety cabinet
25-, 10-, and 5-ml serological pipets
Alpha
Proteobacteria
3A.2.15
Current Protocols in Microbiology
15- and 50-ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
Refrigerated tabletop centrifuge
500-ml beaker for waste
5-cc tuberculin syringes
27-G, 12 -in. (12.7-mm) needles
Additional reagents and equipment for counting cells (Strober, 1997a) and
cytological (see Support Protocol 3) or immunouorescence staining (see
Support Protocol 4) of A. phagocytophilum cultures
NOTE: All solutions and equipment coming into contact with living cells must be sterile
and aseptic technique should be used accordingly.
Pellet cells
4. Using a 10- or 25-ml serological pipet, transfer the appropriate number of A.
phagocytophiluminfected cells in culture medium to a 50-ml conical centrifuge
tube. Prepare a separate tube in a similar manner using uninfected HL-60 cells and
carry through the following steps in parallel as a control.
The number of infected cells can be scaled according to the purpose of the experiment.
For instance, if the resulting semipure preparation of A. phagocytophilum is to be further
puried by density gradient centrifugation (see Support Protocol 6), pellet 2.5 108 cells.
If using the A. phagocytophilum preparation in an in vitro infection assay, pellet a number
of infected cells equal to 1 to 5 times the number of cells to be infected. If host cellfree A.
phagocytophilum recovered from lysed HL-60 cells are to be used in subsequent infection
assays, it is imperative that uninfected HL-60 cells be processed in an identical manner
and used as a control in the subsequent infection assay. This is essential for determining
whether results of the infection assay are inuenced by the presence of contaminating
HL-60 lysate components present in the A. phagocytophilum preparation.
5. Place the 50-ml tubes in a refrigerated tabletop centrifuge. Centrifuge the cells 10
min at 2300 g, 4 C. Discard supernatant in a 500-ml beaker designated for waste.
Be sure the tubes are properly balanced.
7. Place a 50-ml conical centrifuge tube upright in a rack. Remove and place the cap to
the side.
3A.2.16
Current Protocols in Microbiology
Lyse cells
8. Remove the plungers from ve 5-cc syringes (designated nos. 1 to 5). Attach a 27-G,
1
2 -in needle to each.
A set of ve syringes should also be prepared for the uninfected HL-60 cells.
9. Using a 5-ml serological pipet, transfer the cell suspension to syringe no. 1.
10. Uncap the needle of syringe no. 1 and, using constant pressure, expel the suspension
through the needle into the 50-ml tube (see step 7).
For maximum recovery of the suspension, hold the syringe at a 45o angle and place the
end of the needle as close to the inner wall of the tube as possible without touching. This
will allow the suspension to run down the inner wall to the tube bottom and will minimize
the chance of producing an aerosol.
11. Discard syringe no. 1 into a sharps container. Repeat steps 9 and 10 four times.
This process lyses the HL-60 cells without lysing the bacteria.
13. Transfer the supernatants to new 15-ml centrifuge tubes. Discard the tubes containing
the pellets of cellular debris into a biohazard-labeled trash receptacle.
14. Centrifuge the tubes containing the supernatants from step 13 for 5 min at 1000
g, 4 C.
A pellet of considerably smaller size than that generated in step 12 should be visible.
15. Transfer the supernatants to new 15-ml tubes. Discard the tubes containing the pellets
of cellular debris into a biohazard-labeled trash receptacle.
16. Centrifuge the tubes containing the supernatants from step 15 for 10 min at 2300
g, 4 C. Remove and discard the supernatant into the beaker designated for waste.
A small pellet should be visible. The majority of the pellet is composed of host-cell-free
A. phagocytophilum. A minor portion of the pellet consists of HL-60 debris and cellular
components that were not removed in steps 12 and 14.
SUPPORT
PROTOCOL 6
This method is useful for generating A. phagocytophilum preparations that are more highly
puried from contaminating host cell components than those prepared by differential
centrifugation. Such preparations are used for inoculating animals with the purpose of
obtaining antiA. phagocytophilum antiserum that exhibits little to no recognition of
HL-60 cells. Alternatively, such preparations are used for subsequent isolation of A.
phagocytophilum nucleic acids, proteins, or outer membranes. It is best that contaminating
host cellular components be minimized for such procedures. An A. phagocytophilum
Alpha
Proteobacteria
3A.2.17
Current Protocols in Microbiology
preparation obtained via differential centrifugation (see Basic Protocol 3) is layered onto
a discontinuous Renogran gradient and subjected to ultracentrifugation. This results in
the resolution of a band of A. phagocytophilum that is free from host cell components,
with the exception of host cell organelles of a similar density, such as mitochondria.
Materials
5% (v/v) Lysol
70% (v/v) ethanol
A. phagocytophiluminfected HL-60 cells growing in culture (see Basic Protocol 2)
PBS/glucose (see recipe), ice cold
Complete Protease Inhibitor Cocktail Tablets (Roche, cat. no. 1 697 498)
DNase I (from bovine pancreas; Roche cat. no. 776785)
RNase (from bovine pancreas; Roche cat. no. 1 119 915)
30% Renogran (see recipe)
42% Renogran (see recipe)
SPGN buffer (see recipe)
0.5% (v/v) sodium hypochlorite
Class II, Type B2, vertical ow biological safety cabinet
50-ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
Refrigerated tabletop centrifuge
1-liter beaker for waste
10-cc tuberculin syringe
Beckman L7-55 ultracentrifuge and Beckman SW 28 swinging-bucket rotor (or
equivalent)
38.5-ml ultracentrifuge tubes (Beckman Ultra-Clear)
Harvard trip balance
2.0-ml polypropylene microcentrifuge tubes
Additional reagents and equipment for counting cells (Strober, 1997a), cytological
(see Support Protocol 3) or immunouorescence staining (see Support Protocol
4) of A. phagocytophilum cultures, preparing a semipure preparation of A.
phagocytophilum (see Basic Protocol 3), and protein assay (APPENDIX 3A)
NOTE: All solutions and equipment coming into contact with living cells must be sterile
and aseptic technique should be used accordingly.
2. Determine cell density of cultures of interest by examining aliquots using a hemacytometer (Strober, 1997a).
3. Determine the percentage of infected cells in the cultures of interest as described in
Support Protocol 3 or Support Protocol 4.
The percentage of infected cells should be 70%.
Laboratory
Maintenance of
Anaplasma species
4. For each Renogran prep to be prepared, place 2.5 108 infected HL-60 cells in a
(depending on the culture density) of 50-ml conical centrifuge tube.
3A.2.18
Current Protocols in Microbiology
7. Discard supernatant from step 4 into a 1-liter beaker designated for waste.
8. Mechanically lyse the infected cells and recover a semipure preparation of A. phagocytophilum as described in Basic Protocol 3, steps 8 to 16, except use 10-cc syringes
in the lysis steps.
15. Following the nucleic acid digestion in step 11, chill the lysate on ice for 10 min.
16. Using a serological pipet, overlay the lysate on the Renogran gradient. If processing
an odd number of samples, overlay the gradient in the balance tube with deionized
water.
3A.2.19
Current Protocols in Microbiology
Figure 3A.2.5
18. Screw the caps onto all six swinging buckets. Hang each of them at its matched
position on the SW 28 rotor.
CAUTION: Because of the high speeds at which they are used, ultracentrifuge rotors must
be precisely balanced. The SW 28 buckets are numbered 1 through 6. When hanging on
the rotor, 1 must lie opposite 4, 2 must lie opposite 5, and 3 must lie opposite 6. Keep this
in mind when choosing the swinging buckets.
19. Turn the vacuum switch to OFF and wait for the rotor chamber to equilibrate. Open
the rotor chamber door and place the SW 28 rotor onto the drive spindle. Close the
rotor chamber door and turn the vacuum switch to ON. Enter a speed of 22,000 rpm
(87,300 g). Enter a run time of 75 min. Leave the temperature setting at 4 C. Set
the brake at 800 rpm (115 g).
The brake switch of the Beckman L7-55 allows either for full dynamic braking until 0
rpm or for full dynamic braking until 800 rpm followed by no braking to a gentle stop.
Allowing the rotor to come to a gentle stop allows for minimal disruption of the resulting
centrifugation layers. Newer ultracentrifuge models have the option of no brake. Choose
this option if using a newer model. However, keep in mind that it will take considerably
longer for the rotor to come to a full stop without braking.
24. Using a serological pipet, carefully remove the top 18 to 19 ml of the Renogran
gradient and discard into the beaker designated for waste. Be sure to leave 1.5 cm
of Renogran above the enriched A. phagocytophilum layer.
3A.2.20
Current Protocols in Microbiology
SUPPORT
PROTOCOL 7
Materials
Semipure preparation of host cellfree A. phagocytophilum (see Basic Protocol 3)
Phosphate-buffered saline, pH, pH 7.4 (PBS; APPENDIX 2A), chilled
10 M CellTracker Green CMFDA (see recipe)
250-ml beaker for waste
Fluorescence microscope with lters for detecting FITC emission
NOTE: All solutions and equipment coming into contact with living cells must be sterile
and aseptic technique should be used accordingly.
Alpha
Proteobacteria
3A.2.21
Current Protocols in Microbiology
Figure 3A.2.6 Flow cytometric analysis of human neutrophils incubated with CellTracker Green
CMFDAlabeled A. phagocytophilum. Bacteria were incubated with neutrophils for 60 min at ratios
of 0.3 (solid, thick histogram), 3 (dashed histogram), and 30 (dotted histogram) A. phagocytophilum
organisms per cell. Uninfected neutrophils (solid, thin histogram) served as a negative control.
Label A. phagocytophilum
5. Prewarm the 10 M working solution of CellTracker Green CMDFA in a 37 C water
bath for at least 10 min.
CellTracker Green CMFDA is light-sensitive; protect the solution from light.
11. Dilute the labeled bacteria to the desired concentration and incubate with host cells
of choice. Infection can be monitored either by ow cytometry (Fig. 3A.2.6) or
uorescent microscopy using a lter equipped for observing FITC uorescence.
Labeled bacteria should be incubated with recipient host cells for 30 min.
Laboratory
Maintenance of
Anaplasma species
3A.2.22
Current Protocols in Microbiology
Alpha
Proteobacteria
3A.2.23
Current Protocols in Microbiology
COMMENTARY
Background Information
Laboratory
Maintenance of
Anaplasma species
3A.2.24
Current Protocols in Microbiology
Critical Parameters
Preparing slides and culturing A.
phagocytophilum in HL-60 cells
When preparing slides of A. phagocytophiluminfected blood to be subsequently
screened for morulae-positive neutrophils, it
is essential to achieve a good smear. The area
of the smear should nearly cover the slides
entire surface and end in a tongue-shaped arc.
The vast majority of leukocytes will be present
in the periphery of the arc. Conning ones
microscopic examination to this region will
provide the best opportunity to visualize neutrophils. Once an HL-60 culture is inoculated
with A. phagocytophilum, it is important to
maintain a proper HL-60 cell density such that
the culture does not overgrow. Therefore, split
the culture bi-weekly to maintain a density of
25 105 cells/ml. Additionally, it is crucial
to determine the percentage of infected cells
and add uninfected HL-60 cells as needed to
prevent A. phagocytophilum from outgrowing
and lysing the entire host cell population. For
example, if 90% of the cells in a 10-ml culture
of HL-60 cells at a density of 1 106 cells/ml
are infected with A. phagocytophilum, discard
8 ml (8 106 cells) and add 2 106 uninfected
HL-60 cells. Bring the nal volume to 10 ml
with fresh IMDM-10. By day 3, the culture
will again contain 90% infected cells and will
need to be split again. Using this example as a
guide, cultures can be supplemented while still
retaining a high percentage of infected cells.
Lysing infected HL-60 cells
When lysing A. phagocytophiluminfected
HL-60 cells in preparation for obtaining bacterial preparations that are relatively free from
host cellular components, it is crucial to pass
the cells through a 27-G, 12 -in. (12.7-mm) needle ve times to maximize host cell lysis.
It is equally important to thoroughly remove
Alpha
Proteobacteria
3A.2.25
Current Protocols in Microbiology
Troubleshooting
Table 3A.2.1 presents some of the common
problems associated with the protocols in this
unit, as well as their causes and solutions.
Anticipated Results
Inoculation and continual passage of A.
phagocytophilum in HL-60 cell culture
Following inoculation of A. phagocytophiluminfected blood into HL-60 cell
culture, morulae are typically not detectable
until day 5 post-inoculation. However, A.
phagocytophilum has a slow doubling time.
Thus, even on day 5, the percentage of HL-60
cells with morulae remains quite low. By days
12 to 14, morulae should be detectable within
80% of cells.
When viewing HL-60 cells incubated in
the presence of trypan blue under light microscopy, uninfected HL-60 cells are typically
round and have smooth borders. The membranes of infected cells, however, are rougher
in appearance. Moreover, the integrity of the
membranes becomes compromised as the infection progresses, and they become permeable with respect to trypan blue. As a result,
heavily infected cells and cells that have been
lysed upon bacterial exit appear darker than
uninfected cells.
Laboratory
Maintenance of
Anaplasma species
3A.2.26
Current Protocols in Microbiology
Table 3A.2.1 Troubleshooting Guide for Laboratory Maintenance and Staining of Anaplasma phagocytophilum
Problem
Possible cause
Solution
Cultivation of A. phagocytophilum in
HL-60 cells
Entire HL-60 cell population lysed
Pathogenicity of A.
phagocytophilum has waned with
continual passage in
immunocompetent mice
Clumping of cells
Poor blocking
Inoculation of A. phagocytophilum
into mice
Blood from A.
phagocytophiluminfected C3H mice
has low percentage of
morulae-positive neutrophils after
third or fourth passage
Cytological staining
Immunouorescence
Alpha
Proteobacteria
3A.2.27
Current Protocols in Microbiology
Table 3A.2.1 Troubleshooting Guide for Laboratory Maintenance and Staining of Anaplasma phagocytophilum,
continued
Problem
Possible cause
Solution
Laboratory
Maintenance of
Anaplasma species
Time Considerations
With the exception of the protocols for
establishing A. phagocytophilum infection in
HL-60 cell culture (see Basic Protocol 2) or in
mice (see Basic Protocol 1), each protocol described in this unit can be performed in a single
day. Inoculation of HL-60 cells or mice should
take <1 hr, depending on the number of inoculations to be performed. Following inoculation of infected blood into HL-60 cell culture,
it takes 5 days before morulae are evident
in even a minor percentage of cells. By days
12 to 14, however, 90% of the cells should
3A.2.28
Current Protocols in Microbiology
Literature Cited
Borjesson, D.L. and Barthold, S.W. 2002. The
mouse as a model for investigation of human
granulocytic ehrlichiosis: Current knowledge
and future directions. Comp. Med. 52:403-413.
Carlyon, J.A. and Fikrig, E. 2003. Invasion and survival strategies of Anaplasma phagocytophilum.
Cell Microbiol. 5:743-754.
Carlyon, J.A. and Fikrig, E. 2004. Pathogenic strategies of Anaplasma phagocytophilum, a unique
bacterium that colonizes neutrophils. In Sixtythird Symposium of the Society for General
Microbiology (M. Scoureld, ed.) pp. 301329. Cambridge University Press, Cambridge,
U.K.
Carlyon, J.A., Akkoyunlu, M., Xia, L., Yago,
T., Wang, T., Cummings, R.D., McEver, R.P.,
and Fikrig, E. 2003. Murine neutrophils require alpha 1,3-fucosylation but not PSGL-1 for
productive infection with Anaplasma phagocytophilum. Blood 102:3387-3395.
Chen, S.M., Dumler, J.S., Bakken, J.S. and Walker,
D.H. 1994a. Identication of a granulocytotropic Ehrlichia species as the etiologic agent
of human disease. J. Clin. Microbiol. 32:589595.
Donovan, J. and Brown, P. 1995. Parenteral injections. In Current Protocols in Immunology (J.E.
Coligan, A.M. Kruisbeek, D.H. Margulies, E.M.
Shevach, and W. Strober, eds.) pp. 1.6.1-1.6.10.
John Wiley & Sons, Hoboken, N.J.
Donovan, J. and Brown, P. 1998. Anesthesia. In
Current Protocols in Immunology (J.E. Coligan,
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach,
and W. Strober, eds.) pp. 1.4.1-1.4.5. John Wiley
& Sons, Hoboken, N.J.
Dumler, J.S. and Bakken, J.S. 1998. Human ehrlichioses: Newly recognized infections transmitted by ticks. Annu. Rev. Med. 49:201-213.
Dumler, J.S. and Brouqui, P. 2004. Molecular diagnosis of human granulocytic anaplasmosis. Expert Rev. Mol. Diagn. 4:559-569.
Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch,
G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., and
Rurangirwa, F.R. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unication
of some species of Ehrlichia with Anaplasma,
Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi
and HGE agent as subjective synonyms of
Ehrlichia phagocytophila. Int. J. Syst. Evol.
Micr. 51:2145-2165.
Goodman, J.L., Nelson, C.M., Klein, M.B., Hayes,
S.F. and Weston, B.W. 1999. Leukocyte infection by the granulocytic ehrlichiosis agent is
linked to expression of a selectin ligand. J. Clin.
Invest. 103:407-412.
Goodman, J.L., Nelson, C., Vitale, B., Madigan,
J.E., Dumler, J.S., Kurtti, T.J. and Munderloh,
U.G. 1996. Direct cultivation of the causative
agent of human granulocytic ehrlichiosis. New
Engl. J. Med. 334:209-215.
Herron, M.J., Nelson, C.M., Larson, J., Snapp, K.R.,
Kansas, G.S. and Goodman, J.L. 2000. Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P-selectin ligand,
PSGL-1. Science 288:1653-1656.
Hodzic, E., Ijdo, J.W., Feng, S., Katavolos, P., Sun,
W., Maretzki, C.H., Fish, D., Fikrig, E., Telford,
S.R. 3rd, and Barthold, S.W. 1998. Granulocytic
ehrlichiosis in the laboratory mouse. J. Infect.
Dis. 177:737-745.
McCaul, T.F. and Williams, J.C. 1981. Developmental cycle of Coxiella burnetii: Structure
and morphogenesis of vegetative and sporogenic differentiations. J. Bacteriol. 147:10631076.
Cooper, H.M. and Patterson, Y. Production of polyclonal antisera. 1995. In Current Protocols in Immunology ( S.E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, and Warren Strober,
eds.) pp. 2.4.1-2.4.9. John Wiley and Sons,
Hoboken, N.J.
Alpha
Proteobacteria
3A.2.29
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Key References
Borjesson and Barthold 2002. See above.
An excellent review of the use of laboratory mice
for in vivo maintenance and studies of A. phagocytophilum.
Laboratory
Maintenance of
Anaplasma species
3A.2.30
Current Protocols in Microbiology
UNIT 3A.3
ABSTRACT
Human monocytic ehrlichiosis (HME), caused by Ehrlichia chaffeensis, was first recognized in 1986. Infection with this pathogen can be fatal in immune compromised
and elderly humans. E. chaffeensis can also infect dogs and several wild animals. The
clinical symptoms of HME include fever, headache, malaise, myalgia, confusion, rash,
lymphadenopathy, and nausea. White-tailed deer serve as the major reservoir host for the
natural maintenance of E. chaffeensis. E. canis is primarily responsible for the canine
monocytic ehrlichiosis and is endemic throughout the world. It has a significant impact
on the health of dogs. The isolation and growth of Ehrlichia species from vertebrate host
samples is difficult and time consuming. In this unit, methods to recover E. chaffeensis
and E. canis from infected blood samples collected from dogs, deer, and human patients
are described. PCR and RT-PCR methods for sensitive detection of Ehrlichia infection
C 2008 by John Wiley &
are also discussed. Curr. Protoc. Microbiol. 9:3A.3.1-3A.3.16.
Sons, Inc.
Keywords: Ehrlichia isolation r animal blood r PCR r RT-PCR r real-time RT-PCR r
DNA isolation r RNA isolation
INTRODUCTION
Human monocytic ehrlichiosis (HME), caused by Ehrlichia chaffeensis, was first recognized in 1986 (Dawson et al., 1991a). E. chaffeensis can also infect dogs and several
wild animals (Dawson et al., 1996; Magnarelli et al., 1997). The clinical symptoms of
HME include fever, headache, malaise, myalgia, confusion, rash, lymphadenopathy, and
nausea (Rikihisa, 1999). HME can be fatal to the immune compromised and elderly.
White-tailed deer are presumed to be the reservoir hosts of E. chaffeensis (Dawson et al.,
1994; Lockhart et al., 1997). E. canis causes canine monocytic ehrlichiosis and was first
recognized in Algeria in 1935 (Buhles et al., 1974). Wild and domestic dogs with chronic
infection serve as reservoir hosts. During the acute phase of infection, the clinical signs
include fever, anorexia, and lymphadenopathy, and, in the chronic phase of infection, the
dogs may show emaciation, hemorrhage, and peripheral edema (Buhles et al., 1974).
The first successful culture of E. canis was obtained from peripheral blood monocytes
obtained from experimentally infected dogs during the acute phase of the disease (Lovering et al., 1980). Similarly, the first isolation and cultivation of E. chaffeensis in canine
macrophage cell line was reported by Dawson et al. (1991a). The isolation and growth
of Ehrlichia species is difficult and time consuming. Several laboratories described the
isolation of Ehrlichia species from vertebrate animals (Dawson et al., 1991a,b; Lockhart
et al., 1997; Paddock et al., 1997; Breitschwerdt et al., 1998). Protocols for isolating
E. chaffeensis from several human blood samples suspected to be positive for Ehrlichia
are also described (Paddock et al., 1997). Methods of in vitro isolation of Ehrlichia
from naturally infected deer blood are described in Dawson et al. (1994) and Lockhart
et al. (1997). Mice serve as a good model system to understand the host response against
E. chaffeensis (Winslow et al., 1998; Ganta et al., 2002, 2004). In this unit, methods are
Current Protocols in Microbiology 3A.3.1-3A.3.16, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc03a03s9
C 2008 John Wiley & Sons, Inc.
Copyright
Alpha
Proteobacteria
3A.3.1
Supplement 9
described to recover E. chaffeensis and E. canis from blood samples collected from clinically suspected dogs, deer, and human patients. Methods are also described to recover
Ehrlichia organisms from tissue samples collected from experimentally infected mice.
Molecular detection methods, such as PCR, RT-PCR, and real-time RT-PCR, are useful in
detecting Ehrlichia infection, as they are very sensitive and the assays can be performed
rapidly. Molecular methods have been described by several laboratories and most of
them are targeted to either 16S ribosomal DNA or ribosomal RNA (rRNA). 16S rRNA
based tests are 100 times more sensitive than PCR because the target is available in
multiple copies per bacterium (Felek et al., 2001; Sirigireddy and Ganta, 2005). Single
or co-infections with three Ehrlichia species, E. chaffeensis, E. canis, and E. ewingii, can
occur in animals or humans (Breitschwerdt et al., 1998; Murphy et al., 1998; Paddock and
Childs, 2003; Dumler et al., 2007). 16S rRNA-based multiplex real-time RT-PCR is most
efficient in detecting the aforementioned three Ehrlichia species (Sirigireddy and Ganta,
2005). DNA and RNA isolation and their use in detecting Ehrlichia by molecular methods
further aid in pathogen diagnosis and are complementary to culture recovery methods.
CAUTION: E. chaffeensis and E. canis are Biosafety Level 2 (BSL-2) pathogens. Follow
all appropriate guidelines and regulations for the use and handling of these pathogenic
microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more
information.
CAUTION: Some of the methods described in this unit also require the use of Animal
Biosafety Level 2 (ABSL-2) conditions. Follow all appropriate guidelines for the use and
handling of infected animals. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information. Protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.
CAUTION: The protocols described in this unit involve the use of human and/or other
animal blood or tissue samples that may be infected with Ehrlichia species and/or other
potential pathogens. Follow all appropriate guidelines and regulations for the use and
handling of human- and animal-derived materials. See UNIT 1A.1 and other pertinent
resources (APPENDIX 1B) for more information.
CAUTION: Follow strict sterile culture procedures in all steps involving sample processing and during cultivation to minimize contamination.
NOTE: When preparing solutions, use deionized or double distilled water (ddH2 O) and
use reagents of the highest quality. Sterilization by filtration through a 0.22-m filter
or by autoclaving is recommended for most solutions; this is critical for all reagents
used for cell culture work. Discard any reagent that shows evidence of contamination,
precipitation, or discoloration.
BASIC
PROTOCOL 1
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
Materials
Human blood sample in sterile blood collection tube containing EDTA
Hanks balanced salt solution (HBSS; without CaCl2 , MgCl2 , MgSO4 , or phenol
red; APPENDIX 2A)
Histopaque-1083 (Sigma-Aldrich)
3A.3.2
Supplement 9
Figure 3A.3.1 Schematic drawing of the different layers of blood sample before and after centrifugation with Histopaque-1083.
Alpha
Proteobacteria
3A.3.3
Current Protocols in Microbiology
Supplement 9
Materials
Heparinized deer blood samples infected with Ehrlichia chaffeensis
ACE lysing solution (see recipe)
Complete MEM medium (see recipe)
DH82 cells (60% confluent) in 25-cm2 tissue culture flask (UNIT 3A.1)
50-ml tubes
37 C, 5% CO2 incubator
Additional reagents and equipment for DH82 monolayer culture and Cytospin slide
preparation (UNIT 3A.1)
1. Collect 5 to 7 ml of heparinized deer blood infected with E. chaffeensis, transfer to
a sterile 50-ml tube containing 25 ml of ACE lysing solution, and mix gently by
inverting tube several times to form homogeneous solution.
2. Incubate 5 min at room temperature, centrifuge suspension 5 min at 160 g, room
temperature, and discard supernatant.
3. Add 25 ml ACE lysing solution to pellet, mix gently, centrifuge 5 min at 160 g,
room temperature, and then discard the supernatant.
4. Repeat step 3 one additional time.
5. Resuspend final pellet in 5 ml complete MEM medium and transfer contents to a
25-cm2 tissue culture flask containing a 60% confluent monolayer of DH82 cells.
6. Transfer flask to a 37 C, 5% CO2 incubator.
7. Check the infectivity two times a week by preparing a Cytospin slide and evaluating
under a light microscope as described in UNIT 3A.1, Basic Protocol 5. Change 50%
of the medium once every 3 to 4 days.
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
3A.3.4
Supplement 9
BASIC
PROTOCOL 3
This protocol describes a method for isolating E. chaffeensis or E. canis from the blood
of a naturally or experimentally infected dog. This protocol is adapted from Dawson
et al. (1991a) with minor modifications. This revised protocol uses a culture medium
containing 6.5% FBS rather than 12.5% FBS and the cultures are grown in the presence
of 5% CO2 .
Materials
Ehrlichia-infected dog blood in a sterile blood collection tube containing EDTA
Serofuge centrifuge (Becton Dickinson)
37 C, 5% CO2 incubator
Additional reagents and equipment for DH82 monolayer culture and Cytospin slide
preparation (UNIT 3A.1)
1. Collect 6 ml Ehrlichia-infected blood from a dog in a sterile blood collection tube
containing EDTA (purple tube) and use immediately.
Blood samples may be stored up to 1 week at 4 C prior to culture isolation. However, it
is best to use fresh blood to increase the likelihood of recovering Ehrlichia organisms.
BASIC
PROTOCOL 4
This protocol outlines a simple method useful for Ehrlichia isolation from experimentally
infected mice. The mouse model has been used extensively to understand the pathogenicity and to assess host immune response against E. chaffeensis infection (Winslow et al.,
1998; Ganta et al., 2002, 2004, 2007).
Materials
E. chaffeensis-infected mice
Sterile PBS (see recipe), ice cold
24-well sterile culture plates
37 C, 5% CO2 incubator
Additional reagents and equipment for DH82 monolayer culture and Cytospin slide
preparation (UNIT 3A.1)
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3. Resuspend cell pellet in 1/5 volume of DH82 cells harvested from a 25-cm2 tissue
culture flask having a complete monolayer of cells.
4. Transfer cell suspension to a well of a 24-well sterile culture plate and incubate in a
37 C, 5% CO2 incubator.
5. Check the infectivity two times a week by preparing a Cytospin slide and evaluating
under a light microscope as described in UNIT 3A.1, Basic Protocol 5. Change 50%
of the medium once every 3 to 4 days until infection is detected.
The culture may be monitored for up to 10 weeks if the infection is not observed prior
to that. If the cells reach 90% to 100% confluence during this incubation period, replace
the entire volume of medium, detach the cells by gently rocking the culture flask by hand
after adding 15 4-mm sterile glass beads, discard 50% of the culture using a sterile
serological pipet, and replace with an equal volume of fresh culture medium.
BASIC
PROTOCOL 5
Materials
PCR kit (Invitrogen cat. no. 10966-034) containing:
Platinum Taq DNA polymerase
10 PCR buffer
50 mM MgCl2
PCR/RT-PCR primers for E. chaffeensis and E. canis (Table 3A.3.1)
10 mM dNTPs
Nuclease-free water
DNA template
1% agarose gel containing 0.1 g/ml ethidium bromide
1-kb plus DNA ladder (or any other comparable molecular weight DNA standards)
0.2-ml thin-walled PCR/RT-PCR tubes
GenAmp9700 PCR thermal cycler (Applied Biosystems)
1. Prepare PCR master mix by adding the following per reaction (multiply by the
number of samples plus one or two extra reactions to compensate for pipetting
error):
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
3A.3.6
Supplement 9
Table 3A.3.1 Primers and Probes Used in PCR/RT-PCR and Multiplex Ehrlichia Real-Time
RT-PCR
Sequence
5 gcatacttggttataaata
E. chaffeensis-reverse primer
5 gtattaccgcggctgctggcac
E. canis-forward primer
5 cctctggctataggaaattg
E. canis-reverse primer
5 gtattaccgcggctgctggcac
5 ctcagaacgaacgctgg
5 catttctaatggctattcc
5 TET/cttataaccttttggttataaataatgttag/TAMRA
5 FAM/tatagcctctggctataggaaattgttag/TAMRA
5 ROX/ctaaatagtctctgactatttagatagttgttag/BQH2
For example, if preparing a master mix for ten reactions, add twelve times the suggested
volumes of each reagent and then transfer 23-l aliquots of the homogeneous mixture to
each reaction tube.
3. Transfer the reaction tubes to a GeneAmp9700 PCR thermal cycler or similar instrument and initiate the PCRs by setting up the machine for the following program:
1 cycle:
35 cycles:
1 cycle:
4 min
30 sec
30 sec
1 min
5 min
94 C (denaturation)
94 C (denaturation)
55 C (annealing)
72 C (extension)
72 C (extension).
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Figure 3A.3.2 PCR product analysis of E. chaffeensis and E. canis. Lanes 1 and 3 have the
PCR products in the reactions containing E. chaffeensis-specic PCR primers with and without the
templates, respectively. Similarly, lanes 2 and 4 included PCR products with E. canis primers with
and without the DNA template, respectively. A molecular weight marker (1-kb plus DNA ladder from
Promega) is included in lane M. Predicted molecular sizes for the PCR products of E. chaffeensis
and E. canis are 0.44 and 0.42 kb, respectively.
SUPPORT
PROTOCOL 1
Materials
ACE lysing solution (see recipe)
Whole blood
DNA extraction buffer (see recipe)
20 mg/ml proteinase K in 50 mM TrisCl, pH 7.5
Phenol, pH 8.0
25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
24:1 (v/v) chloroform/isoamyl alcohol
70% and 100% ethanol, ice cold
TE buffer (see recipe)
1.7-ml microcentrifuge tubes
Table top high-speed microcentrifuge
65 C water bath
1. Add 1 ml ACE lysing solution to 0.2 ml whole blood in a 1.7-ml microcentrifuge
tube, shake gently by inverting tube several times to create a homogenous mixture,
and incubate 5 min at room temperature.
2. Centrifuge lysate 5 min at 160 g, room temperature, and discard supernatant.
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
3. Add 1 ml ACE lysing solution to carefully rinse the white blood cell pellet.
This procedure aids in the lysis of red blood cells. Steps 1 to 3 are not needed when using
this protocol for isolating genomic DNA from a cell culture pellet.
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Supplement 9
4. Add 0.5 ml DNA extraction buffer containing 12.5 l proteinase K (20 mg/ml) to
the cell pellet, mix gently by pipetting up and down, and incubate 2 hr in a 65 C
water bath.
Incubation may be extended for up to 20 hr.
5. Add 0.5 ml phenol and gently vortex or mix the contents by inverting the tube several
times.
CAUTION: Phenol is extremely toxic, highly corrosive, and can cause severe burns. It
may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves,
goggles, and protective clothing. Always use in a chemical fume hood. Rinse any areas of
skin that come in contact with phenol with a large volume of water and wash with soap
and water; do not use ethanol. Also see UNIT 1A.3.
BASIC
PROTOCOL 6
Materials
SuperScript III, one-step RT-PCR system with Platinum Taq DNA polymerase and
2 buffer (Invitrogen cat. no. 11732-020)
PCR/RT-PCR primers for E. chaffeensis and E. canis (Table 3A.3.1)
RNAsin (RNase inhibitor; Promega cat. no. N2515)
Nuclease-free water
RNA template
1.5% agarose gel containing 0.1 g/ml ethidium bromide (Voytas, 2000)
1-kb plus DNA ladder (or any other comparable molecular weight DNA standards)
0.2-ml thin-walled PCR/RT-PCR tubes
GenAmp9700 PCR thermal cycler (Applied Biosystems)
1. Prepare an RT-PCR master mix by adding the following for a total volume of 23 l
per reaction (multiply each volume with the number of samples plus one or two
reactions for pipetting error):
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2. Add 2 l RNA template or water (for negative control) to each reaction tube containing 23 l of master mix.
3. Transfer reaction tubes to a thermal cycler and initiate RT-PCR by performing the
parameters described in Basic Protocol 5, step 3, except add a 1-hr incubation cycle
at 48 C at the start of the PCR cycles to aid in synthesizing cDNA.
4. Resolve 5 l PCR products (along with DNA markers) on a 1.5% agarose gel
containing 0.1 g/ml ethidium bromide (Voytas, 2000).
Molecular weight markers should also be resolved on agarose gels to aid in the identification of the predicted 0.4-kb DNA amplicon (Fig. 3A.3.2).
BASIC
PROTOCOL 7
Materials
SuperScript III, one step RT-PCR system with Platinum Taq DNA polymerase and
2 buffer (Invitrogen cat. no. 11732-020)
10 mM dNTPs
Real-time RT-PCR primers and TaqMan probes for E. chaffeensis, E. canis, and
E. ewingii (Table 3A.3.1)
Nuclease-free water
RNA (see Support Protocol 2)
1.7-ml microcentrifuge tubes
PCR tubes for Smart Cycler (Cepheid cat. no. 11-400-3)
Smart Cycler real-time PRC machine (Cepheid)
1. Prepare a master mix in a 1.7-ml microcentrifuge tube by adding the following
reagents for a total volume of 23 l per reaction (multiply each volume by the
number of samples plus one or two reactions for pipetting errors):
For example, if preparing a master mix for ten reactions, add twelve times suggested
volume of each reagent and then transfer 23-l aliquots of the homogeneous mix to each
reaction tube.
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Supplement 9
2. Dispense 23 l of master mix into real-time RT-PCR tubes and add 2 l of RNA or
water (negative control) as templates.
The experiment should include a positive control (DNA or RNA template from known
positives) and a negative control that contains all the reagents except a DNA or RNA
template.
3. Perform real-time RT-PCR in a Smart Cycler system (or similar instrument that is
designed for use in performing TaqMan probe-based real-time RT-PCR assays) using
the following parameters:
1 cycle:
1 cycle:
45 cycles:
30 min
2 min
15 sec
30 sec
1 min
48 C (reverse transcription)
94 C (initial denaturation)
94 C (denaturation)
50 C (annealing)
60 C (extension)
The optics of Smart Cycler should be set to turn on during the extension phase of each
amplification cycle and, similarly, the threshold fluorescence intensity should be set to
10 U. The dye set should be selected as FTTR (FAM, TET, TAMRA, and ROX). At
the completion of the setup cycles, the results are displayed on a computer attached to the
machine. If a sample tests positive, a Ct-value (the cycle at which the fluorescence crossed
the threshold of 10 U for a specific pathogen probe) will be displayed for the positives
(Fig. 3A.3.3). A reaction for which the Ct-value is displayed as 39 is considered as
positive. If a blood sample has more than one pathogen, real time RT-PCR analysis is
expected to yield positive results for more than one pathogen.
Figure 3A.3.3 Real-time data generated for Ehrlichia triplex real-time RT-PCR using a Smart
Cycler system is presented. Equal molar concentrations of in vitrosynthesized templates of
E. chaffeensis, E. canis, and E. ewingii are used to demonstrate the detection by real-time assay
(Sirigireddy and Ganta, 2005). Negative controls contained all components of a real-time RT-PCR
mixture with the exception of the templates (template volume replaced with water). The Ct values
generated by the Smart Cycler are presented as a table format generated by the machine.
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SUPPORT
PROTOCOL 2
Materials
Ehrlichia-infected blood, tissue samples, or culture organisms
Tri-reagent and Tri-reagent BD (Sigma-Aldrich) or similar reagents (available from
other manufacturers)
TE buffer (see recipe) or nuclease-free water
RNAsin (RNase inhibitor; Promega cat. no. N2515)
1.7-ml microcentrifuge tubes
Table-top high-speed microcentrifuge
1. Isolate total RNA from Ehrlichia-infected blood, tissue samples, or culture organisms
using the Tri-reagent or Tri-reagent BD (from blood) method by following the
manufacturers instructions.
2. Dissolve RNA pellet in 100 l TE buffer or nuclease-free water.
3. Add 40 U RNAsin to purified RNA solution and store up to 2 years at 80 C until
use.
4. Use 2 l of each DNA or RNA in PCR or RT-PCR assays, respectively, to determine
the presence of Ehrlichia.
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
3A.3.12
Supplement 9
COMMENTARY
Background Information
Several emerging tick-borne rickettsial infections have been reported in recent years
and have shown to significantly impact
the health of humans and other vertebrates
(Paddock et al., 1993; De Silva et al., 1996;
Dumler et al., 2001). These include infections by E. chaffeensis, a human monocytic
agent, E. ewingii, a human ewingii ehrlichiosis agent, and Anaplasma phagocytophilum,
a human granulocytic anaplasmosis agent
(Dawson et al., 1991a; Chen et al., 1994;
De Silva et al., 1996; Caspersen et al., 2002;
Dumler et al., 2007). E. canis has been identified as the major cause of tick-borne infections
in dogs in nearly all parts of the world for several decades (Haas and Meyer, 1986).
Culture methods that serve as the gold standard for pathogen diagnosis are also described
for recovering Ehrlichia species from human,
deer, and dog blood and mouse tissue samples
(Dawson et al., 1991a,b; Lockhart et al., 1997;
Paddock et al., 1997; Breitschwerdt et al.,
1998; Ganta et al., 2002, 2004, 2007). Simple step-by-step protocols are described in this
unit to aid in isolating Ehrlichia species from
clinically suspected patient samples.
Accurate diagnosis aids considerably in
defining treatment regimes for infections with
Ehrlichia species in humans or animals. In this
unit, various protocols for DNA and RNA isolation and molecular methods for rapid diagnosis are presented. Various laboratories have
described similar methods (Felek et al., 2001;
Sirigireddy and Ganta, 2005) and the methCurrent Protocols in Microbiology
Critical Parameters
Isolation and cultivation of Ehrlichia from
infected animal samples
Isolating Ehrlichia from human and/or animal blood or tissue samples can be very challenging; thus one should carefully follow all
the steps for culture isolation as outlined in
this unit.
The volume of blood used is important for
culture isolation. Consider using a large volume of blood sample (up to 30 ml) for culture recovery experiments, particularly when
a blood sample is obtained from an animal
suspected to be having a chronic phase of infection.
If an animal is under antibiotic treatment,
the probability of culture recovery of Ehrlichia
significantly decreases.
Culture must be maintained by following
strict sterile culture techniques to avoid bacterial and fungal contamination. Cultures should
be monitored for up to 10 weeks before considering a sample to be negative.
Nucleic acid isolation and molecular
detection methods
It is important to prevent the introduction
of previously amplified products into PCR/RTPCR reactions to avoid false positives.
It is critical to have two independent
laboratories to physically separate the prePCR assembly (which includes nucleic acid
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Supplement 9
Table 3A.3.2 Troubleshooting Guide for Ehrlichia Isolation and Its Molecular Applicationa
Problem
Possible cause
Solution
No Ehrlichia-positive cells
observed despite the identification
of morulae-like structure in a blood
smear
One or more of the ingredients of master Carefully follow the protocols for PCR
mix are contaminated with previously
assembly and analysis as described
amplified products or genomic DNA or
RNA templates
a For additional troubleshooting information related to culture methods, see Table 3A.1.1.
Troubleshooting
Table 3A.3.2 presents some of the common
problems associated with the protocols in this
unit.
Anticipated Results
Isolating Ehrlichia from infected vertebrate
host
If starting with a sufficient volume of fresh
blood (which may range from 5 to 30 ml),
then the Ehrlichia inclusions in macrophage
cell culture are expected to be seen within
7 days to 3 weeks. If the bacterial load is
very low, detection of culture positives may
take up to 6 to 10 weeks. After 10 weeks of
culture monitoring, a sample may be considered negative for Ehrlichia.
Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
Molecular methods
Both PCR and RT-PCR methods are extremely sensitive in detecting low levels of infection; however, RT-PCR is 100 times more
sensitive as the 16S rRNA target is present in
hundreds of copies per cell. If a sample is positive for Ehrlichia, a predicted size amplicon
is seen following resolution of PCR/RT-PCR
Acknowledgments
This study was supported by the National
Institutes of Health grants AI055052 and
AI070908. The manuscript is published as
Kansas Agricultural Experiment Station contribution no. 08-223-B.
Literature Cited
Breitschwerdt, E.B., Hegarty, B.C., and Hancock,
S.I. 1998. Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia
chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or
Bartonella vinsonii. J Clin. Microbiol. 36:26452651.
Buhles, W.C., Huxsoll, D.L., and Ristic, M. 1974.
Tropical canine pancytopenia: Clinical, hematologic, and serologic response of dogs to
Ehrlichia canis infection, tetracycline therapy, and challenge inoculation. J. Infect. Dis.
130:357-367.
Caspersen, K., Park, J.H., Patil, S., and Dumler,
J.S. 2002. Genetic variability and stability of
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Isolation and
Molecular
Detection of
Ehrlichia from
Vertebrate
Animals
3A.3.16
Supplement 9
UNIT 3A.4
3A.4.1
Supplement 5
BASIC
PROTOCOL
Materials
Wolbachia-infected and -uninfected Drosophila embryos (Support Protocol 1)
Apple juice agar plates (Support Protocol 2; Fig. 3A.4.1)
50% bleach solution (Clorox bleach recommended)
Halocarbon oil 700 (Sigma)
Uninfected Drosophila males
Camel hair paint brush (Fig. 3A.4.1) or alternate preferred tool for manipulating
embryos
Fine mesh for embryo collection and washing
Filter paper, damp
Double-sided tape (e.g., Scotch, #666)
Glass slides
Microinjector, microinjection needles (Support Protocol 3; Fig. 3A.4.2D,E) and
micromanipulator
Dissecting microscope
Compound microscope with movable stage
Prepare Drosophila embryos
1. After female flies oviposit in the yeast paste coating of apple juice agar plates (Fig.
3A.4.1), add 2 l water and agitate using a soft paint brush to suspend embryos.
Embryos that are inserted into the agar are relatively difficult to remove and may be
ignored since their extraction is not an efficient use of time.
A fine camel hair brush works well, but brush preference varies between users. Thus,
several brush types may be purchased and tested. Metal probes are also useful tools for
manipulating embryos.
2. Filter embryos suspended in the yeast through mesh and rinse thoroughly with water
to remove yeast.
Figure 3A.4.1 shows an inexpensive design for embryo collection.
Transfection of
W. pipientis into
Drosophila
Embryos
3A.4.2
Supplement 5
Figure 3A.4.1 Tools for embryo collection. (A) Brush for egg manipulation, apple juice plate,
funnel, and disassembled mesh sieve. (B) Drosophila media bottles, a disassembled egg collection
chamber, and an assembled egg collection chamber. (C) Assembled mesh sieve with water bottle
and brush for suspending embryos off the apple juice plates.
6. Align embryos on a piece of apple juice agar (Fig. 3A.4.2B) using a wet brush or
another preferred tool.
The anterior of embryos may be recognized by its tapering end and the projecting point of the micropyle (site of sperm entry). Embryos should be oriented in the
same anterior/posterior direction, touching laterally and flush along the posterior axis
(Fig. 3A.4.2C). Groups of 30 embryos work well.
7. Place a piece of double-sided tape along the edge of a clean glass slide and invert over
the row of embryos on agar. Slowly, lower the tape into contact with the embryos,
such that the embryos adhere to the tape.
Note that the tape may be toxic to embryos. The suggested brand was observed to work
well with Drosophila embryos. If it cannot be obtained, alternate brands should be tested
empirically for toxicity effects.
8. Lift the tape carefully from the slide so as not to damage the embryos and replace
onto the center of the same slide, such that the tape no longer overhangs the slide.
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Figure 3A.4.2 Dechorionated embryos on mesh (A) are transferred and aligned on agar (B). (C)
Aligned embryos on tape under oil. Note that some embryos are too old (marked with asterisk)
or are oriented in the incorrect direction (within box marked with a cross). The latter would be
destroyed prior to microinjection. (D) Microinjection needle with (E) detail of the needle tip. (F) A
sequence of penetrating and injecting an embryo. The smallest increment of the scale shown in
(D) and (E) is 10 m.
Transfection of
W. pipientis into
Drosophila
Embryos
Desiccation is an important step: over-desiccation will reduce survivorship while underdesiccation will result in embryo leaking upon microinjection. As each laboratory differs
in its conditions, the desiccation time must be determined empirically for each location.
Slides with embryo groups can be desiccated for varying lengths of time and then microinjected. The appropriate time will be based upon observations during injection (e.g.,
leaking) and observed survivorship. While embryos may be desiccated in ambient room
conditions, room conditions may change daily in some locations. Thus, a desiccator may
be used to promote uniformity (see http://en.wikipedia.org/wiki/Desiccator for additional
information).
3A.4.4
Supplement 5
10. Cover the embryos with water-saturated halocarbon oil (Fig. 3A.4.2C) and store at
18 to 22 C.
12. Insert the needle into the posterior of a donor embryo and withdraw 10% of the
yolk. Withdraw needle and repeat with an adjacent embryo. Repeat with ten or more
embryos to obtain sufficient Wolbachia-infected cytoplasm for injections.
In this step and subsequently, it is important not to expose the needle to air for extended
periods, which will allow yolk at the needle tip to dry and clog the needle. Instead, keep
the needle tip submerged in oil.
13. Exchange slides, replacing infected donor embryos with uninfected recipient embryos. Orient the needle as described in step 11. Prepare for embryo injection.
Embryo injection (step 14) should be an abrupt event (Fig. 3A.4.2F). The needle should
be quickly plunged into the embryo. If properly desiccated, the embryo should dimple
only slightly during injection. Effort should be made to prevent the needle tip from going
too deep (>20% of the embryo length).
If the embryo has not been sufficiently desiccated, cytoplasm can leak from the embryo.
Often the leaking cytoplasm can be observed flowing into the needle. If the latter occurs,
the needle should be withdrawn from the embryo, and the recipient embryo cytoplasm
should be expelled from the needle before proceeding. The cytoplasm may be expelled
into the oil away from embryos.
14. Use the microinjector to pump cytoplasm from the needle into the embryo. Withdraw
the needle slowly as the embryo is filled.
The embryo should visibly swell during the injection.
The pump technique depends upon the type of microinjector. Different needles will also
vary in the amount of pressure required. Even using the same needle, the pressure required
will change depending upon the amount of cytoplasm remaining in the needle and periodic
clogging. Thus, the pressure must be constantly readjusted during injection. A useful
strategy permitted by some microinjectors that allow the user to set both the pressure and
time of injections is to set the time for a period that is shorter than needed. In this way,
the user can adjust for embryo variation by simply adjusting the number of pumps into
the recipient embryo.
Needle clogging is a frequent problem during injections. Many microinjectors include a
clearing function that allows for a brief pulse of high pressure to clear clogs. Clearing of
clogs should be done away from embryos in the oil.
15. Once finished injecting embryos on the slide, transfer to a dissecting microscope and
use a probe to crush or tease away and remove uninjected embryos to simplify the
downstream screening of injected lines.
Killing of embryos should not be done with the injection needle due to the risk of damaging
the needle. Instead, while injecting, mentally keep track of the embryos to be killed by
their position in the line.
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Problem embryos occur commonly for several reasons including: (1) embryo overdeveloped, attempting to inject an embryo after cell formation increases the risk of damaging
the needle; (2) embryos not attached firmly to tape and move away from the needle when
try to inject.
17. Observe slides frequently (multiple times per day) for larvae. When observed, gently
remove larvae from the oil and transfer onto Drosophila media (e.g., Jazz Mix;
Applied Scientific).
If allowed sufficient time, larvae will crawl out of the oil to become lost and die.
The surface of fly media will dry and develop a skin that is difficult for a single larva
to penetrate, resulting in mortality of transferred larvae. Thus, media quality should be
examined. The surface of media can be scratched prior to adding larvae and the larvae
placed into the resulting groove. Larvae may be gently manipulated on the media to
reduce the amount of oil on the larvae.
18. Once pupae are observed (6 days), monitor frequently to obtain virgin females.
Injected individuals are often not observed again until pupation, when they migrate to the
surface and commonly attach to the container wall.
Females will not mate for 8 hr post-eclosion.
Females and males should be removed when observed and transferred individually into
vials with media. The rationale for preventing injected females and males from mating is
that their Wolbachia-infection status is unknown.
19. Mate injected females (G0 ) with males known to be Wolbachia uninfected and then
place individually in vials to oviposit. Once sufficient embryos (G1 ) are obtained,
sacrifice the female and perform PCR to detect Wolbachia infection.
Based upon prior work (Xi and Dobson, 2005), G0 females that are negative via PCR
assay are unlikely to result in transfected lines and may be discarded with their progeny.
G0 females that are positive via PCR assay do not necessarily result in transfected lines.
Thus, PCR assays of the G1 progeny are required for the latter. Isofemale lines with G1
PCR positive individuals were observed to be stably infected.
Appropriate PCR primers will depend upon the Wolbachia infection. However, PCR with
the general wsp primers (Zhou et al., 1998) have been used by the authors for the general
detection of the presence/absence of infections.
SUPPORT
PROTOCOL 1
Transfection of
W. pipientis into
Drosophila
Embryos
3A.4.6
Supplement 5
embryos in the late afternoon or evening. Thus, it may be advantageous to maintain the
flies on a shifted light-dark cycle.
To maximize embryo production, it is advantageous to know the age of flies used to
generate embryos. Therefore, cohorts of similarly aged flies can be generated by emptying
adults from rearing bottles that contain many immatures and recording the date. Adults
that are subsequently removed from the same bottles fall within a known time period.
A variety of designs for embryo collecting apparati have been described (Wieschaus
and Nusslein-Volhard, 1986; Santamaria, 1987). An inexpensive version consists of
polypropylene Drosophila media bottles (AS-355, Fisher Scientific) inverted over apple
juice plates with yeast paste (Fig. 3A.4.1B).
Approximately 2 days prior to embryo collection, adults are transferred from culture bottles with fly food media such as Jazz Mix from Applied Scientific (media bottles; Fig.
3A.4.1B) into embryo collection chambers to become accustomed to the environment.
The flies may remain in the collection chambers for more than a week for continued
collections, but the apple juice plates must be exchanged at least once per day. Therefore,
if embryo collections are not to be repeated for several days, it may be convenient to
return the flies into media bottles when finished with embryo collection.
When collecting embryos, disturbing the flies can reduce oviposition. Therefore, to
reduce disturbances, they can be covered with a black cloth. A notation system may be
used to identify unproductive collection chambers, which can be replaced with groups
of females that are more productive. Ten productive collection chambers should yield a
sufficient number of embryos for a two person team. However, if collection is to continue
for several hours, additional chambers should be made ready to substitute for exhausted
females.
SUPPORT
PROTOCOL 2
Materials
Agar
Sucrose
Apple juice
10% Tegosept (optional, Fisher Scientific)
Live bakers yeast
35 10mm petri plates (BD Falcon)
1. Dissolve 12 g agar and 12.5 g sucrose in 375 ml distilled water by stirring with heat.
Stir fast enough to maintain the suspension but do not create bubbles and do not
allow to boil over.
2. Once dissolved, remove from heat and continue to stir until the temperature drops to
80 C.
3. While stirring, add 125 ml apple juice followed by 3.5 ml of 10% Tegosept.
Tegosept (an antifungal agent) is not required, but facilitates long-term storage of plates.
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Current Protocols in Microbiology
Supplement 5
4. Immediately divide into aliquots in small petri plates (35 10mm; BD Falcon; see
Fig. 3A.4.1). Add at least enough to cover the bottom of the plate.
Plating should be done quickly, before the solution solidifies.
The petri plates work well with the embryo collection chamber that is described above.
Materials
Micropipet puller (Sutter Instrument Co., P-87)
Borosilicate glass capillaries (World Precision Instruments, TW100F-4)
Micropipet beveller (Sutter Instrument Co., BV-10)
NOTE: Microinjection needles may be purchased commercially (e.g., Eppendorf). The
author does not have experience with purchased needles. Alternatively, needles may
be prepared if appropriate equipment is available. The program described here should
serve as a starting point only. The parameters will require adjustment depending upon
the Puller model, ambient conditions, and the age of the heating element.
1. Using an automated micropipet puller, pull needles to be used for microinjection
(Fig. 3A.4.2D,E) from capillaries using the two-step program in Table 3A.4.1.
Step 1
Step 2
Heat
405
370
Pull
0
150
Velocity Time
10
150
40
50
Pressure
0
300
2. Bevel needle tips to a 20 angle using the micropipet beveller (Fig. 3A.4.2E).
COMMENTARY
Background Information
Transfection of
W. pipientis into
Drosophila
Embryos
3A.4.8
Supplement 5
protocol for the transfer of Wolbachia infections (transfection) via embryonic microinjection is described.
Breeuwer, J.A. and Werren, J.H. 1990. Microorganisms associated with chromosome destruction
and reproductive isolation between two insect
species. Nature 346:558-560.
Anticipated Results
Once appropriate technique is developed,
>30% hatch rate can be obtained, with 50%
of larvae reaching adulthood (Xi and Dobson, 2005). Transferring from Wolbachiainfected Drosophila simulans to aposymbiotic
Drosophila simulans, >60% infection can be
detected by PCR of injected individuals surviving to adult (G0 ). However, a subset of the
G0 females stably transmit the infection to offspring (Xi and Dobson, 2005).
Time Considerations
With a team of two people and assuming that the flies are laying sufficient eggs,
>500 eggs may be injected in a day. Screening slides for survivorship and transferring survivors requires only a few minutes and should
be done multiple times per day until the slides
are discarded. Once larvae are transferred to
media bottles, the development of injected flies
may be significantly reduced relative to uninjected flies. Thus, media bottles should be
observed for >2 weeks at 27 C.
Literature Cited
Bandi, C., Anderson, T.J.C., Genchi, C., and
Blaxter, M.L. 1998. Phylogeny of Wolbachia in
filarial nematodes. Proc. R. Soc. Lond. [Biol.]
265:2407-2413.
Boyle, L., ONeill, S.L., Robertson, H.M., and
Karr, T.L. 1993. Interspecific and intraspecific
horizontal transfer of Wolbachia in Drosophila.
Science 260:1796-1799.
Alpha
Proteobacteria
3A.4.9
Current Protocols in Microbiology
Supplement 5
3A.4.10
Supplement 5
Zhou, W., Rousset, F., and ONeil, S.L. 1998. Phylogeny and PCR based classification of Wolbachia strains using wsp gene sequences. Proc.
R. Soc. Lond. [Biol.] 265:509-515.
Alpha
Proteobacteria
3A.4.11
Current Protocols in Microbiology
Supplement 5
UNIT 3B.1
This unit presents simple methods for growth and maintenance of Brucella abortus. B.
abortus grows more slowly than E. coli K-12; therefore, experiments with this organism
require advance planning to have it ready and in the correct growth phase. Since brucellosis is one of the most frequent laboratory-acquired infections, avoidance of infection
requires careful microbiological technique. All work with B. abortus must be performed
at Biosafety Level 3 (BSL-3), and experiments with B. abortus cultures are performed in
a Class II biosafety cabinet (BSC; see UNIT 1A.1). In the U.S., laboratories working with
this bacterium are required to seek prior approval from the Select Agent Program of either the Department of Agriculture (USDA) or the Center for Disease Control (CDC; see
Internet Resources), and individual laboratory personnel must receive security clearance
from the CDCs Select Agent Program to access cultures of B. abortus. Other countries
may have similar regulations. Be sure to check with the appropriate authorities before
starting work with Brucella (e.g., see APPENDIX 1B). Receipt of Brucella cultures from
interstate sources is also controlled and requires a separate permit from the USDA for
transfer of organisms that may be harmful to livestock or poultry.
Basic Protocol 1 describes how to grow B. abortus in liquid culture. Many Brucella
genes have been shown to be regulated by growth phase; therefore, particular attention is
given to growth phases to allow reproducibility between experiments. Basic Protocol 2
describes how to generate laboratory stocks of B. abortus for long-term frozen storage and
discusses regulations pertaining to maintaining a Brucella strain collection. The Alternate
Protocol describes how to generate stocks for long-term storage by lyophilization. Basic
Protocol 3 gives a method for detecting undesirable rough colonies, which result from
spontaneous variants appearing after extended in vitro passages without selection in an
animal host.
CAUTION: B. abortus is a BSL-3 pathogen. Follow all appropriate guidelines for the use
and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
NOTE: B. abortus has been classified as a Select Agent by the United States Government. Refer to the CDCs Select Agent Program for more information (http://www.
cdc.gov/od/sap). Also refer to UNIT 1A.1 and other pertinent resources (APPENDIX 1B).
BASIC
PROTOCOL 1
Materials
B. abortus strain
Agar plates:
Tryptic soy agar (TSA) plates (see recipe)
TSA blood agar plates (see recipe)
Brucella agar plates (e.g., BBL, Oxoid, EMD), prepare according to
manufacturers instructions
Potato infusion agar plates (PIA; see recipe)
Tryptic soy broth (TSB; see recipe)
70% (v/v) ethanol in a spray bottle
Contributed by Yao-Hui Sun, Andreas B. den Hartigh, Renee M. Tsolis, and Thomas A. Ficht
Current Protocols in Microbiology (2005) 3B.1.1-3B.1.11
C 2005 by John Wiley & Sons, Inc.
Copyright
Alpha
Proteobacteria
3B.1.1
Figure 3B.1.1
circles OD600 .
Growth of Brucella abortus 2308 in tryptic soy broth. Solid circles, CFU/ml; empty
2. Aliquot 5 ml sterile tryptic soy broth (TSB) to a sterile, disposable, 15-ml polypropylene centrifuge tube
3. Using a 1-l inoculating loop, inoculate the tube with a loopful of bacteria from the
plate. Close the tube securely, and vortex to suspend the bacteria.
4. To measure the optical density, add 4 ml TSB into a 10-mm, screw-capped, glass tube
that will fit into a Spectronic 20 spectrophotometer. Add 5 ml of TSB to an empty
tube as a blank for the spectrophotometer. Pipet 1 ml of the B. abortus suspension
into the tube with 4 ml TSB (1:5 dilution), close the cap securely, and vortex to mix.
Laboratory
Maintenance of
Brucella abortus
CAUTION: All work with open B. abortus cultures must be performed within a Class II
BSC (see UNIT 1A.1).
3B.1.2
Current Protocols in Microbiology
The authors recommend use of a Spectronic 20 spectrophotometer with holder for 10-mm
screw-capped glass cuvettes, or a Klett colorimeter (fitted with a 660-nm filter), which
will also hold the screw-capped glass tubes. The advantage of these instruments is that
the cuvettes can be tightly closed, allowing the instrument to be located outside the BSC.
However, any other spectrophotometer that uses plastic cuvettes without lids can also be
used within the BSC. This protocol specifies the Spectronic 20, but can be modified to fit
another spectrophotometer by scaling the volumes up or down to fit the capacity of the
cuvettes used. The Klett colorimeter uses arbitrary units (Klett units) to measure culture
density, so a growth curve similar to the one shown in Figure 3B.1.1 can be established
for this instrument using the Klett meter.
5. Place the dilution tubes into a rack. Spray the outside of the tubes and the rack with
70% ethanol and place them into a second, closed container (e.g., a large plastic box
with lid) for transport to the spectrophotometer.
CAUTION: If there has been any leakage or spillage of bacteria on the outside of the tubes,
spray them first with 10% bleach or 1% Virkon S, then with 70% ethanol, to decontaminate
them.
6. Wipe the 70% ethanol off the surface of the tubes and measure the optical density
at 600 nm (OD600 ) against the blank tube containing 5 ml TSB. To be accurate,
the OD600 reading must be between 0.1 and 0.4; if the OD600 is outside this range,
make a second dilution of the original suspension from step 3 and repeat the OD600
measurement.
While the screw-capped tubes require a large (5-ml) sample volume, compared to plastic
cuvettes, they have the advantage that the sample is contained in a closed tube, which
allows the spectrophotometer to be located outside the BSC, saving valuable workspace.
7. Calculate the OD600 by multiplying the measured OD600 by the dilution factor (e.g.,
measured OD600 of 0.2 1:5 dilution = OD600 of 1.0 in the original suspension).
An OD600 of 1.0 is equivalent to 2 to 4 109 CFU/ml of B. abortus.
8. Calculate the dilution factor of the original culture needed to generate a culture with
an OD600 of 0.01, and dilute accordingly into a sterile, disposable 50-ml polypropylene centrifuge tube containing 5 ml TSB.
9. Place the tubes in a 1-liter polypropylene straight-sided jar that has been fitted with
styrofoam tube racks (see Fig. 3B.1.2) and close. Place the jar in a shaking incubator
and incubate overnight (16 hr) with shaking at 180 rpm.
10. Remove the jar from the shaker and open it in the BSC.
Figure 3B.1.2 Double containment jar for incubating Brucella cultures. To construct, cut the
styrofoam package insert from the 50-ml conical tubes to t a 1000-ml straight-sided jar (e.g.,
Nalgene) and insert. This will secure the 50-ml conical tubes during incubation.
Alpha
Proteobacteria
3B.1.3
Current Protocols in Microbiology
11a. For immediate use: Dilute the culture to the required concentration for the application.
11b. For short-term storage: Place the culture on ice for 1 to 2 hr.
Liquid cultures of B. abortus will rapidly lose viability if stored at 4 C. Viability can
be improved by centrifuging the cells 20 min at 5000 g, room temperature, and
resuspending the pellet in peptone saline maintenance medium before storage.
11c. For long-term storage: Proceed to Basic Protocol 2 or the Alternate Protocol.
BASIC
PROTOCOL 2
Materials
B. abortus strain to be stored
TSA blood agar plates (see recipe)
TSB/50% (v/v) glycerol (see recipe)
15-ml disposable polypropylene centrifuge tubes, sterile
1.5-ml freezer vials
1. In a Class II BSC, streak the Brucella strain to be stored onto a TSA blood agar plate
and incubate 3 to 4 days at 37 C, 5% CO2 , until colonies are visible.
Colonies will be colorless and domed (convex), and will reach 1 to 2 mm in diameter. B.
abortus is nonhemolytic on TSA-blood agar plates. PIA, Brucella agar, or TSA can also
be used, but B. abortus will grow more slowly on TSA.
Not all Brucella strains require CO2 for growth, so the growth requirements for each
individual strain should be tested.
Laboratory
Maintenance of
Brucella abortus
4. Aliquot 1 ml of the bacterial suspension to each of two freezer vials. Close the vials
securely, spray the outside of the vials with 70% ethanol, and wipe off the ethanol
with a paper towel before storing at 80 C.
3B.1.4
Current Protocols in Microbiology
In the absence of repeated freeze-thaw cycles, the freezer stocks should maintain viability
indefinitely. For frequently used strains, it is a good policy to use one of the freezer vials
as a working stock to inoculate cultures, as this vial may lose viability over time with
repeated thawing and freezing due to removal from the freezer. The second vial should be
held in reserve in case the working stock suffers said loss of viability.
Other methods of long-term storage, such as storage in liquid nitrogen or lyophilization
(see Alternate Protocol), will also maintain viability of Brucella stocks; however, liquid
nitrogen storage of vials has the disadvantage that the vials may explode on removal,
creating infectious aerosols.
ALTERNATE
PROTOCOL
CAUTION: This protocol is not the best choice for preservation of virulent Brucella
stocks, as it requires transport of loosely stoppered vials between the BSC and the
lyophilizer. Extra respiratory protection should be used (e.g., a HEPA-filtered respirator)
for this step to prevent infection in case of a spill.
Alpha
Proteobacteria
3B.1.5
Current Protocols in Microbiology
CAUTION: Be sure the tubes are balanced properly, as imbalance can lead to centrifuge
accidents, resulting in aerosolization of Brucella.
5. Carefully pipet off the supernatant and discard into a beaker containing 50 ml
undiluted bleach. Resuspend each pellet in 10 ml peptone saline maintenance medium
by vortexing.
6. Pellet the cells and resuspend in peptone saline maintenance medium a total of three
times, using the same centrifugation conditions as in step 4.
7. After the third wash, resuspend cells in stabilizing medium at a concentration of
1010 CFU/ml.
The concentration can be estimated by measuring in a spectrophotometer (see Basic
Protocol, steps 4 to 8).
The cell suspension may be stored up to 3 days at 4 C before lyophilizing.
8. Aliquot 1 ml of the bacterial suspension aseptically into sterile 10-dram glass vials.
Set autoclaved stoppers loosely on top of the vials and place them onto a tray that
will fit on a shelf inside the lyophilizer.
CAUTION: Use special care when transporting the loosely stoppered vials to the
lyophilizer, as potential exists for spilling viable Brucella outside the BSC.
9. Run the lyophilizer according to the manufacturers instructions so that the final
temperature does not exceed room temperature and the residual moisture content is
1% to 2%.
The lyophilizer should be located inside the BSL-3 lab.
The authors routinely employ a temperature range of between 20 and 0 C in the
evaporating chamber and 50 to 60 C in the condensing chamber.
10. Use the freeze dryers internal stoppering device to seal all of the vials at the end of
the run and prior to returning the evaporation chamber to atmospheric pressure.
Failure to properly seal the vials will result in condensation, resuspension of the bacteria,
and reduction in viability.
11. Place metal crimp closures on the vials to prevent the rubber stoppers from being
removed inadvertently.
12. Check viability of one sample vial by resuspending the contents in 1 ml peptone
saline maintenance medium and performing ten-fold serial dilutions. Spread 0.1 ml
of each dilution on a TSA plate and incubate for 3 to 4 days at 37 C, 5% CO2 , until
individual colonies can be counted.
Better than 99.9% recovery is expected. Vials may be stored at 4 C for up to 2 years
without loss of viability.
BASIC
PROTOCOL 3
Laboratory
Maintenance of
Brucella abortus
3B.1.6
Current Protocols in Microbiology
individual colonies on the surface of solid medium with crystal violet, which will stain
rough but not smooth colonies.
Materials
B. abortus growing on solid medium
Crystal violet stock solution (see recipe)
Agar plate containing well-spaced individual colonies of Brucella
10% (v/v) bleach solution in a beaker
1. Dilute the crystal violet stock solution 1:40 with sterile distilled, deionized water.
2. In a Class II BSC, flood the surface of each plate with 1 ml of diluted crystal violet
solution. Allow the plates to stand for 30 sec to 1 min.
Allowing the liquid to stand too long on the plates may cause the colonies to float off the
surface of the agar.
3. Carefully pipet off the crystal violet solution into a beaker of 10% bleach solution.
4. Examine the colonies on the plate. Restreak individual colonies for isolation to make
a stock culture.
Rough colonies are stained dark blue while smooth colonies resist staining and remain
white.
Alpha
Proteobacteria
3B.1.7
Current Protocols in Microbiology
Stabilizing medium
2.5% (w/v) Bacto Casitone (Difco)
5% (w/v) sucrose
1% (w/v) sodium glutamate
Sterilize by passing through a 0.2-m filter unit
Store indefinitely at 4 C
Tryptic soy agar (TSA)
Dissolve the following in a 2-liter Erlenmeyer flask containing 1 liter H2 O:
15 g pancreatic digest of casein
5 g Bacto Soytone (Difco)
5 g NaCl
15 g agar
Leave the stir bar in the flask and autoclave 20 min at 121 C to sterilize. Prepare and
preincubate the plates as described above for PIA (see recipe). Store up to 1 month
at 4 C.
TSA is widely used in microbiology, and it may prove more economical to purchase
the powdered prepared TSA from Difco or Oxoid than to prepare this medium from
scratch.
Laboratory
Maintenance of
Brucella abortus
3B.1.8
Current Protocols in Microbiology
COMMENTARY
Background Information
Brucella abortus is a small, gram-negative
coccobacillus. The bacteria are nonmotile and
do not form spores. Although many Brucella
strains can be cultivated aerobically in a bacteriological incubator, some strains require 5%
to 10% carbon dioxide for growth. For primary
isolation of Brucella from clinical specimens,
media should be incubated in a 5% to 10%
carbon dioxide atmosphere; under these conditions bacteria may take up to 30 days to grow.
However, laboratory strains of B. abortus will
form visible colonies on solid media within 3
to 4 days. B. abortus colonies are colorless, 1 to
2 mm in diameter, and have a convex surface.
They are nonhemolytic on blood agar. The six
species of Brucella are designated B. abortus, B. canis, B. melitensis, B. neotomae, B.
ovis, and B. suis. In addition, two new species,
B. pinnepediae and B. cetaceae, have been
proposed, based on molecular characterization
of Brucella isolates from dolphins, porpoises,
whales, and seals. While it has been proposed
that the different Brucella species should be
considered as biovars of a single species, Brucella melitensis, adoption of this proposal has
met with resistance from the Brucella research
community, which is seeking to maintain the
species designations. Therefore, in the scientific literature, both the biovar and the species
designations are currently in use. For a discussion of the controversy see Moreno et al.
(2002).
B. abortus is a pathogen of cattle, in
which it causes abortion late during gestation.
In addition, B. abortus has been found in
wildlife species in the U.S., including bison
and elk. This has presented a challenge for
wildlife management, particularly in areas
of the United States such as Wyoming, as
the seropositivity rates have been found
to be close to 50% for bison and 28% for
elk (for more information on this problem,
see
http://wyagric.state.wy.us/relatedinfo/
wybrucellosisinfo.htm). Based on these
surveillance results, there is some concern
about transmission between these wildlife
species and cattle, or transmission to hunters
handling infected carcasses.
B. suis, which is closely related to B. abortus, is a pathogen of swine, while B. melitensis infects goats and sheep. B. melitensis, B.
suis, and B. abortus can be transmitted to humans via contact with infected animals or their
tissues, or by consumption of unpasteurized
dairy products. Brucellosis is rare in the U.S.
as a result of its eradication in cattle and pasteurization of dairy products, with fewer than
200 (mostly imported) cases reported per year
to CDC. Worldwide, however, over 500,000
cases of human brucellosis are reported to
the World Health Organization, and the disease is widespread, especially in Africa, South
America, the Middle East, and Russia. Humans generally are considered a dead end
host and do not transmit the infection. During the 1950s, both the U.S. and the former
U.S.S.R. developed technology for use of Brucella species as a bioweapon, and, for this reason, B. abortus, B. suis, and B. melitensis are
regulated as Select Agents in the U.S.
Some individuals infected with Brucella
may initially be asymptomatic. Within 8 weeks
of exposure, acute onset of symptoms including fever, weakness, headache, malaise, fatigue, night sweats, and muscle and back pain
may be reported. Other patients may experience a more gradual development of these
symptoms over months. If the illness goes untreated, chronic brucellosis, which is characterized by recurring (undulant) fevers, can develop. Since these symptoms are nonspecific,
history of possible exposure will aid in the
diagnosis. Blood cultures often fail to yield
Brucella, hence a diagnosis is made by determining serum agglutination titers with Brucella antigen. The recommended treatment for
brucellosis is a 6-week course of doxycycline,
in combination with rifampin. Treatment with
a single antibiotic has been associated with a
high rate of relapse of brucellosis.
Alpha
Proteobacteria
3B.1.9
Current Protocols in Microbiology
Laboratory
Maintenance of
Brucella abortus
expensive TSB is generally used. One consideration for the choice of medium is the intended application. For experiments such as
enumeration of CFU obtained from tissue culture or animal experiments, where the plates
will be discarded after counting, TSA is a
good choice because of the cost and the large
number of plates used. However, serial passage of Brucella on this medium can result
in attenuation. In fact, serial passage in vitro
was originally used to derive the live, attenuated animal vaccines currently in use, and
it has been shown that for vaccine production, use of more nutrient-rich media, such as
TSA blood agar or PIA, reduces the appearance of spontaneous rough mutants lacking
a complete lipopolysaccharide (Alton et al.,
1975). Therefore, for construction of strains
to be used for virulence studies, or for strains
that will be added to the laboratory stock, it is
advisable to keep the number of in vitro passages to a minimum and to maintain stocks on
the more nutrient-rich PIA or TSA blood agar.
Other media, such as chocolate agar and modified Thayer-Martin medium, will also support
growth of B. abortus and are routinely used in
clinical microbiology laboratories.
Brucellosis is the most frequently reported
laboratory-acquired infection, so it is important to follow established laboratory protocols for avoidance of contamination and infection. Disinfectants such as 70% ethanol,
1% Virkon S, or 10% bleach should always
be within reach while working in the BSC.
Aerosols, cuts, needle sticks, or splashes in
the eye have been implicated as causes of
laboratory-acquired infections. Use of sharps
and glass items should be minimized to reduce the risk of infected wounds. In particular,
procedures that can generate aerosols, such as
homogenization of tissues, vortexing, electroporation, and centrifugation require special
care to ensure containment of the aerosols.
Many centrifuge manufacturers now offer special rotors that will contain aerosols in case of
a spill: these rotors are highly recommended
for centrifugation of samples containing Brucella, since centrifuge accidents are a frequent
cause of laboratory infection (e.g., see Fiori
et al., 2000).
When working in the BSL-3 lab, it is a
good idea to plan in advance and, before starting the experiment, make a checklist to ensure
that all required materials and equipment are
present. A frequent cause of lost time is having to change out of the personnel protective
equipment, leave the lab, and fetch whatever
is missing in the middle of an experiment. An
Anticipated Results
B. abortus grows in TSB with a doubling
time of 3 to 3.5 hr. Under these conditions,
a culture of B. abortus 2308 inoculated as described in Basic Protocol 1 will be in logarithmic growth phase between 8 and 20 hr,
at late log/early stationary phase between 20
and 24 hr, and in stationary phase after 24 hr
(Fig. 3B.1.1). If a different B. abortus strain is
used, then a growth curve should be prepared
for each individual strain. Unlike E. coli or
Salmonella, which grow to a density of 5
109 CFU/ml, B. abortus can grow to culture
densities exceeding 1 1010 CFU/ml. For B.
abortus cultures, an OD600 of 1.0 is log phase
and is equivalent to 24 109 CFU/ml, which
is two to four times higher than an E. coli
culture with the same optical density. This is
likely due to the small size of B. abortus, which
is 0.5 m in diameter and 0.6 to 1.5 m in
length.
Time Considerations
Three days in advance of the planned experiment, the B. abortus strain should be streaked
out from the frozen stock culture. Cultures on
blood agar plates, if sealed with Parafilm, can
be maintained at 4 C for up to 4 weeks and
still maintain viability; however, the bacteria
from these plates will grow more slowly initially on inoculation into a liquid culture, and
this should be taken into consideration when
the bacterial culture is to be harvested at a particular growth phase. Alternatively, one can
use a plate that has been stored at 4 C for
up to 4 weeks to inoculate an overnight culture (thickly), then use this saturated overnight
culture to reinoculate a new starting culture to
an OD600 of 0.01.
For work in the BSL-3 lab, always plan extra time (at least 30 min.) for disinfection, and
BSL-3 entry and exit procedures. It is wise
to wipe the outer and inner surfaces of the
BSC with 70% ethanol before starting work,
and after completing work, all surfaces in the
BSC and the lab (e.g., door handles, incubator
3B.1.10
Current Protocols in Microbiology
Literature Cited
Key References
http://serine.urbm.fundp.ac.be/seqbruce/
GENOMES
http://bbp.vbi.vt.edu/index.php
http://www.moag.gov.il/brunet
The BruNet contains epidemiological information
about brucellosis and its control
http://www.cdc.gov/od/sap
The CDC site contains information about its SelectAgent Program
http://www.aphis.usda.gov/vs/cvb/selectagent.htm
This site contains information about the USDAs
Select Agent Registration Program
Internet Resources
http://www.cdc.gov/od/ohs/biosfty/bmbl4/
bmbl4toc.htm
This site contains the CDCs Biosafety in Microbiological and Biomedical Laboratories (BMBL).
Alpha
Proteobacteria
3B.1.11
Current Protocols in Microbiology
UNIT 3C.1
ABSTRACT
Trench fever is the common name for the acute febrile syndrome associated with a
Bartonella quintana bacterial infection. The focus of this unit is to describe reliable
methods for cultivation and cryopreservation of B. quintana and can be applied to
cultivation and preservation of all Bartonella. Detailed recipes for preparation of three
types of semisolid media are also included. Curr. Protoc. Microbiol. 10:3C.1.1-3C.1.13.
C 2008 by John Wiley & Sons, Inc.
Keywords: Bartonella, trench fever r culture r HIB-B r BB-H r chocolate agar r
cryopreservation
INTRODUCTION
B. quintana infection has affected millions of people during war and is currently reemerging in inner cities throughout the world and in AIDS patients (Maurin and Raoult, 1996;
Ohl and Spach, 2000). Of all bacteria, B. quintana has the greatest known requirement
for exogenous heme, a large cyclic organic molecule (protoporphyrin) containing an iron
atom (Vinson, 1966; Myers et al., 1969, 1972). It is generally accepted that this extraordinary supplement requirement is similar for all Bartonella species, as erythrocytes,
hemoglobin, or hemin are essential for cultivation (Birtles et al., 1995).
The overall function of this unit is to describe reliable methods for cultivation and preservation of B. quintana (previously known as Rochalimaea quintana). Bartonella species
are relatively slow growing and require nutrient-rich media; therefore there is a high
risk of contamination. Thus, this unit includes a biosafety cabinet preparation protocol
(Support Protocol) that should be followed for all procedures. Procedures for inoculation
of semisolid media from three sources are described: glycerol stock (Basic Protocol 1),
liquid suspension (Basic Protocol 2), and solid plate culture (Basic Protocol 3). A unique
harvesting method (Basic Protocol 4) is used to collect bacteria from the surface of a
plate, and long-term storage of B. quintana is accomplished by cryopreservation with
glycerol (Basic Protocol 5).
CAUTION: B. quintana is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate
guidelines and regulations for the use and handling of pathogenic microorganisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: Follow all appropriate guidelines and regulations for the use and handling
of human-derived materials. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
STRATEGIC PLANNING
Culture Media
Although several liquid media have been shown to facilitate replication of B. quintana
(Huang, 1967; Mason, 1970; Maggi et al., 2005), medium solidified with 1.5% agar
Alpha
Proteobacteria
Current Protocols in Microbiology 3C.1.1-3C.1.13, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc03c01s10
C 2008 John Wiley & Sons, Inc.
Copyright
3C.1.1
Supplement 10
Medium
Comments
HIB-B
Heart infusion broth with 4% blood (v/v), 2% serum (v/v), and 1.5%
agar. A good general purpose medium for culturing Bartonella.
CHOCO
BB-H
H2 Ob
Hemin chloridec
Final concentration
496.8
3.2
0.05 mM
490.3
9.7
0.15 mM
337.7
162.3
2.5 mM
Simulated niche
provides the most reliable growth substrate. Three separate semisolid formulations containing alternate heme sources are provided in this unit (see Table 3C.1.1): (1) heart
infusion broth with 4% (v/v) sheep blood (HIB-B), (2) chocolate agar containing 1%
(w/v) hemoglobin (Koehler et al., 1992), and (3) Brucella broth supplemented with a
variable concentration of hemin (BB-H). Both HIB-B and chocolate agar plates can be
used for routine passage of B. quintana. Alternatively, BB-H plates are used to study
the effect of heme concentration on gene expression and physiology of B. quintana (see
Table 3C.1.2 and Background Information).
Culture Conditions
B. quintana is considered a microaerophile (only a small amount of oxygen is required for
growth) and a capnophile (growth is enhanced in the presence of increased CO2 tension).
This atmosphere is typically achieved using a candle extinction jar. Alternatively, a 5%
CO2 incubator (at atmospheric O2 ) can be used. The standard incubation temperature
for B. quintana is 35 to 37 C in a humidified CO2 -rich environment. Alternatively,
incubation can be performed at 30 C to simulate conditions experienced by B. quintana
in the body louse vector (see Background Information).
Isolation of B. quintana
Isolation of B. quintana from nature is challenging even for an experienced researcher,
and a detailed explanation of the methods employed is beyond the scope of this unit.
Although cell coculture systems are considered superior, chocolate agar and HIB-B can
be used to isolate B. quintana from human samples (Koehler et al., 1992; La Scola et al.,
1999), as well as decontaminated body lice (La Scola et al., 2001).
Laboratory
Maintenance
of B. quintana
3C.1.2
Supplement 10
BASIC
PROTOCOL 1
There are three procedures depending on the source of inoculum; (1) glycerol stock,
(2) liquid suspension, and (3) agar plate culture. The type of media used is based on the
particular experiment (see Strategic Planning). Be sure to properly label the plates with
a fine-tipped permanent marker on the agar-side of the petri plate.
Materials
B. quintana glycerol stock (Basic Protocol 5)
Petri plates with solidified medium of choice (see Strategic Planning and Reagents
and Solutions)
250 ml glass beaker containing 100 ml 95% ethanol
Ice bucket
Kimwipes, moistened with 70% (v/v) ethanol
Aerosol-barrier pipet tip
Glass culture spreader, sterilized with ethanol
5% CO2 incubator with a pan of H2 O to maintain humidity
Additional reagents and equipment for preparing a biological safety cabinet
(Support Protocol) and using the spread technique (APPENDIX 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after
preparation of workspace and tools (see Support Protocol and APPENDIX 4D).
1. Place glycerol freezer stock in ice bucket in a prepared biological safety cabinet (see
Support Protocol) and allow contents to thaw completely.
Thawing can be expedited by warming the tube between the palms. The tube should be
gently inverted to resuspend bacteria and ensure a consistent inoculum.
Keep the frozen stock tube in an ice bucket when not in use.
Figure 3C.1.1 Bent inoculation wire. This tool is useful for inoculation of plates from a conuent
lawn of a clonal population. A Brucella agar plate containing 0.15 mM hemin chloride is shown.
For color version of this gure see http:www/currentprotocols.com.
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2. Sterilize the outside of the screw-capped tube by gently wiping with a 70% (v/v)
ethanol-moistened Kimwipe.
This is to ensure that the freezer stock does not become contaminated. The moistened
Kimwipe can also be used to partially unscrew the lid. Do not rub too hard as you are
likely to erase the label.
Materials
Liquid suspension of B. quintana (Basic Protocol 4)
Petri plates with solidified medium of choice (see Strategic Planning and Reagents
and solutions)
Aerosol-barrier pipet tip
Glass culture spreader, sterilized with ethanol
5% CO2 incubator with a pan of H2 O to maintain humidity
Additional reagents and equipment for using the spread technique (APPENDIX 4A) and
preparing a biological safety cabinet (Support Protocol)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after
preparation of workspace and tools (see Support Protocol and APPENDIX 4D).
1. Using aseptic technique, obtain 10 to 150 l of the liquid suspension using an
aerosol-barrier pipet tip.
2. Use the spread technique (see APPENDIX 4A) to evenly distribute the liquid on the
surface of the solidified medium using an ethanol-sterilized glass culture spreader.
Laboratory
Maintenance
of B. quintana
Volume used for inoculation is based on empirical determination of the viable density
by plating. However, if the volume exceeds 150 l, the plates should be dried agar-side
down in the cabinet. Return the glass culture spreader to the ethanol and re-flame prior
to spreading each separate plate.
3C.1.4
Supplement 10
BASIC
PROTOCOL 3
Materials
B. quintana colony or confluent lawn (from Basic Protocol 1 or 2)
Standard inoculation loop or bent inoculation wire (see Fig. 3C.1.1)
Kimwipe, moistened with 70% (v/v) ethanol
Petri plates with solidified medium of choice (see Strategic Planning and Reagents
and solutions)
5% CO2 incubator with a pan of H2 O to maintain humidity
Additional reagents and equipment for sterilizing the inoculation loop or bent wire
using a flame (APPENDIX 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after
preparation of workspace and tools (see Support Protocol and APPENDIX 4D).
1. Sterilize the handle of an inoculation loop or bent wire with a Kimwipe moistened
with 70% (v/v) ethanol.
2. Sterilize the inoculation loop or bent wire using a flame (see APPENDIX 4A) and cool it
by touching the agar surface of the un-inoculated plate near the outer perimeter.
3. Touch the colony (with a loop) or confluent lawn (with bent wire) and gently streak
across the surface of a new agar plate.
Avoid using too much pressure when streaking as gouges in the surface result in inefficient
harvesting (Basic Protocol 4).
BASIC
PROTOCOL 4
Materials
Plate(s) containing B. quintana clonal populations (Basic Protocols 1, 2, or 3)
Heart infusion broth (HIB; see recipe)
Single-edged razor blades with grooved handle (Smith Brand, cat. no. 67-0238)
Test tube holder (see Fig. 3C.1.2)
250-ml glass beaker containing 100 ml 95% ethanol
1000-l micropipettor
Sterile collection tube
Additional reagents and equipment for sterilizing harvesting tool using a flame
(APPENDIX 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after
preparation of workspace and tools (see Support Protocol and APPENDIX 4D).
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Figure 3C.1.2 Harvesting B. quintana from a chocolate agar plate. The harvesting tool, consisting of a single-edged razor blade and a test tube holder, is used to scrape the bacterial lawn
toward the outer perimeter of the plate.
3. Soak the blade portion of the harvesting tool in a beaker containing 95% ethanol and
briefly pass through a flame to sterilize as described for a glass culture spreader (see
APPENDIX 4A).
4. Hold the plate agar-side-up with one hand, and the sterilized harvesting tool with the
other. With the blade slightly angled toward the surface of the agar, gently scrape
the bacteria into a single clump close to the outer perimeter of the plate. Return the
harvesting tool to the beaker of 95% ethanol.
CAUTION: Dispose of the contaminated blade(s) in a sharps biohazard container.
Avoid movements of the blade that gouge the surface or create fragments of agar.
The bacteria have a tendency to stick to each other and the blade. A single blade can be
used to harvest >10 plates.
5. Hold the plate agar-side up with one hand, and a 1000-l micropipettor containing
300 l sterile HIB with the other. Slightly tilt the plate such that the clump is facing
you and is toward the bottom.
6. Moisten the clump by slowly ejecting this 300 l HIB liquid medium directly
onto it.
The liquid decreases the stickiness of the bacteria. The HIB should flow over the clump
of bacteria and form a pool in the space between the agar surface and the inner wall of
the plate.
Laboratory
Maintenance
of B. quintana
7. Holding the pipet tip nearly horizontal to the surface of the plate, moisten the pipet
tip in the pool of HIB and push clump of bacteria into this pool.
3C.1.6
Supplement 10
The goal is not to resuspend the clump, but rather collect it from the surface of the plate
such that it can be aspirated and transferred. Avoid movements of the pipet tip that gouge
the surface or create fragments of agar.
CRYOPRESERVATION OF B. QUINTANA
Cryopreservation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see Support Protocol). Long-term storage is achieved by
freezing at 80 C in a final concentration of 12.5% (v/v) glycerol. Prepare at least two
vials of each strain and if possible, store in separate freezers.
BASIC
PROTOCOL 5
Materials
Cryopreservation solution (see recipe)
1-ml screw-capped cryovials (Nunc, cat. no. 375353)
Fine-tipped permanent marker (to label the cryovials)
80 C freezer
Additional reagents and equipment for growing and harvesting a clonal population
of B. quintana (Basic Protocol 4)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after
preparation of workspace and tools (see Support Protocol and APPENDIX 4D).
1. Grow a clonal population of B. quintana and harvest (see Basic Protocol 4) into
liquid HIB.
2. Combine equal volumes of the cryopreservation solution and the liquid suspension
in a properly labeled 1-ml screw-capped cryovial.
3. Mix by gentle inversion and store >10 years in 80 C freezer.
SUPPORT
PROTOCOL
Materials
Laminar-flow type biosafety cabinet equipped with germicidal UV bulb
Spray bottle with 70% ethanol (v/v)
Kimwipes
Latex, vinyl, or nitrile protective gloves
Laboratory coat
Touch-O-Matic (Hanau) bunsen burner
10-l, 100-l, and 1000-l micropipettors
Aerosol-barrier micropipet tips, sterile
Biohazard bag
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1. Using the spray bottle of 70% ethanol, lightly mist the bench surface of the biosafety
cabinet and wipe with a Kimwipe.
2. With the exception of live bacterial cultures, place all items required for a particular
procedure in the cabinet, and lightly mist with 70% ethanol paying special attention
to the barrels of the micropipettors.
3. Turn on the UV bulb for 10 min to allow ethanol to evaporate and germicidal action
to occur.
Do not sit in front of the cabinet when the UV light is on as damage to skin and eyes can
occur.
5. Outside the cabinet, gently mist from the fingertips of gloved hands to elbows with
70% ethanol.
Use caution when using ethanol spray bottle around open flame and avoid direct contact
with eyes or skin. Do not place spray bottle in the cabinet.
8. Remove live bacteria and ensure that gas to the burner is off.
Live bacterial cultures should be immediately placed in an incubator. Used culture media,
micropipet tips, and any other or potentially contaminated material should be discarded
in a biohazard bag and autoclaved.
9. Lightly mist micropipet barrels and the surface of the cabinet bench with 70%
ethanol.
10. As in step 5 (above), lightly mist hands and arms. Dispose of gloves in biohazard
bag.
11. Finally, expose the cabinet to the germicidal bulb for 10 min as in step 3 (above).
Laboratory
Maintenance
of B. quintana
Dissolve 14 g BBL Brucella Broth (Becton Dickinson, cat. no. 211088) with 496.8
down to 337.7 ml H2 O (see Table 3C.1.2) in a 1000-ml Pyrex flask. Add 7.5 g Difco
Agar (Becton Dickinson, cat. no. 214530) and autoclave for 15 min at 121 C. Cool
for 45 min in a 56 C H2 O bath. Lightly mist outside of flask with 70% ethanol
continued
3C.1.8
Supplement 10
and place in prepared biosafety cabinet. Add 3.2 to 162.3 ml 5% hemin solution
(see recipe) to concentration appropriate for specific simulated environment (see
Table 3C.1.2). Pour into sterile petri plates in the cabinet, then pass flame over the
surface of agar using a standard bunsen burner to remove bubbles. Replace lid.
Allow plates to dry overnight (agar-side down) in the cabinet. Re-sleeve, invert,
and store up to 1 month at 4 C.
The approximate formula per liter is 10.0 g pancreatic digest of casein, 10.0 g peptic digest
of animal tissue, 1.0 g dextrose, 2.0 g yeast extract, 5.0 g sodium chloride, 0.1 g sodium
bisulfite, and 15 g agar.
Also see Strategic Planning.
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of flasks with 70% ethanol and place in cabinet. Add hemoglobin and IsoVitaleX
enrichment to GC Agar. Swirl to mix, and dispense 28 ml into petri plates with
a sterile graduated pipet. Allow plates to dry overnight in the cabinet. Re-sleeve,
invert, and store up to 1 month at 4 C.
Prepare 202 ml batches rather than 1010 ml batches as they are easier to dispense in petri
plates in the cabinet. Also see Strategic Planning.
The approximate formula per liter is 7.5 g enzymatic digest of casein, 7.5 g enzymatic digest
of animal tissue, 1.0 g corn starch, 4.0 g dipotassium phosphate, 1.0 g monopotassium
phosphate, 5.0 g sodium chloride, 10.0 g agar, 10.0 g hemoglobin, 0.01 g Vitamin B12 ,
10.0 g L-glutamine, 1.0 g adenine, 0.03 g guanine hydrochloride, 0.013 g p-aminobenzoic
acid, 0.25 g nicotinamide adenine dinucleotide, 0.1 g thiamine pyrophosphate, 0.02 g ferric
nitrate, 0.003 g thiamine hydrochloride, 25.9 g L-cysteine hydrochloride, 1.1 g L-cystine,
and 100.0 g dextrose.
COMMENTARY
Background Information
Laboratory
Maintenance
of B. quintana
3C.1.10
Supplement 10
Figure 3C.1.3 Conuent lawn of B. quintana. A chocolate agar plate inoculated from a liquid
suspension by the spread plate method and incubated in a candle jar for 7 days is shown. Formation
of colonies of various sizes within a grainy background sheen is typical. For color version of this
gure see http:www/currentprotocols.com.
Anticipated Results
The growth rate of B. quintana can vary
greatly depending on the strain, passage number, inoculation source, inoculation density,
and culture media. Initial isolation from mammalian blood or decontaminated lice usually
requires several weeks of incubation on chocolate agar or HIB-B. Alternatively, colony formation is usually evident in 5 to 7 days from
strains that have been grown at least once on
HIB-B or chocolate agar.
The colony morphology of B. quintana is
small (0.2 to 1 mm) and circular, with a convex
elevation, even margin, and a translucent-tan
color. The color of the colonies can vary with
different media. A confluent lawn typically exhibits an opaque sheen with a variety of colony
sizes and contains 1 107 to 1 1010 bacteria
(see Fig. 3C.1.3).
Time Considerations
The major portion of hands-on time to complete the basic protocols in this unit is devoted
to preparation of growth media. Dissolving
the media components (0.5 hr), autoclaving
(1.0 hr), cooling (0.75 hr), and dispensing
media into plates (0.25 hr) requires 2.5 hr.
The cooling time can be extended (to 3 hr)
and the overnight drying time can be reduced
(to 3 hr). The major portion of total time
for completion of the basic protocols is dedicated to incubation (5 to 21 days; see Anticipated Results). Following preparation of the
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Literature Cited
Alsmark, C.M., Frank, A.C., Karlberg, E.O.,
Legault, B.A., Ardell, D.H., Canback, B.,
Eriksson, A.S., Naslund, A.K., Handley, S.A.,
Huvet, M., La Scola, B., Holmberg, M., and
Andersson, S.G. 2004. The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae. Proc. Natl. Acad. Sci. U.S.A.
101:9716-9721.
Battisti, J.M., Sappington, K.N., Smitherman,
L.S., Parrow, N.L., and Minnick, M.F.
2006. Environmental signals generate a differential and coordinated expression of the
heme receptor gene family of Bartonella
quintana. Infect. Immun. 74:3251-3261.
Battisti, J.M., Smitherman, L.S., Sappington, K.N.,
Parrow, N.L., Raghavan, R., and Minnick,
M.F. 2007. Transcriptional regulation of the
heme binding protein gene family of Bartonella
quintana is accomplished by a novel promoter
element and iron response regulator. Infect.
Immun. 75:4373-4385.
Birtles, R.J., Harrison, T.G., Saunders, N.A., and
Molyneux, D.H. 1995. Proposals to unify the
genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov.,
Bartonella peromysci comb. nov., and three
new species, Bartonella grahamii sp. nov.,
Bartonella taylorii sp. nov., and Bartonella
doshiae sp. nov. Int. J. Syst. Bacteriol. 45:1-8.
Boonjakuakul, J.K., Gerns, H.L., Chen, Y.T., Hicks,
L.D., Minnick, M.F., and Koehler, J.E. 2007.
Proteomic and immunoblot analysis of Bartonella quintana total membrane proteins identifies antigens recognized by serum from infected
patients. Infect. Immun. 75:2548-2561.
Breitschwerdt, E.B. and Kordick, D.L. 2000.
Bartonella infection in animals: Carriership,
reservoir potential, pathogenicity, and zoonotic
potential for human infection. Clin. Microbiol.
Rev. 13:428-438.
Eremeeva, M.E., Gerns, H.L., Lydy, S.L., Goo,
J.S., Ryan, E.T., Mathew, S.S., Ferraro, M.J.,
Holden, J.M., Nicholson, W.L., Dasch, G.A.,
and Koehler, J.E. 2007. Bacteremia, fever, and
splenomegaly caused by a newly recognized
Bartonella species. N. Engl. J. Med. 356:23812387.
Fournier, P.E., Minnick, M.F., Lepidi, H., Salvo,
E., and Raoult, D. 2001. Experimental model of
human body louse infection using green fluorescent protein-expressing Bartonella quintana.
Infect. Immun. 69:1876-1879.
Laboratory
Maintenance
of B. quintana
3C.1.12
Supplement 10
Key References
Anderson, B.E. and Neuman, M.A. 1997. Bartonella spp. as emerging human pathogens. Clin.
Microbiol. Rev. 10:203-219.
Dehio, C. 2004. Molecular and cellular basis of
bartonella pathogenesis. Annu. Rev. Microbiol.
58:365-390.
Two reviews regarding molecular pathogenesis of
several Bartonella species.
Maurin, M. and Raoult, D. 1996. Bartonella
(Rochalimaea) quintana infections. Clin. Microbiol. Rev. 9:273-292.
An extensive and comprehensive review of the epidemiology, clinical manifestations, diagnosis and
treatment of B. quintana infections.
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UNIT 4A.1
ABSTRACT
Neisseria gonorrhoeae is a human pathogen of mucosal surfaces, thus laboratory manipulations must include appropriate safety measures. The growth requirements and
behavior of the gonococcus are significantly different from many bacteria, necessitating
modifications of common laboratory techniques. A fastidious organism, N. gonorrhoeae
requires enriched media in a CO2 atmosphere at 35 to 37 C for growth. In addition,
N. gonorrhoeae expresses potent autolysins whose activity increases following glucose
depletion during stationary phase, leading to cell death. Long believed to be an obligate
aerobe, the gonococcus is capable of anaerobic growth when provided with a suitable
electron acceptor. This unit provides information for both aerobic and anaerobic growth,
basic long-term and daily maintenance of gonococcal cultures, as well as safety considC 2008 by
erations for laboratory studies. Curr. Protoc. Microbiol. 8:4A.1.1-4A.1.26.
John Wiley & Sons, Inc.
Keywords: Neisseria gonorrhoeae r bacterial growth r Neisseria media r
anaerobic bacterial growth r frozen stock
INTRODUCTION
Neisseria gonorrhoeae (gonococcus) is an obligate human pathogen, thus all strains
are derived from human infections. Best known as the agent of the sexually transmitted
disease gonorrhea, this organism readily infects other mucosal surfaces, including the
pharynx and eyes, and may cause invasive disease if inoculated into the bloodstream.
Thus, appropriate laboratory precautions are needed to prevent laboratory acquired infections following accidental contact with broken skin or procedures that may promote
splashes or produce aerosols from gonococcal suspensions. Safety issues specifically
related to common laboratory manipulations of gonococci are discussed, including CDC
recommendations for N. gonorrhoeae.
While N. gonorrhoeae can be readily grown in the laboratory, it is a fastidious organism
that requires enriched media along with a 3% to 10% CO2 atmosphere at 37 C for
growth, conditions commonly used for clinical isolates. The organism is sensitive to
desiccation and cannot survive temperatures <25 C for extended periods of time. In
addition, the viability of N. gonorrhoeae decreases rapidly in stationary phase, due to
enhanced autolysis following the depletion of glucose from the medium. Thus, many
techniques used for both long- and short-term growth and storage of most microbes
are unsuitable for N. gonorrhoeae, such as overnight broth cultures or refrigeration of
plate-grown colonies. Detailed protocols are presented for the laboratory growth of N.
gonorrhoeae under both aerobic and anaerobic conditions. A protocol for a chemically
defined medium is also provided for those whose work requires the rigor of exact medium
composition.
Neisseria gonorrhoeae is a well-adapted human pathogen, which is demonstrated by
the high degree of phase and antigenic variation observed for many surface features of
the organism. See UNIT 4A.3 for detailed information on N. gonorrhoeae pilin and Opa
variations and their effects on colony morphology.
Current Protocols in Microbiology 4A.1.1-4A.1.26, February 2008
Published online February 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc04a01s8
C 2008 John Wiley & Sons, Inc.
Copyright
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4A.1.1
Supplement 8
STRATEGIC PLANNING
Biosafety Considerations
Laboratory associated infections with N. gonorrhoeae from contaminated clinical material have been reported in the United States. The primary risk factor centers on the direct or
indirect contact of mucous membranes with infectious materials, though the importance
of aerosols has not yet been determined. Therefore, the most probable sites of laboratory
acquired infections include the pharynx (throat), the conjunctiva (eye surface), and invasive disease acquired through contact with broken skin. Neisserial pharyngitis is often
asymptomatic, while infection of the conjunctiva rapidly produces a severe, purulent
response that can lead to scarring and permanent damage. Infection of the bloodstream,
called disseminated gonococcal infection (DGI), initially gives rise to fevers, migratory
polyarthralgia (aching joints), and petechial, maculopapular, or pustular rash (skin rash).
Without treatment, joints may become infected, leading to septic arthritis often of the
elbow or knee. Thus, it is important to seek medical treatment for known accidental
exposures or symptoms that may indicate exposure, as gonococcal infections respond
readily to standard antibiotic therapy.
In addition, since the risk of aerosols is not yet known, the CDC recommends that
production quantities of N. gonorrhoeae be handled under BSL-3 containment. The CDC
does not qualify what constitutes production level quantities, but does make reference
to vaccine production. Thus, the determination for any given set of experiments must be
determined through review of the proposed study by the individual Institutional Biosafety
Committee.
NOTE: Examples of common laboratory precautions and emergency laboratory procedures for posting in the laboratory are given in Figures 4A.1.1 through 4A.1.3.
Laboratory
Maintenance of
N. gonorrhoeae
Many gonococcal clinical isolates are amino acid and/or nucleic acid precursor auxotrophs, traits that are used as epidemiological markers. Due to the known needs of
many common laboratory strains of N. gonorrhoeae, and the unknown needs of clinical
isolates, a rich medium is used for the isolation and growth of gonococci.
4A.1.2
Supplement 8
Figure 4A.1.1 An example of a specic biosafety information form required to be posted in the
laboratory to comply with the safety requirements of the Institutional Biosafety Committee at the
University of Rochester for additional special practices for work with Neisseria gonorrhoeae.
CO2 conditions
The need for enhanced CO2 to initiate growth of many microbes is well known, especially
with the mucosal pathogens. It has been suggested that this mimics the host environment,
and is only required initially until the number of microbes in the colony grow sufficiently
for their metabolism to generate the needed CO2 . There are several ways to generate
the needed CO2 to initiate gonococcal growth. Since most liquid cultures are grown in
benchtop shaking incubators under room air, a sodium bicarbonate supplement is added
to the culture broth to provide the needed CO2 . Pre-incubating the medium in a CO2
incubator can also provide the needed CO2 ; however, this results in more variation than
supplementing the medium with sodium bicarbonate. For growth on a solid medium,
CO2 can be provided by the use of a CO2 incubator, a CO2 -generating GasPak (BBL),
or a candle jar. Wheaton Science (http://www.wheatonsci.com/) offers a large range of
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Figure 4A.1.2 An example of a specic biosafety information form required to be posted in the
laboratory to comply with the safety requirements of the Institutional Biosafety Committee at the
University of Rochester for laboratory specic biosafety information.
suitable, wide-mouth glass jars for use as candle jars. The GasPak system (BBL) is
commercially available from BD (http://www.bd.com/). It involves the generation of
CO2 from the mixing of sodium bicarbonate with citric acid in a foil-package that is
activated by the addition of water. The plates and the CO2 -generating package are placed
into an air-tight container (GasPak container). The closed containers are placed into a
37 C incubator overnight to allow gonococcal growth. The candle jar is the precursor to
the GasPak. It involves placing the plates into a large, fire-resistant container, traditionally
a large glass jar. A small, white, votive candle (unscented) is placed on top of the plates.
The candle is lit and the lid tightly closed. The flame will burn until the oxygen in
the jar is consumed. At this time, the closed container is placed in a 37 C incubator.
The burning candle generates 3% CO2 , which is sufficient to initiate gonococcal
growth.
Laboratory
Maintenance of
N. gonorrhoeae
4A.1.4
Supplement 8
Figure 4A.1.3 An example of a specic biosafety information form required to be posted in the
laboratory to comply with the safety requirements of the Institutional Biosafety Committee at the
University of Rochester for emergency procedures for spills involving Neisseria gonorrhoeae.
be used to scrape the frozen stock. Experience has shown that the larger and sturdier
applicator stick (i.e., the opposite end of the sterile cotton-tipped swab) does not break as
often as toothpicks. The longer applicator stick also appears to minimize contamination
of the GCMB plate with Staphylococci sp. Therefore, if using toothpicks, wear gloves.
Minimize the time the stock vial is held to limit the warming of the vial. The expected
viability of gonococcal frozen stocks is not as high as with most other organisms, and
the viability decreases with freeze-thaw cycles. This lower viability often necessitates
the use of a larger inoculum.
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the desiccant as soon as the color indicator shows any sign of change is recommended,
while the palladium catalyst is dried in a similar manner, at least weekly.
This need for dry conditions means spills in the anaerobe chamber must be cleaned
immediately. Spills of media can be as damaging to the functioning of the chamber as
spills of cultures. Thus, all chambers should contain disinfectant, either 10% bleach or
70% ethanol in a squeeze bottle, along with towels for blotting and biohazard bags to
facilitate removal of contaminated materials through the entry portal.
Measuring function
It is recommended to measure proper functioning of the anaerobe chamber. This can be
done by the use of a combined hydrogen and oxygen monitor or anaerobic indicator strips.
The use of a dual monitor allows for the assessment of chamber function (oxygen level)
as well as monitoring the reducing agent (hydrogen). If sufficient levels of hydrogen are
present, the oxygen will be reduced if the palladium catalyst is active. This information
aids in troubleshooting of the system if anaerobic conditions are not achieved. Anaerobic
indicator strips, which contain methylene blue, are available from BD as part of the
GasPak systems. The anaerobic strips become decolorized after 4 to 6 hr in anaerobic
conditions.
GCMB Solid Medium
Plate media used for clinical isolation of N. gonorrhoeae, including Thayer-Martin,
modified Thayer-Martin, Lewis-Martin, and GC-Lect, are chocolate-based media that
contain antibiotics to inhibit growth of yeast and bacteria other than gonococci. Most
research laboratories use a commercially prepared base media (Difco GC mediumbased
or BBL GC- or BBL GCII agarbased) for their studies. These recipes are all very similar,
using an enzymatic digest of meat proteins or a combination of milk and meat proteins
as sources of nitrogen and amino acids. They also include corn starch to bind free fatty
acids released by growing gonococci, as these tend to inhibit gonococcal growth at high
concentrations. Di- and mono-potassium phosphates are used to buffer the pH of the
medium with sodium chloride to maintain osmotic balance. Agar is included in these
preparations at a final concentration of 1%, which is a lower concentration than used for
most plate media, but empirically determined to enhance the isolation of N. gonorrhoeae.
See the BD Website (http://www.bd.com), maintained by the manufacturer of both Difco
and BBL products, for further information on these products.
General considerations
The following is a list of useful tips for preparing GCMB solid medium for culture of
N. gonorrhoeae.
1. It is important to completely mix GC base media with water prior to autoclaving,
as clumps of media stuck to the container tend to burn during the autoclave cycle.
Likewise, it is important to remove GCMB from the autoclave at the end of the
cycle. Leaving it in the hot autoclave for a prolonged time tends to damage critical
components in the media, and the resulting plates will not support gonococcal growth.
The soluble corn starch in the media tends to form small flecks. This is not unusual
and may be mistaken for contamination. Check by incubating a plate at 37 C to see
if the flecks increase in size, as would be expected for microbial contamination.
Laboratory
Maintenance of
N. gonorrhoeae
4A.1.6
Supplement 8
Kelloggs supplement
Douglas S. Kellogg, among others, examined the correlation of colony morphology
with virulence. Since colony morphology is dependent on growth media, these studies
often included comparisons of media and the effects of different supplementation on N.
gonorrhoeae growth. One supplement formulation was found to enhance the growth of
all tested strains and is now identified as Kelloggs supplement (Kellogg et al., 1963).
Often, 500 ml of Kelloggs supplement is made and dispensed into aliquots of multiple
bottles during filter sterilization. Kelloggs supplement can be easily contaminated, thus
preparing smaller aliquots limits loss due to inadvertent exposure. This 100 solution
can be stored at least 6 months at 4 C without loss of activity. The solution is close to
saturation for glucose, which makes it somewhat difficult to prepare. It is important to
use a large (1-liter) beaker and stir bar to quickly help the glucose into solution and to
add the glucose slowly, allowing it to go into solution before adding more. If too much
is added at once, the glucose will form a rock-like clump on the bottom of the beaker,
which will take a long time to go into solution. Patience is required.
Chemically Dened Medium (CDM)
General considerations
In some cases, it is desirable to determine the exact nutrient requirements of a given strain
or to manipulate the type of iron source that can be utilized by a particular strain. The
chemically defined medium (CDM) is made from a mixture of several solutions that are
prepared and sterilized independently (Morse and Bartenstein, 1980). The CDM stock is
often made at 2 concentration, which allows it to be diluted 1:1 with sterile, distilled,
deionized water for broth, or 1:1 with sterile 2% to 4% agar for plates. It is recommended
to use a high-quality agar to preserve the known concentration of the various components.
While this is a chemically defined medium, it is not a minimal medium and will support
the growth of many microbes. Thus, it is advisable to either prepare the exact amount
needed, plus allowance for errors, or dispense the 2 CDM into commonly needed
volume aliquots during the filter sterilization process.
Iron
Chemically defined medium is often used to test the effect of individual components on
bacterial growth. Iron is often a limiting factor in bacterial growth. Thus, testing various
types of iron sources addresses the ability of pathogens to utilize iron from the human
host.
There are many potential sources of iron that can be used to supplement CDM.
Two commonly used iron sources are ferric chloride (FeCl3 . 6H2 O) and ferric nitrate
(Fe(NO3 )3 . 9H2 O). Regardless of the source, a 10 mM stock solution, used at a final
concentration of 0.01 mM can be tested with 1 CDM. See Reagents and Solutions for
recipes.
Beta
Proteobacteria
4A.1.7
Current Protocols in Microbiology
Supplement 8
Solid medium
It is important to use a high-quality agar when preparing solid medium, such as low-EEO
agar for IEF immunological assays, or Noble agar, to prevent the introduction of trace
chemicals often found in less-purified preparations.
CDM plates may be soft. It is acceptable to increase the final agar concentration to 2%
(w/v) and to chill the plates after pouring to enhance gelling. Warm the hardened plate
to room temperature before using to plate N. gonorrhoeae.
BASIC
PROTOCOL 1
Laboratory
Maintenance of
N. gonorrhoeae
Figure 4A.1.4 Overnight growth of N.gonorrhoeae strain F62 on GCMB plate. The 16-hr culture
of F62 shows regions of colony growth that are considered very closely spaced but not conuent.
Cells in these colonies tend to have high viability and thus are preferred for use in subsequent
experiments.
4A.1.8
Supplement 8
able to grow as fast as those that produced well-isolated colonies, due to competition for
nutrients. Thus, the majority of cells in these small but not confluent colonies have not
yet reached stationary phase, and maintain viability. The ability to grow large numbers
of viable colonies on plates is critical, as broth cultures of gonococci will not survive
an overnight incubation. Thus, scraping cells off plate cultures is the only method to
generate large numbers of gonococci for study.
Materials
Gonococci inoculum
GCMB plates (see recipe for GCMB solid medium), at room temperature
Microbiological loop or sterile cotton-tipped applicator
37 C incubator with 3% to 10% CO2 flow or CO2 -generating GasPak or candle jar
(Wheaton cat. no. W216938) with candle
Sterile wooden applicators (opposite end of cotton-tipped swab) or flat tooth-picks,
sterilized
1. Using a sterile loop or cotton-tipped applicator, place a small inoculum of gonococci
on the edge of the medium. Spread to cover no more than 1/6 of the plate, keeping
it near the edge.
2. Without heating the loop, start the second quadrant streak, entering the initial inoculum multiple times. Turn the plate and repeat two times to streak the entire plate. See
Figure 4A.1.5.
Oftentimes, it is not necessary to perform the standard microbiological four-quadrant
streak method (see APPENDIX 4A), as there are few colonies found in the fourth quadrant.
Thus, depending on the style of the individual, streaking in thirds often generates more
viable gonococcal colonies per plate.
Figure 4A.1.5 Optimal streaking pattern for N. gonorrhoeae studies. Standard microbiological
plating instructions are designed to provide well-isolated colonies. Since N. gonorrhoeae rapidly
die following localized depletion of glucose due to rapid growth, streaking patterns are modied
to increase the regions of growth constrained colonies to provide a large number of colonies with
high viability.
Beta
Proteobacteria
4A.1.9
Current Protocols in Microbiology
Supplement 8
3. If using the cotton-tipped swab, turn the applicator to use a fresh portion of the tip
to streak out each of the remaining quadrants.
4. Invert the plates and incubate overnight at 37 C with 3% to 10% CO2 .
The gonococcal growth will be viable the next day, but the viability will decline rapidly
over the next 12 to 24 hr, depending on the strain. It is best to use fresh gonococcal plate
cultures for each day.
A technique to generate large amounts of small but not confluent colonies is to sub-culture
late in the afternoon. Spread the initial inoculum, followed by filling the rest of the plate
with tightly spaced streaking, each entering the initial inoculum. The majority of these
colonies will be small but not confluent if used within 15 to 18 hr of growth. The relatively
short incubation time coupled with the close spacing of the cells, to generate competition,
will result in a high proportion of the growth with good viability.
Laboratory
Maintenance of
N. gonorrhoeae
4A.1.10
Supplement 8
Figure 4A.1.6 Oxidase assay used for presumptive identication of Neisserial colonies. A shows
a GCMB plate of N. gonorrhoeae with a small drop of tetramethyl-p-phenylenediamine solution.
The oxidase-positive colonies rapidly turn pink-purple. B shows both oxidase-positive (+) and
oxidase-negative () colony types. This presumptive assay is used to identify oxidase-positive
colony types. Note that tested colonies cannot be used for subculturing or other assays but serve
to conrm that a particular colony morphology type has a high probability of being N. gonorrhoeae.
For a color version of this gure, see http://www.currentprotocols.com
Current Protocols in Microbiology
OXIDASE TEST
Colony morphology can vary significantly in N. gonorrhoeae, and can be mistaken for
contamination by other organisms. A quick method to presumptively identify a colony
as N. gonorrhoeae is the oxidase test. A freshly prepared 1% di- or tetramethyl-pphenylenediamine solution will react with Neisseriae, and turn the colony color pink
to purple (see Fig. 4A.1.6). This rapid assay can be performed in a variety of ways. A
colony can be picked up onto a swab and then a drop of reagent is applied directly onto
the colony on the swab and monitored for color change. Alternatively, a small amount of
the reagent can be added directly to the questioned colony type on the culture plate, being
careful to limit the spread of the reagent on the plate. Once an oxidase-positive colony
type has been identified, the other identical colonies on the plate from non-tested regions
can be used with high level of assurance that they are N. gonorrhoeae. While some
protocols call for a freshly prepared 1% solution, a toothpick full of di- or tetramethylp-phenylenediamine in 0.5 ml deionized water in a microcentrifuge tube has been found
to work well. In addition, some methods require DMSO to solubilize di- or tetramethylp-phenylenediamine and 2 to 3 days bacterial growth; these protocols are not specific for
GC, which are strongly oxidase positive.
CATALASE TEST
A common contaminant that gives rise to colonies very similar in appearance to GC
is Streptococcal sp. A rapid method to distinguish these contaminants from GC is the
catalase test, as GC are strongly catalase positive while Streptococcal sp. are catalase
negative. Catalase is a protective enzyme that converts hydrogen peroxide into water and
oxygen. In this very simple test, a drop of hydrogen peroxide (3% to 30%) is placed onto
a slide and a sample of colony is dragged through this drop, and the immediate formation
of bubbles indicates the presence of catalase (see Fig. 4A.1.7). Care must be taken if the
colony is obtained from blood or chocolate plates as erythrocytes contain catalase and
carryover from medium can give rise to a false-positive test.
SUPPORT
PROTOCOL 1
SUPPORT
PROTOCOL 2
Figure 4A.1.7 Slide catalase assay. Samples of two different colonies were spread on a glass
microscope slide. A drop of hydrogen peroxide was placed on each streak. Note the extensive
bubbling from the N. gonorrhoeae (+) sample.
BASIC
PROTOCOL 2
Beta
Proteobacteria
4A.1.11
Supplement 8
Broth cultures require a relatively high initial inoculum, which may be related to the
need for CO2 , as the time in lag-phase is dependent on CO2 concentration (see Strategic
Planning for a discussion of CO2 conditions). In addition, the growth pattern of different
gonococcal strains varies, depending on the expression of pili and other surface adherence
factors. Thus, some strains will generate smooth suspensions while others tend to form
grainy cultures.
Materials
Supplemented GCP (see recipe)
Overnight plate culture of N. gonorrhoeae (see Basic Protocol 1)
3- to 10-ml sterile tubes
Sterile culture flask (volume five times that of broth culture)
Sterile cotton-tipped swab
Spectrophotometer capable of reading at 600 nm and cuvettes
37 C shaking incubator
1. Transfer 1 to 3 ml of supplemented GCP into a 3- to 10-ml sterile tube, and the
desired amount into the culture flask.
2. Use a sterile cotton-tipped swab to remove gonococci from the overnight plate(s).
Be sure to use the small but not confluent colonies from the overnight plate culture to
ensure high viability in the inoculum. Some prefer to pre-moisten the swab in the GCP
before scraping up the gonococci, as it may make it easier to resuspend the cells from the
swab.
3. Vigorously agitate the swab in the tube containing the 1 to 3 ml GCP to create an
inoculating suspension of gonococci.
Do not use a tube that is filled more than 1/3 total height by the GCP, as head-space
is needed to allow for vortexing without splashing the gonococcal suspension out of the
tube. A capped tube is highly preferred for extra protection.
4. Vortex the suspension at high speed to make a smooth mixture of the gonococci,
taking precautions to avoid splashing.
5. Add the inoculating suspension, in small additions, to the GCP in the culture flask
until the desired density is reached.
Many strains of gonococci will not grow if the initial concentration of gonococci, as
measured by OD600 of the culture, is <0.1, and some may require much higher initial
inoculums. Keep the volume of culture to no more than 20% of the flask volume to ensure
appropriate aeration of the culture. Successful exponential growth of F62 with initial
starting cultures at OD600 = 0.07 to 0.09 has been obtained, reaching mid-log (OD600 =
0.5) within 2.5 hr growth.
6. Place the flask on a 37 C shaking incubator at relatively high speed (200 to 250 rpm).
7. Allow the culture to grow until the desired density is reached. Acquire intermediate
OD600 readings (every hour) to ensure that the culture is growing appropriately, i.e.,
starting exponential phase within 3 hr of growth.
Laboratory
Maintenance of
N. gonorrhoeae
The culture will start to lyse if allowed to go into stationary phase for any length of time
(often within 1 hr). For the laboratory strain F62, OD600 readings of 1 to 1.2 are typical
of early stationary phase. The length of time in which it takes a broth culture to reach
this level of growth depends both on the strain and on the viability and size of the initial
inoculum. Quite often, stationary phase is obtained within 8 to 10 hr of growth from an
initial culture OD600 of 0.1. Failure to enter exponential growth may indicate that the
initial inoculum was too small, or that its viability was low.
4A.1.12
Supplement 8
8. Subculture growing broth cultures into fresh complete GCP, however, avoid overdiluting new cultures. Keep the density over OD600 > 0.05, usually no more than a
1:20 dilution. Grow with shaking at 37 C.
BASIC
PROTOCOL 3
An effective method to grow N. gonorrhoeae anaerobically in broth has not yet been
discovered. There have been studies using microaerobic broth, established by either
completely filling the culture container with broth (to minimize air space) or capping
the culture or by initiating the culture aerobically, then stopping all agitation of the
culture (Knapp and Clark, 1984). This loss of motion coupled with the poor solubility of
oxygen in liquids will establish an oxygen gradient in the culture. Neither system has been
particularly reproducible, therefore, they are not provided in this unit. Contact the authors
for more information concerning these techniques (ginny-clark@urmc.rochester.edu).
Materials
GCMB solid medium (see recipe)
Fresh (1-day-old) N. gonorrhoeae plate culture
GCP broth (see recipe; non-supplemented GCP is suitable for the temporary
resuspension of GC)
70% ethanol
20% sodium nitrite solution, sterilized (see recipe)
100-mm plates
3- to 10-ml sterile tubes with caps
Sterile cotton-tipped swabs
Sterilized glass spreaders (APPENDIX 4A)
Tweezers or forceps
Bunsen burner
Paper discs, 1/2 -in. diameter, sterilized (Schleicher and Schuell 740-E)
Anaerobe chamber containing 37 C incubator (highly preferred) or 37 C incubator
with H2 /CO2 -generating GasPak (BBL) (BD cat. no. 260678)
1. Pour thick GCMB plates using 30 ml GCMB solid medium per 100-mm diameter
plate (30 to 36 plates per liter).
It is important to use fresh (1-day-old), room-temperature, thickly poured GCMB plates,
as the anaerobic environments tend to be dehydrating, and normal GCMB plates dry out
too much to support gonococcal growth. Allow plates to sit on the benchtop overnight,
do not bag or refrigerate.
2. Start a subculture of the gonococci, grow overnight aerobically on GCMB (see Basic
Protocol 1).
Results are more reproducible if the gonococci have been subcultured at least once from
frozen stock.
3. Place 1 to 3 ml GCP into a 3- to 10-ml sterile tube, preferably one with a cap.
Beta
Proteobacteria
4A.1.13
Current Protocols in Microbiology
Supplement 8
4. Use a sterile cotton-tipped swab to remove the small but not confluent colonies from
the overnight aerobic plate culture of gonococci.
If the swab is pre-moistened in the GCP before scraping up the gonococci, it may be
easier to resuspend the cells from the swab.
5. Vigorously agitate the cotton-tipped swab with gonococci colony in the tube containing the 1 to 3 ml GCP to produce an inoculating suspension of gonococci.
6. Vortex the suspension at high speed to create a smooth mixture of the gonococci,
taking precautions to avoid splashing.
It is desirable to have the concentration at an OD600 of 1.0 to 1.5.
7. Add 100 l of the gonococci suspension to each plate. Evenly distribute the inoculum
by spreading with a sterile glass spreader.
This method is preferred because spreading the gonococcal suspension with a swab
often scratches the medium. These scratches make recovery of the anaerobically prepared
gonococci more difficult, as the organisms appear to stick in the scratches.
Alternatively, heavily inoculate the plates by dipping a fresh, sterile swab into the gonococcal suspension and use it to spread a heavy inoculum evenly across the fresh GCMB
plate.
Spread the inoculum over the plate by streaking the wet swab completely over the plate
in one direction, turn the plate 120 and spread again, then turn and spread a third time.
This will provide an even suspension of gonococci on the plate. A wet swab is used to limit
scratches in the medium and provide a suitable number of gonococci. Allow the plates to
dry until the gonococcal suspension is completely absorbed.
8. Dip the tweezer tips into 70% ethanol and flame to sterilize.
9. Use the tweezers to place a sterile 1/2 -in. diameter paper disc onto the center of the
inoculated GCMB plate.
Gently tap the disc onto the surface with the tweezers tip to achieve good contact with
the medium. This is beneficial if the medium surface is very wet and the disc is sliding on
the inoculum. Alternatively, tap the disc onto the plate with a pipet tip used to dispense
the nitrite solution.
10. Slowly place 40 l of 20% sodium nitrite solution onto the paper disc.
Care must be taken to avoid adding the fluid faster than the disc can absorb it, or the disc
will float on the surface of the liquid and shift position on the plate. It is also important to
add the nitrite to the disc before it absorbs too much water from the medium, which then
limits nitrite absorption. Setting discs onto no more than five to ten plates before adding
nitrite is recommended. Allow the nitrite to fully absorb into the disc prior to inverting
the plates to prevent the discs from moving.
11. Place all the prepared plates (inverted) into the container for incubation in the
anaerobe chamber and load into the entry portal. Alternatively, place the inverted
plates into the GasPak container, and activate the H2 /CO2 -generating GasPak.
In some cases, the gas flow into the entry portal is fast and can move the items in the
portal. If the plates are in a container, they are less likely to be tipped. However, make
sure the lid is placed on the container in an offset manner, as the lid can become vacuum
sealed to the container during the evacuation portion of the cycle. A permanent solution
to the vacuum-lock problem is to place a small hole in the lid of the container to allow
for gas exchange.
Laboratory
Maintenance of
N. gonorrhoeae
12. Cycle entry portal to switch the atmosphere to 85% N2 , 10% H2 , and 5% CO2 ,
according to manufacturers instructions.
4A.1.14
Supplement 8
Figure 4A.1.8 Plate of anaerobically grown N. gonorrhoeae. A GCMB plate was inoculated with
N. gonorrhoeae and incubated anaerobically as described in Basic Protocol 3. Arrows indicate the
ring of strong N. gonorrhoeae growth at the optimal nitrite concentration surrounding the paper
disc. Note how the region between the disc and the denoted growth ring is clear and devoid of
growth. N. gonorrhoeae growth typically extends outward beyond the ring of optimal growth for
0.5 to 1 in. This is best observed by tipping the plate to nd the optimal lighting to show the
differences in the medium surface reectivity due to bacterial growth.
13. Open the internal portal door, remove the plates from the entry portal and place in
the 37 C incubator.
Placing the plates into a container allows easier group handling in the glove box. The
container also helps to limit dehydration of the plates during incubation.
BASIC
PROTOCOL 4
Beta
Proteobacteria
4A.1.15
Supplement 8
Set up the anaerobe chamber so that the instruments and reagents are easily reached. The
working space in the anaerobe chamber is often limited, so it is important to maintain a
clutter-free area for viewing and manipulating the cultures.
Materials
24- to 42-hr anaerobic gonococcal culture
GCP stock (see recipe)
GCMB plates (see recipe for GCMB solid medium)
20% sodium nitrite solution, sterilized (see recipe)
Squeeze bottle with 70% ethanol or 10% bleach
Anaerobe chamber containing 37 C incubator stocked with:
Pipets and sterile tips
Waste container lined with biohazard bag
Sterile swabs
Paper discs, 1/2 -in. diameter, sterilized (Schleicher and Schuell 740-E)
Tweezers or forceps
Rack to hold tubes, tweezers, marker
Pipet aide
Container for holding inoculated plates
Lab-wipes or paper towels
3- to 5-ml sterile tubes
Mechanical vortexer
Individually wrapped sterile glass spreaders or disposal sterile plastic
Bacteriological loops, optional
1. Remove 24- to 42-hr anaerobic gonococcal plate cultures from the incubator, and
identify the ring of gonococcal growth on each plate.
2. Place 1 to 1.5 ml GCP stock broth into a 3- to 5-ml sterile tube.
3. Use a sterile swab to remove the growth ring and resuspend gonococci in the tube
containing GCP stock. Agitate swab vigorously and vortex to create a smooth suspension of gonococci, being careful to avoid splashing.
4. Add 100 l of this GC suspension to a fresh thickly poured GCMB plate.
5. Gently spread with a fresh sterile swab, evenly spreading the suspension in three
directions as described in Basic Protocol 3, step 7.
Alternatively, use autoclaved, individually wrapped glass spreaders or pre-sterilized plastic spreaders. Glass spreaders cannot be sterilized by dipping in ethanol and burning off
the alcohol, as the anaerobic environment will not support a flame, therefore, place a
sufficient number of sterilized glass spreaders in the chamber for the procedure. One
spreader can be used to spread multiple plates inoculated with the same gonococcal
suspension if care is taken to avoid contamination.
An alternative is to use sterile disposable bacteriological loops. These plastic loops can
be bent into spreaders by gently pressing the loop end onto the inside of the plate top.
This will cause the thinner, 2 to 3 in. of the loop end to bend at an angle to the handle,
which can then be used to evenly spread the inoculum.
Laboratory
Maintenance of
N. gonorrhoeae
The rate of anaerobic growth is much slower than aerobic growth. To approximate the
number of anaerobic plates needed for a given protocol, assume that under anaerobic
conditions, 5% to 10% of the gonococci will be obtained as compared to aerobic growth.
4A.1.16
Supplement 8
7. Use the tweezers to place a sterile paper disc onto the center of the inoculated GCMB
plate. Tap to set the disc.
The tweezers must be sterilized to avoid contaminating the plates. Options include the
use of a ceramic incinerator to heat-sterilize the tweezer tips in the anaerobe chamber or
to sterilize the tweezers prior to entering the anaerobe chamber, and maintaining sterility
by placing them into sterile tube.
Do not add the discs to too many plates at once, as the paper will absorb water from the
inoculated plate. Once the paper is saturated, it cannot absorb the nitrite solution, which
will then run out onto the plate.
8. Slowly add 40 l of 20% sodium nitrite solution onto the paper disc.
9. Let the 20% sodium nitrite solution absorb into the disc before inverting plates so
that the disc does not slip from the center of the plate.
10. Invert plates and place into the incubator for 24 to 42 hr.
A quick and easy way to place inverted plates into the container is to place the container
lid on the floor of the anaerobe chamber, top down. Place the plates on the lid, right side
(tops) up. Place the bottom of the container over the plates and onto the lid. Slide a hand
under the lid and carefully turn the container right-side up. This works best if the plates
fill the container. If there are not enough to fill the container, add empty plates to the stack.
BASIC
PROTOCOL 5
Variations in piliation and expression of opa proteins give rise to significant differences
in colony morphology (see Fig. 4A.1.9). It is often desirable to maintain the complete
range of colony types in stock cultures. However, the efficiency of transformation is
substantially higher in piliated strains, thus their inclusion in stock cultures is essential
if the strain is to be used for genetic experiments. While it is possible to select for
non-piliated variants from a predominantly piliated stock, in many strains, the reverse
is not feasible, as loss of pili is often due to loss of the pilin expression site (PE ) in the
chromosome. See UNIT 4A.3 for information on discriminating piliated from non-piliated
N. gonorrhoeae.
Never allow the frozen stock sample to thaw, as the viability of the gonococci will rapidly
decline. Always place the frozen stock vial in dry ice when taken out of the freezer, and
minimize the time that the vial is held in hand to limit thawing while removing samples
to maintain a suitable viability of the stock culture. When the viability appears to be
significantly diminished, either switch to a second stock vial or prepare additional stocks
from a fresh culture prepared from the original stock.
Materials
GCP stock (see recipe)
18- to 24-hr culture of N. gonorrhoeae
50% glycerol (see recipe), autoclaved
Dry ice
Current Protocols in Microbiology
Beta
Proteobacteria
4A.1.17
Supplement 8
Figure 4A.1.9 Photograph of piliated and non-piliated N. gonorrhoeae. Pilin expression can
undergo phase variation and is reected in variation in colony morphology. Piliated (P+) cells form
colonies that tend to be smaller, taller, and darker (gumdrop-like) when viewed under a stereoscope
while non-piliated (P) cells form colonies that take a atter, more wafer-like appearance (Kellogg
et al., 1968).
8. Place the cryovial into pulverized dry ice to snap-freeze the sample.
An ethanol- or acetone-dry-ice mixture can also be used to snap freeze the sample, but
the organics tend to remove the ink labeling from the cryovial.
How long any given stock remains viable varies significantly with the quality of the initial
frozen culture, the number of times the frozen stock is accessed, and the degree of thawing
that occurred with each handling. Viable N. gonorrhoeae from frozen stocks that were
well over 10 years old have been retrieved.
4A.1.18
Supplement 8
CDM, 2
For 1 liter, mix the following:
50 ml CDM stock solution 1 (see recipe)
360 ml CDM stock solution 2 (see recipe)
20 ml CDM stock solution 3 (see recipe)
40 ml CDM stock solution 4 (see recipe)
50 ml 20% (w/v) glucose solution (see recipe)
Add ddH2 O to 800 ml total volume
Adjust pH to 7.4 with 10 N NaOH
Add 2 ml 10 mM ferric chloride (FeCl3 6H2 O) or ferric nitrate (Fe(NO3 )3 9H2 O)
(see recipes)
Add ddH2 O to a final volume of 1 liter
Add 2 ml 250 mM CaCl2 (see recipe; final concentration 0.25 mM)
Sterilize by passing through a 0.2-m filter sterilization system with sterile storage
container
Transfer into appropriate aliquots and store up to 1 week at 4 C
Refer to Table 4A.1.1 for a brief explanation of the component stock solutions.
It is important to add the CaCl2 to diluted CDM as it has a tendency to precipitate. Adding
2 ml to 1000 ml does not significantly alter the concentrations of the final medium.
There are many potential sources of iron that can be used to supplement CDM.
Two commonly used iron sources are ferric chloride (FeCl3 6H2 O) and ferric nitrate
(Fe(NO3 )3 9H2 O); see Strategic Planning. Regardless of the source, a 10 mM stock solution, used at a final concentration of 0.01 mM can be tested with 1 CDM.
Table 4A.1.1 CDM Stock Solutions and their Purpose
Stock solution
Purpose
Salts solution
Vitamins/biotin solution
Phosphate solution
Beta
Proteobacteria
4A.1.19
Current Protocols in Microbiology
Supplement 8
CDM, liquid
Dilute 2 CDM 1:1 with sterile ddH2 O and add 1/100 volume 4.2% NaHCO3 .
Prepare fresh.
CDM, solid
For plates, prepare 3% agar (3 g agar to 100 ml H2 O), autoclave, and cool to 55 C.
Mix the agar 1:1 with 2 CDM stock (see recipe; warmed to 55 C), then pour
plates.
4A.1.20
Supplement 8
Component
Amount
Solvent
Stock concentration
(mM)
Final concentration
(mM)
240.3
0.15
540 mg
50 ml 1.0 N HCl
hypoxanthine
750 mg
100 ml 1.0 N
NaOH
136.1
0.37
L-tyrosine
1065 mg
100 ml 1.0 N
NaOH
181.2
0.39
L-tryptophan
200 mg
100 ml 1.0 N
NaOH
204.3
0.39
Uracil
750 mg
100 ml 1.0 N
NaOH
112.1
0.45
Neutral-soluble amino acid solution: add all listed amino acids to 2000 ml ddH2 O
L-alanine
1500 mg
2000 ml ddH2 O
89.9
1.1
L-arginineCl
250 mg
2000 ml ddH2 O
210.8
0.71
L-asparagineH2 O
385 mg
2000 ml ddH2 O
150.1
0.17
L-aspartic
9.65 g
2000 ml ddH2 O
171.2
3.8
L-cysteineCl
825 mg
2000 ml ddH2 O
157.6
0.35
L-glutamic
23.3 g
2000 ml ddH2 O
169.1
8.8
L-glutamine
750 mg
2000 ml ddH2 O
146.1
0.34
Glutathione (reduced)
690 mg
2000 ml ddH2 O
307.3
0.15
Glycine
375 mg
2000 ml ddH2 O
75.1
0.33
DL-histidineClH2 O
375 mg
2000 ml ddH2 O
209.6
0.12
DL-isoleucine
450 mg
2000 ml ddH2 O
131.2
0.23
DL-leucine
1365 mg
2000 ml ddH2 O
131.2
0.69
DL-lysineCl
750 mg
2000 ml ddH2 O
182.0
0.27
DL-methionine
225 mg
2000 ml ddH2 O
149.2
0.1
DL-phenylalanine
375 mg
2000 ml ddH2 O
165.2
0.15
L-proline
750 mg
2000 ml ddH2 O
115.1
0.43
DL-serine
750 mg
2000 ml ddH2 O
105.1
0.48
DL-threonine
750 mg
2000 ml ddH2 O
119.1
0.42
L-valine
900 mg
2000 ml ddH2 O
117.2
0.51
acid (K salt)
acid (Na salt)
Component
Stock concentration
(mM)
Final concentration
(mM)
ThiamineCl
0.593
0.59
Co-carboxylase
0.104
0.01
Biotin
1.2
0.12
mg D-Pantothenic acid
0.40
0.004
Beta
Proteobacteria
4A.1.21
Current Protocols in Microbiology
Supplement 8
Ferric chloride, 10 mM
Add 27 to 40 mg FeCl3 6H2 O to a 15-ml conical
Divide the weighed amount by the molecular weight (270.3)
Multiply the result by 100, and add that volume of ddH2 O in milliliters
Mix and filter sterilize (using a 0.2-m filter)
Store in 1-ml aliquots up to 1 year at 4 C
This iron solution is clear yellow. Discard the solution if the yellow color intensifies or if
precipitates form, as that indicates that the iron is oxidized and no longer available for
gonococcal growth.
Since the amount of ferric chloride needed to make a 10 mM solution is small, weighing
out the reagent first and then adding the appropriate amount of ddH2 O to the reagent is
recommended.
Ferric nitrate, 10 mM
Add 40 to 60 mg Fe(NO3 )3 9H2 O to a 15-ml conical tube
Divide the weighed amount by the molecular weight (404.0)
Multiply the result by 100, and add that volume of ddH2 O in milliliters
Mix and filter sterilize (using a 0.2-m filter)
Store in 1-ml aliquots up to 1 year at 4 C
This iron solution is clear yellow. Discard the solution if the yellow color intensifies or if
precipitates form, as that indicates that the iron is oxidized and no longer available for
gonococcal growth.
Since the amount of ferric nitrate needed to make a 10 mM solution is small, weighing
out the reagent first and then adding the appropriate amount of ddH2 O to the reagent is
recommended.
Laboratory
Maintenance of
N. gonorrhoeae
After the medium has cooled to 55 C (30 min, depending on the number of flasks
and the size of the water bath), transfer the cooled medium to a stir plate set on low
and add 5 ml (i.e., 1/100 volume of the medium) Kelloggs supplement (see recipe).
If needed, add antibiotics at this time. Pour plates using 20 to 25 ml per 100
15mm diameter plate (20 to 22 plates per 500 ml medium). After the plates have
completely set and cooled, invert on the benchtop. For long-term storage (>3 days),
place the plates into bags and store, inverted, at 4 C. It is convenient to store poured
plates in the bags used to package the Petri plates, as they are sterile and the perfect
diameter for the plates. However, it is critical to let the poured plates dry before
bagging, as the moisture will condense in the cold and can lead to contaminated
plates due to the growth of mold, which gets transferred into the plates via water
droplets. As a rule, allow poured media to sit at least overnight, and preferably closer
to 48 hr before packaging for long-term storage. If plates must be stored early, it
may be beneficial to put small vent holes into the Petri dish bags to allow for air flow.
Refer to Strategic Planning for additional details.
4A.1.22
Supplement 8
GCP stock
To 1 liter of ddH2 O, add:
15 g proteose peptone no. 3 (Difco)
1 g soluble starch
4 g K2 HPO4
1 g KH2 PO4
5 g NaCl
Stir until well mixed, 5 to 10 min. Note that the solution may not be clear, as
the starch may not go fully into solution. Distribute the well-mixed medium into
200-ml bottles, filling no more that half-full. Loosely attach the bottle lids and
autoclave. When the medium has cooled to room temperature, tighten the lids and
store in 100-ml aliquots up to 1 year at room temperature.
Kelloggs supplement
To 200 ml ddH2 O, slowly add 200 g dextrose (glucose) in portions, and allow each
portion to dissolve before adding more. Mix until all the glucose is in solution. Add
5.0 g glutamine, 250 mg ferric nitrate, and 10 mg Co-carboxylase (Sigma-Aldrich
cat. no. C-8754). Continue stirring until all solids have gone into solution. Transfer
to a 500-ml graduated cylinder and add ddH2 O to make 500 ml total volume. Return
to beaker and stir for additional 5 to 10 min. Filter sterilize using a 0.22-m filter
into sterile bottles. Store up to 6 months at 4 C.
Refer to Strategic Planning for additional details.
Beta
Proteobacteria
4A.1.23
Current Protocols in Microbiology
Supplement 8
750 mg DL-lysineCl
225 mg DL-methionine
375 mg DL-phenylalanine
750 mg L-proline
750 mg DL-serine
750 mg DL-threonine
900 mg L-valine
Prepare fresh and use immediately for CDM stock solutions
Sodium bicarbonate, 4.2% (w/v)
Add 4.2 g NaHCO3 to 100 ml ddH2 O
Stir to dissolve
Sterilize by filtration using a 0.22-m filter
Store up to 1 year at room temperature
Sodium nitrite solution, 20% (w/v)
Add 20 g of NaNO2 to 100 ml ddH2 O
Stir to dissolve
Sterilize by filtration using a 0.22-m filter
Store up to 1 year at room temperature
Supplemented GCP
To GCP stock, add:
1/100 volume 4.2% sodium bicarbonate (see recipe)
1/100 volume Kelloggs supplement (see recipe) or IsoVitaleX (BBL; BD cat.
no. 211875-211876)
Prepare only the amount needed for the day
COMMENTARY
Background Information
Laboratory
Maintenance of
N. gonorrhoeae
They can also use transferrin- and lactoferrinbound iron in the human host, as well as heme
and hemoglobin (reviewed in Schryvers and
Stojiljkovic, 1999). N. gonorrhoeae can use
three carbon sources: glucose, pyruvate, and
lactate (Morse and Bartenstein, 1974). Aerobically grown gonococci primarily utilize glucose via the Entner-Douderoff pathway, with
minor use of the pentose phosphate pathway
(Hebeler and Morse, 1976). When grown on
pyruvate, the use of the pentose phosphate
pathway accounts for one-third of carbon
metabolism.
For many years, N. gonorrhoeae was considered an obligate aerobe, even when isolated
from mixed infections with other known obligate anaerobic bacteria. However, the gonococcus can grow anaerobically when provided
with nitrite as a terminal electron acceptor
(Knapp and Clark, 1984). Under anaerobic
conditions, the Entner-Douderoff pathway is
used exclusively, forming lactate and acetate
in equivalent amounts (Morse et al., 1974).
Many clinical gonococcal isolates show
some degree of auxotrophy, which necessitates the use of rich media for isolation
4A.1.24
Supplement 8
Beta
Proteobacteria
4A.1.25
Current Protocols in Microbiology
Supplement 8
Time Considerations
Gonococcal autolysis significantly impacts
the laboratory growth of N. gonorrhoeae, as
cells do not retain viability on culture medium
after reaching stationary phase. Thus, planning for any experiment with gonococci must
include 1 day to obtain growth from frozen
stock along with a second overnight incubation
to obtain suitable numbers of viable organisms for experimental purposes. Clones from
genetic studies must be recultured for continued viability every 1 to 2 days, thus being
able to rapidly analyze clones is necessary for
procedures that may require screening of large
numbers of colonies for low-frequency events.
Literature Cited
Dillard, J.P. and Seifert, H.S. 2001. A variable genetic island specific for Neisseria gonorrhoeae
is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates. Mol. Microbiol. 41:263277.
Hebeler, B. and Morse, S.A. 1976. Physiology
and metabolism of pathogenic Neisseria: Tricarboxylic acid cycle activity in Neisseria gonorrhoeae. J. Bacteriol. 128:192-201.
Kellogg, D.S. Jr., Peacocky, W.L. Jr., Deacon, W.E.,
Brown, L., and Pirkle, C.I., 1963. Neisseria
gonorrhoeae. I. Virulence genetically linked to
clonal variation. J. Bacteriol. 85:1274-1279.
Kellogg, D.S. Jr., Cohen, I.R., Norins, L.C.,
Schroeter, A.L., and Reising, G., 1968. Neisseria gonorrhoeae. II. Colonial variation and
pathogenicity during 35 months in vitro. J. Bacteriol. 96:596-605.
Key References
Morse, S.A. 1978. The biology of the gonococcus.
CRC Crit. Rev. Microbiol. 7:93-189.
Stephen A. Morse played a major role in elucidating
N. gonorrhoeae physiology.
Perspectives on Pathogenic Neisseriae. 1989. Clin.
Microbiol. Rev. 2: Supplement.
This entire journal supplement is dedicated to
pathogenic Neisseriae, with solicited review articles on major areas of Neisserial research.
U.S. Department of Health and Human Services
Centers for Disease Control and Prevention and
National Institutes of Health. 2007. Biosafety in
Microbiological and Biomedical Laboratories,
5th Edition (BMBL). U.S. Government Printing
Office, Washington.
The document in its entirety is available online at
http://www.cdc.gov/od/ohs
Laboratory
Maintenance of
N. gonorrhoeae
4A.1.26
Supplement 8
UNIT 4A.2
Neisseria gonorrhoeae is a Gram-negative coccus and the causative agent of the sexually
transmitted disease, gonorrhea. The disease generally manifests as localized urethritis in
men and cervicitis in women. In women, the infection is often asymptomatic initially, but
may spread to the uterus and fallopian tubes, causing pelvic inflammatory disease, chronic
pelvic pain, and infertility. Untreated infections in men or women may also cause systemic
disease (disseminated gonococcal infection), which may be manifested as tenosynovitis,
meningitis, or endocarditis. For laboratory workers, it is most important to remember
that N. gonorrhoeae causes eye infections. Normally gonococcal eye infections occur in
newborns infected during passage through the birth canal of an infected mother; these
infections often lead to blindness if left untreated. Laboratory-acquired N. gonorrhoeae
eye infections have been reported; therefore, it is important to wear safety glasses when
performing procedures that may cause gonococci to be propelled into the air and to seek
medical treatment if the eyes or other mucosal surfaces are exposed to N. gonorrhoeae.
Gonococci are naturally transformable and this property has greatly facilitated its study.
Gonococci readily take up DNA containing the 10-bp Neisseria DNA uptake sequence
(DUS) GCCGTCTGAA (Goodman and Scocca, 1988). The DUS is found frequently
in the chromosome of N. gonorrhoeae and other Neisseria species, often in two copies
as an inverted repeat. Plasmids used for transformation of N. gonorrhoeae must contain
the DUS to be taken up efficiently. Unlike many transformable bacteria, N. gonorrhoeae
does not regulate transformation: gonococci are transformable at all stages of growth
(Sparling, 1966). However, only piliated gonococci are naturally transformable, and
piliation is a phase-variable phenotype. Therefore, care must be taken to choose piliated
colonies for transformation.
This unit describes methods for genetic manipulation of N. gonorrhoeae including various
methods of mutagenesis and complementation, as well as methods for growing and
purifying DNA from these bacteria.
CAUTION: Neisseria gonorrhoeae is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. In particular, wear safety glasses as exposure of the eyes to N. gonorrhoeae
will cause conjunctivitis (see Critical Parameters and Troubleshooting). See UNIT 1A.1 and
other pertinent resources (APPENDIX 1B) for more information.
IMPORTANT NOTE: Unless otherwise indicated, all incubations are to be performed at
37 C, 5% CO2 .
STRATEGIC PLANNING
Growth on Agar or in Liquid Medium
Gonococci are fastidious. They have multiple nutritional requirements and are generally
grown in rich medium, either gonococcal base medium (GCB) or chocolate agar. They
require CO2 in the atmosphere for growth on agar plates or sodium bicarbonate in
liquid medium (Morse and Bartenstein, 1974). Medium must be supplemented with
Kelloggs supplements (Kellogg et al., 1963) or IsoVitalex (Becton Diconson). Recipes
for gonococcal base medium and supplements are found in the Reagents and Solution
section below. Gonococci should be inoculated from an agar plate into GCBL at a density
Beta
Proteobacteria
Contributed by Joseph P. Dillard
Current Protocols in Microbiology (2005) 4A.2.1-4A.2.19
C 2005 by John Wiley & Sons, Inc.
Copyright
4A.2.1
Antibiotic
MS11 (g/ml)
FA1090 (g/ml)
Chloramphenicol
10
Erythromycin
10
Kanamycin
80
ND
Nalidixic Acid
1.5
1.5
Penicillin G
0.3
ND
Spectinomycin
75
75
Streptomycin
100
100
Tetracycline
0.2
no less than 107 CFU/ml to reliably obtain a viable liquid culture. The medium must be
prewarmed to 37 C. In general, inoculating to a density of 5 107 to 1 108 (OD540 of
0.2) is the most reliable method. Transfer may be done using a sterile Dacron swab. Do
not use cotton, as these swabs may contain fatty acids which are inhibitory to some strains
of N. gonorrhoeae. Optimal numbers of live gonococci for inoculation are obtained from
agar plates 18- to 20-hr old. The doubling time for laboratory strains in liquid medium is
60 min.
Genetic
Manipulation of
N. gonorrhoeae
4A.2.2
Current Protocols in Microbiology
Alternatively, the ability of N. gonorrhoeae to incorporate plasmids into the chromosome can be used for making mutations (Hamilton et al., 2001). A single fragment of
gonococcal DNA can be cloned into a plasmid in E. coli. Transformation with the circular plasmid, followed by selection for the antibiotic resistance marker on the plasmid
will result in the isolation of transformants that have integrated the plasmid by singlecrossover recombination. Since this method results in duplication of the cloned sequence,
the method is often referred to as insertion-duplication mutagenesis.
Due to the high efficiency of transformation observed with this method, it is possible to
introduce mutations that do not contain an antibiotic resistance gene or other selectable
marker. Plasmids or PCR products containing deletions, insertions, or point mutations
can be used for transformation. If plasmids are used, they should be linearized to prevent
single-crossover recombination. Potential transformants can be screened by PCR for an
increase or decrease in the size of the PCR product amplified from the gene of interest. If
small mutations are introduced (e.g., point mutations, epitope tag insertions), it is helpful
to also introduce or eliminate a restriction site, allowing potential transformants to be
identified by PCR and restriction digest.
Materials
Recipient N. gonorrhoeae strain, frozen
GCB plates (see recipe), room temperature and 37 C
10 ng/l plasmid DNA
Appropriate restriction enzyme and buffer (for double-crossover only)
GCB plate containing appropriate antibiotic (Table 4A.2.1; for fragments
containing selectible markers only)
Dacron swabs (Fisher Scientific), sterile
Additional reagents and equipment for PCR (see Support Protocol)
1. Streak recipient N. gonorrohoeae strain from frozen stock onto a GCB plate. Incubate
overnight (<24 hr).
Prepare DNA
2a. For inserting a construct into the chromosome by double-crossover event: Linearize
the plasmid unless it is large (>8 kb). Be sure not to cut the transforming DNA away
from the gonococcal DNA uptake sequence.
2b. For performing insertion-duplication mutagenesis: Do not linearize the plasmid.
3. Draw two 1-cm circles on the back of a 37 C GCB agar plate, where, upon streaking,
the colony density will be medium and thin, respectively.
Appropriate places would be one-third and two-thirds of the way down the middle of the
plate.
4. Spot 10 to 20 l DNA solution (step 2a or 2b) on the agar inside the circles.
Maximize the DNA concentration but do not exceed 10 g. Allow DNA to soak into
the plate.
Amounts of DNA in the hundreds of nanograms are appropriate.
If the spots are not dry within 10 min, it may be necessary to dry the plate in a biosafety
hood with the lid of the plate removed, 20 min or less.
5. Pick a piliated colony of the recipient from the plate prepared in step 1. Streak
through the DNA spots and incubate overnight.
See Background Information for a discussion of appraising the piliation state of N.
gonorrhoeae.
Beta
Proteobacteria
4A.2.3
Current Protocols in Microbiology
6a. For transformations using fragments containing selectable markers: Using a sterile
Dacron swab, transfer and uniformly spread the colonies that grew on the spots onto
a GCB plate containing the appropriate antibiotic.
6b. For transformations using fragments without selectable markers: Streak individual
colonies to be screened onto GCB plates using an inoculating loop.
CAUTION: See Critical Parameters and Troubleshooting for important safety
considerations.
8. Streak individual transformants onto GCB plates and screen for nonselectable mutations by PCR (see Support Protocol).
Due to transformation occurring within only some members of a colony, nonselected
mutants identified by this method should be regarded as likely mixed populations and
should be restreaked; the presence of the mutation in individual colonies should be
verified.
BASIC
PROTOCOL 2
Materials
Recipient N. gonorrhoeae strain, frozen
GCB plate (see recipe)
10 ng/l plasmid DNA
GCBL medium (see recipe) containing 5 mM MgSO4 , room temperature and 37 C
GCBL medium containing Kelloggs supplements (see recipe)
60% (v/v) glycerol
Dacron swabs, sterile
T-25 tissue culture flask, 37 C
1. Streak recipient N. gonorrhoeae strain from frozen stock onto a GCB plate. Incubate
overnight.
2. Pick a piliated colony. Streak onto a GCB plate and incubate overnight (18 to 20 hr).
See Background Information for a discussion of appraising the piliation state of N.
gonorrhoeae.
4. Using a Dacron swab, prepare a recipient cell suspension by transfering 1/4 to 1/2 of
all of the colonies resulting from step 2 into a 1.5-ml microcentrifuge tube containing
1 ml GCBL medium/5 mM MgSO4 , 37 C. Swirl the swab to suspend bacteria.
4A.2.4
Current Protocols in Microbiology
5. Prepare transformation mix by adding 20 l recipient cells to the DNA solution (step
3). Incubate 5 to 15 min at 37 C.
6. If it is possible this strain will again be transformed, add 60% glycerol to a final
concentration of 15% (v/v) to the remaining recipient cell suspension (without DNA).
Flash freeze using liquid nitrogen or a dry ice/ethanol bath.
7. Transfer entire transformation mix to 2 ml GCBL containing Kelloggs supplements
in a T-25 tissue culture flask (already at 37 C). Incubate flask with the lid loosened
2 to 6 hr.
Plate transformants
8a. If selecting for antibiotic resistance: Prepare 10-fold serial dilutions in GCBL over
a range of 100 to 104 . Plate 100 l of each dilution onto an individual GCB plate
with the appropriate antibiotic. Incubate until colonies are observed (24 to 48 hr).
8b. If screening without selection: Prepare 10-fold serial dilutions in GCBL over a range
of 103 to 106 . Plate 100 l of each dilution onto individual GCB plates without
antibiotics. Incubate until colonies are observed (18 to 24 hr).
9. Screen for desired mutation by PCR (Support Protocol).
BASIC
PROTOCOL 3
Materials
Recipient N. gonorrhoeae strain, frozen
GCB plates (see recipe) with and without antibiotic (Table 4A.2.1)
0.3 M sucrose: pass through a 0.22-m filter to sterilize; store up to 1 year at room
temperature
10 mg/l DNA solution
GCBL medium with Kelloggs supplements (see recipe)
GCBL medium (see recipe)
Dacron swabs (Fisher Scientific), sterile
Electroporation cuvette, 2-mm gap length
Electroporatator
1. Streak recipient N. gonorrhoeae strain from frozen stock onto a GCB plate. Incubate
overnight.
2. Using a sterile Dacron swab, transfer 50% to 100% of the colonies on the plate into
1 ml of 0.3 M sucrose at room temperature.
Sucrose provides osmotic stabilization. Suspension of N. gonorrhoeae in low osmotic
strength buffer results in autolysis and cell death.
Beta
Proteobacteria
4A.2.5
Current Protocols in Microbiology
Materials
Genetic
Manipulation of
N. gonorrhoeae
4A.2.6
Current Protocols in Microbiology
2. Microcentrifuge desired volume of cells (generally 1.5 ml) for 30 sec at 12,000
rpm. Resuspend cells in 1 ml low ionic strength buffer, such as sterile 1 mM HEPES,
pH 7. Repeat wash two more times.
3. Resuspend cells in 40 l 1mM HEPES per planned electroporation.
4. Add target plasmid to cell suspension.
5. Transfer cell/DNA mixture into an electroporation cuvette. Electroporate with the
pulse controller set at 2.5 kV, 200 , and 25 F.
6. Plate 10 and 100 l electrotransformation mix onto LB plates containing 40 g/ml
kanamycin. Incubate overnight at 37 C.
7. Use sterile toothpicks to transfer several potential transformants onto LB plates
containing 40 g/ml kanamycin and 12 g/ml tetracycline.
Transformants should be resistant to both antibiotics and show two plasmids when examined by whole-cell lysis (cracking) gel (Kado and Lin, 1981; Dillard and Yother, 1994).
Such transformants will serve as the primary recipient.
9. Plate 100, 10, and 1 l onto individual LB plates containing 40 g/ml kanamycin
and 25 /ml chloramphenicol to select for KnR CmR transformants. Incubate 16 hr
at 37 C.
Any RDP146 cells not carrying all three plasmids will be killed in this step.
10. Grow NS2114Sm to mid-log phase (OD600 of 0.5) in LB medium without antibiotics
(4 hr).
This step can be started towards the end of the incubation described in step 9.
11. Wash or scrape transconjugates from plates into LB medium and mix 1:1 with
mid-log phase NS2114Sm.
12. Mate 15 min in a 37 C water bath. Incubate 30 min in rotator at 37 C.
13. Plate 100, 10, and 1 l onto individual LB plates containing 100 g/ml streptomycin,
40 g/ml kanamycin, and 25 g/ml chloramphenicol. Incubate 16 hr at 37 C.
14. If desired, screen for transposon location and orientation by PCR (see Support
Protocol).
Beta
Proteobacteria
4A.2.7
Current Protocols in Microbiology
15. Transform plasmid DNA into gonococci by spot transformation (Basic Protocol 1).
Select for transformants on GCB plates containing 25g/ml chloramphenicol.
BASIC
PROTOCOL 5
CHEMICAL MUTAGENESIS
Chemical mutagenesis has been used in N. gonorrhoeae. Its use is infrequent since
directed mutagenesis is highly efficient. Chemical mutagens that directly act on the DNA
are effective (Campbell and Yasbin, 1984). However, chemical mutagenesis methods
that rely on error-prone repair are not, since N. gonorrhoeae has no SOS-type system
(Black et al., 1998). The procedure below describes use of ethylmethane sulphonate
(EMS). N-Methyl-N -nitro-N-nitrosoguanidine (MMNG) could be used instead, at 5 g
per 108 cfu.
Materials
N. gonorrhoeae strain, frozen
GCB plates (see recipe)
GCBL medium with Kelloggs supplements and sodium bicarbonate (see recipe),
37 C
Ethyl methanesulfonate (EMS)
GCBL medium
GCBL medium containing 15% glycerol
GCB plates containing nalidixic acid (Table 4A.2.1)
Dacron Swab
1. Streak N. gonorrhoeae strain from frozen stock onto a GCB plate. Incubate overnight.
2. Using a sterile Dacron swab, transfer cells from 1/4 to 1/2 of the plate into 3 ml
GCBL medium with Kelloggs supplements and sodium bicarbonate, 37 C, to an
OD540 of 0.2. Retain a 0.5-ml aliquot.
3. Add EMS to a final concentration of 1% (v/v), e.g., 20 l to a 2-ml culture.
4. Incubate with aeration and remove 0.5-ml aliquots at 10, 20, 30, and 40 min. For each
time point, immediately microcentrifuge aliquot 30 sec at 12,000 rpm and remove
supernatant. Resuspend cells with 0.5 ml GCBL.
Do not forget to include the untreated culture (0-min time point) taken in step 2.
5. Prepare serial dilutions over a range of 100 to 106 . To determine killing frequency,
plate 100 l of each dilution onto an individual GCB plate for quantification of
colony forming units. Plate 100 l of each dilution onto an individual GCB plate
containing nalidixic acid to determine mutation frequency.
Point mutations in the gyrB gene result in nalidixic acid resistance (NalR ). Thus, measuring
the frequency of mutations conferring nalidixic acid resistance gives a measure of the
effectiveness of the chemical mutagenesis procedure.
Genetic
Manipulation of
N. gonorrhoeae
8. Streak bacteria onto GCB plates from the frozen, mutagenized stock from step 6 that
gave the highest mutation frequency or 99% to 99.9% killing frequency, then screen
for phenotype.
4A.2.8
Current Protocols in Microbiology
BASIC
PROTOCOL 6
Materials
Gene of interest
pKH35 (Hamiltan et al., 2005) or similar plasmid
E. coli
N. gonorrhoeae mutant of interest
GCB plate (see recipe) containing chloramphenicol (Table 4A.2.1)
1 M IPTG solution
Dacron swab, sterile (Fisher Scientific)
Additional reagents and equipment for preparing a miniprep (Engebrecht et al.,
1991), spot transformation (Basic Protocol 1), and PCR (Support Protocol)
1. Clone gene of interest into pKH35 or similar plasmid and transform into E. coli.
2. Purify plasmid using standard miniprep procedure (Engebrecht et al., 1991).
3. Use 200 to 500 ng plasmid and the N. gonorrhoeae mutant of interest to perform
spot tranformation (Basic Protocol 1) on a GCB plate.
4. Use a sterile Dacron swab to transfer all of the resulting colonies from a DNA spot
onto a GCB plate containing chloramphenicol. Incubate 24 to 48 hr.
5. Pick individual colonies and screen by PCR for presence of gene of interest adjacent
to lctP (see Support Protocol).
6. Induce expression of the gene of interest by adding of 1 M IPTG to a final concentration of 1 mM either in liquid culture or on solid medium.
Beta
Proteobacteria
4A.2.9
Current Protocols in Microbiology
ALTERNATE
PROTOCOL 1
BASIC
PROTOCOL 7
Materials
N. gonorrhoeae, overnight culture
GCB plates (see recipe)
TES buffer (see recipe)
10 mg/ml RNase A
10% SDS
Tris-buffered phenol (APPENDIX 2A)
Chloroform
3 M sodium acetate
95% and 70% ethanol
Dacron swab, sterile
13-ml snap-cap polypropylene tubes
1-ml pipette tips with wide bore
1. Using an inoculating loop, streak three to five colonies of N. gonorrhoeae onto each
of eight to ten GCB plates. Incubate overnight.
2. Using one sterile Dacron swab per plate, tranfer and suspend cells in 5 ml TES buffer
in a 13-ml snap-cap polypropylene tube.
Genetic
Manipulation of
N. gonorrhoeae
3. Add 1 l of 10 mg/ml RNase A (10 mg/ml) and 50 l of 10% SDS. Incubate 10 min
at 37 C to digest RNA.
Lysis should occur instantly.
4A.2.10
Current Protocols in Microbiology
4. Extract with an equal volume of tris-buffered phenol. Mix by gentle inversion twenty
times. Centrifuge 10 min at 6000 g (7000 rpm), 4 C.
5. Transfer aqueous (top) phase to a new tube using a micropipette equipped with a
wide-bore 1-ml pipette tip and repeat extraction.
6. Extract once with chloroform. Invert and centrifuge as in step 4.
7. Transfer aqueous (top) phase to a new tube. Add 1/10 vol 3 M sodium acetate and
2 vol of 95% ethanol. Mix and incubate 30 min at 20 C.
8. Centrifuge 10 min at 12,000 g, 4 C.
9. Wash pellet twice with 70% ethanol.
10. Invert tube and allow pellet to dry until white or clear.
11. Suspend DNA in 200 to 500 l water.
ALTERNATE
PROTOCOL 2
Growth of N. gonorrhoeae in liquid culture makes the preparation steps for DNA purification easier than those for preparing DNA from bacteria grown on plates. The bacteria
can be harvested by centrifugation (instead of swabbing) and the recovery of bacteria
is more efficient. The drawback to this method is that the cultures must be monitored
closely to ensure that the bacteria are still in the growth phase and have not begun to
autolyse before chromosome purification begins. Autolysed gonococcal cultures still
exhibit significant optical density (OD540 of 1.2), but purification of DNA from these
cultures gives low yields and mostly degraded DNA. However, if growth is monitored
to ensure growing cells are used, preparation of DNA from 200 ml of late-log phase
gonococci (in this protocol) provides significantly more DNA than the preparation from
ten agar plates. This method does not have an RNase step. Rather, the RNA functions
as carrier nucleic acid, allowing the DNA and large RNA to be easily recovered with a
glass hook.
Materials
N. gonorrhoeae, frozen stock
GCB plate (see recipe)
GCBL medium with Kellogs supplements and sodium bicarbonate (see recipe),
37 C
TE buffer (APPENDIX 2A)
10% SDS
5 M potassium acetate
95% and 70% ethanol
Dacron swab
13-ml polypropylene tube
Glass rod
65 C water bath
1. Streak N. gonorrhoeae from frozen stock onto a GCB plate and incubate overnight.
2. Using a sterile Dacron swab, transfer 1/4 to 1/2 of colonies on the plate to 3 ml
GCBL medium, 37 C, with Kelloggs supplements and sodium bicarbonate.
3. Incubate until culture reaches late log phase (OD540 of 1.0 1.4; 4 to 6 hr). Transfer
to 200 ml GCBL, 37 C, with supplements and sodium bicarbonate.
Beta
Proteobacteria
4A.2.11
Current Protocols in Microbiology
4. Incubate until culture reaches late log phase (OD540 of 1.4; <20 hr).
5. Centrifuge culture 10 min at 3,000 g, 4 C. Resuspend in 2.5 ml TE buffer on ice.
6. Transfer to a 13-ml polypropylene tube. Add 10% SDS to 1% (300 l of 10%) and
lyse the cells 15 min at 65 C.
7. Add 1/5 vol of 5 M potassium acetate (700 l) and incubate an additional 15 min
in a 65 C water bath. Transfer to ice and incubate 60 min.
8. Remove cell debris by centrifuging 15 min at 12,000 g, 4 C. Add supernatant to
2 vol of 95% ethanol. Hook out the DNA with a glass rod.
Addition of the cleared lysate to ethanol will cause the DNA and RNA to come out of
solution and appear as long white strands or gobs floating in the liquid. A glass hook
can easily be fashioned from a Pasteur pipette using a Bunsen burner. The hook should
be sealed and contain at least a slight bend. If the DNA cannot be removed with a hook
it can still be recovered by centrifugation.
9. Wash DNA once with 70% ethanol, allow it to dry, and resuspend in 200 to 500 l
TE.
SUPPORT
PROTOCOL
Materials
N. gonorrhoeae growing on solid medium
Colony lysis solution (see recipe)
Appropriate restriction enzyme(s) and buffer(s) (optional)
Thermocycler and appropriate PCR tubes
Additional reagents and equipement for PCR (Kramer and Coen, 2001)
1. Scrape an amount of N. gonorrhoeae equal to one to five colonies and transfer to 50
to 100 l colony lysis in a PCR tube.
2. Lyse cells 15 min at 94 C in a thermocycler followed by incubation for 5 min at
25 C.
3. Use 2.5 l lysate as template to a 25-l PCR reaction (Kramer and Coen, 2001).
4. Use 5 l PCR product in 30 l restriction digest, if identification of a restriction site
is necessary.
See Bloch (1995) for more information about restriction mapping.
Genetic
Manipulation of
N. gonorrhoeae
4A.2.12
Current Protocols in Microbiology
9.66 ml H2 O
Store up to 1 year at room temperature
Scale as needed.
GC medium base
42% (w/w) Proteose peptone #3 (15.10 g)
3% (w/w) cornstarch (1.01 g)
11% (w/w) K2 HPO4 (4.03 g)
3% (w/w) KH2 PO4 (1.01 g)
14% (w/w) NaCl (5.03 g)
28% (w/w) agar (10.07 g)
Weight amounts given in parentheses indicate appropriate mix for preparing GCB plates
(see below).
GCB plates
36.25 g GC Medium Base (Difco; also see recipe)
1.25 g agar
1 liter H2 O
Autoclave with stir bar
Cool to 50 to 60 C
Add 10 ml filter-sterilized Kelloggs supplements I (see recipe)
Add 1 ml filter-sterilized Kelloggs supplement II (see recipe)
Pour into 100-mm plates using 20 ml per plate
Store plates sealed in bags up to 6 months at 4 C
GCBL medium
Dissolve the following in 1 liter H2 O:
15 g proteose peptone #3
4 g K2 HPO4
1 g KH2 PO4
1 g NaCl
Adjust pH to 7.2 with HCl if necessary
Autoclave and store indefinitely at room temperature
Add Kelloggs Supplements I & II to 1 each from individual or a combined stock
(see recipe)
Add sodium bicarbonate to 0.042% (final) from a 100 stock (0.42 g in 10 ml
H2 O; filter sterilize)
Unless the protocol step specifcally indicates that Kelloggs supplements or sodium bicarbonate are to be included in the medium, these reagents should be omitted.
It is only necessary to add Kelloggs supplements when the bacteria are to be grown in the
medium. It is not necessary to add Kelloggs supplements when the bacteria are diluted
or transformed in GCBL, i.e., steps when the bacteria are only in the medium for a short
period of time. Sodium bicarbonate is added only when the bacteria are to be grown in
liquid culture with aeration (in sealed tubes or flasks) and not when they are grown in static
culture in a CO2 incubator. Unless the protocol step specifically indicates that Kelloggs
supplements or sodium bicarbonate are to be included in the medium, these reagents can
be omitted.
Kelloggs supplements
Supplements I and II may be mixed (10 ml supplement I + 1 ml supplement II)
and frozen in 11-ml aliquots. One such aliquot is sufficient to supplement 1 liter of
GCB or GCBL medium (see recipe). Filter sterilize before use.
Beta
Proteobacteria
4A.2.13
Current Protocols in Microbiology
Supplement I, 100
40 g glucose
1 g glutamine
2 mg cocarboxylase (thiamine pyrophosphate)
Adjust volume to 100 ml with H2 O
Sterilize by passing through a 0.2-m filter
Store indefinitely at 20 C
Supplement II, 1000
50 mg Fe(NO3 )3 9H2 O
Adjust volume to 100 ml with H2 O
Sterilize by passing through a 0.2-m filter
Store indefinitely at 20 C
An alternative to preparing supplements is to purchase them lyophilized. Supplement IsoVitalex (Becton Dickinson) can be used in place of Supplements I & II.
Do not store Kelloggs supplements at 4 C or room temperature.
TES
250 l 1 M TrisCl, pH 8 (APPENDIX 2A)
200 l 0.5 M EDTA
50 l 5 M NaCl
4.5 ml H2 O
Store up to 1 year at room temperature
COMMENTARY
Background Information
Genetic
Manipulation of
N. gonorrhoeae
resulting nonpiliated bacteria are not transformable. Therefore, care should be taken to
choose colonies of piliated N. gonorrhoeae for
transformation procedures. See Figure 4A.2.1.
The gonococcal genome sequence was
completed for strain FA1090 and is accessible in GenBank (accession number
AE004969). A useful annotation is available at the STD genomes website of
the Los Alamos National Laboratories
(http://www.stdgen.lanl.gov). Although there
are some significant differences in the
genomes of N. gonorrhoeae strains (Gibbs
and Meyer, 1996; Hamilton et al., 2005),
the sequence information obtained for strain
FA1090 is generally useful for searching for
genes of interest and for designing primers for
cloning gonococcal genes.
4A.2.14
Current Protocols in Microbiology
Figure 4A.2.1 Gonococcal colony morphologies. Gonococci expressing pili (P+ ) form smaller
colonies that appear to have a dark ring around the edge. This effect is due to the cells aggregating
to form a mound. Nonpiliated cells form a pancake-shaped colony that has a greater diameter
and a fuzzier edge. In addition, when the colonies are viewed with oblique substage lighting, a
range of colony opacities will be detected. Colony opacity is due to presence of outer membrane
opacity (Opa) proteins. Translucent (Opa ) colonies appear clear or somewhat blue, whereas
opaque colonies may appear yellow and refract the light to give an almost crystalline or granular
appearance.
Beta
Proteobacteria
4A.2.15
Current Protocols in Microbiology
Genetic
Manipulation of
N. gonorrhoeae
Colony variants
Gonococci undergo spontaneous highfrequency variation of surface molecules by
well described mechanisms (Seifert, 1996;
Stern et al., 1986). This variation is seen in
colony variants that appear on agar plates.
For the purposes of growing gonococci, it
should be noted that expression of pili and/or
opacity proteins results in clumping during
growth in liquid culture. The clumps are difficult to disperse and make it difficult to follow culture growth using OD measurements or
plating. Therefore it is advisable to use nonpiliated (P ), transparent (Opa ) variants
unless these molecules are required for other
purposes (e.g., transformation or adhesion
and invasion assays). Piliated variants form
colonies that are small and convex and thus
appear to have a dark ring around them (Kellogg et al., 1963). Nonpiliated variants form
larger, flatter colonies. Opacity protein expression is judged using a stereo dissecting microscope and oblique light. Opa variants may appear transparent or slightly blue. Opa+ variants
are opaque, somewhat yellow, and refractory
(Swanson et al., 1971). Nonpiliated variants
will come to predominate in liquid culture
due to a slightly faster growth rate. Nearly all
colonies will appear opaque on an old (>24-hr)
plate due to variation occurring in a proportion
of the cells in a colony at an appreciable rate.
4A.2.16
Current Protocols in Microbiology
Protocol
Description
Time required
4-5 days
Basic Protocol 3
Electroporation
4 days
Basic Protocol 4
Shuttle mutagenesis
12-14 days
Basic Protocol 5
Chemical mutagenesis
2 days
4-5 days
Basic Protocol 7
2 days
Chromosome preparation
Anticipated Results
Transformation of N. gonorrhoeae is highly
efficient. If using the spot transformation
method to introduce mutations into the gonococcal chromosome (Basic Protocol 1), frequencies of 10% to 50% transformant/total
CFUs or greater may be expected (Gunn and
Stein, 1996). Screening of ten potential transformants is generally sufficient to identify the
mutant of interest, even without selection. Using liquid transformation (Basic Protocol 2),
frequencies 1% may be expected. In general,
the frequency will depend on the length of the
homologous region targeting the mutation and
the length of any heterologous sequence being inserted. Lengths of homologous DNA of
500 to 1 kb on each side of the mutation or
insertion to be introduced are sufficient to give
efficient transformation. Long lengths of heterologous sequence to be inserted will greatly
reduce the transformation frequency, although
insertions >6 kb have been obtained (BoyleVavra and Seifert, 1993). The insertion of plasmids into the chromosome (Basic Protocol 1)
also depends on the length of the homologous
sequence with homologous regions of 300 to
500 bp resulting in transformation frequencies
in the 107 to 106 range (Hamilton et al.,
2001).
The insertion of plasmids into the chromosome also depends on the length of the homologous sequence with homologous regions of
300 to 500 bp resulting in transformation frequencies in the 107 to 106 range (Hamilton
et al. 2001). The frequency of transformation by electroporation (Basic Protocol 3) is
very low (109 ) and will only produce a few
transformants per electroporation. Shuttle mutagenesis (Basic Protocol 4) works well and
has been used to produce multiple mutations
in single genes or random mutations in the
gonococcal chromosome (Haas et al., 1993;
Boyle-Vavra and Seifert, 1993, 1994; Mehr
and Seifert, 1997). The constructs for complementation on the chromosome by doublecrossover recombination (Basic Protocol 6)
are effective for complementation; however, as
these contain significant stretches of heterologous DNA, the transformation frequencies are
significantly lower than those of simple insertion mutations. The lac promoter used in some
of the constructs gives relatively high levels of
transcription of introduced genes, approaching
those of strong gonococcal promoters (Seifert,
1997).
Time Considerations
Although the transformation step can take
as little as 5 min, the entire procedure from
streaking the bacteria to freezing down the
transformed strain may take five days. Most
of the time is spent waiting for the bacteria to grow. In particular, selection with
chloramphenicol or erythromycintwo of
the most useful antibiotics for gonococcal
manipulationsignificantly slows growth of
the bacteria such that transformants will take
one and a half to two days to form a colony.
Table 4A.2.2 provides time requirements
for many of the protocols presented in this
unit.
Literature Cited
Belland, R.J. 1991. H-DNA formation by the coding
repeat elements of neisserial opa genes. Mol.
Microbiol. 5:2351-2360.
Beta
Proteobacteria
4A.2.17
Current Protocols in Microbiology
Black, C.G., Fyfe, J.A., and Davies, J.K. 1998. Absence of an SOS-like repair system in Neisseria
gonorrhoeae. Gene 208:61-66.
Bloch, K.D. 1995. Mapping by Multiple Endonuclease Digestions. In Current Protocols in
Molecular Biology (F.M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J.G. Seidman, J.A.
Smith, and K. Struhl, eds.) pp. 3.2.1-3.2.5. John
Wiley & Sons, Hoboken, N.J.
Boyle-Vavra, S. and Seifert, H.S. 1993. Shuttle mutagenesis: Two mini-transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae. Gene 129:51-57.
Boyle-Vavra, S. and Seifert, H.S. 1994. Shuttle
mutagenesis: A minitransposon for producing
PhoA fusions in Neisseria gonorrhoeae. Gene
155:101-106.
Campbell, L.A. and Yasbin, R.E. 1984. Mutagenesis of Neisseria gonorrhoeae: Absence of error
prone repair. J. Bacteriol. 160:288-293.
Chen, I. and Dubnau, D. 2004. DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 2:241-249.
Cooke, S.J., Jolley, K., Ison, C.A., Young, H.,
and Heckels, J.E. 1998. Naturally occuring isolates of Neisseria gonorrhoeae, which display
anomalous serovar properties, express PIA/PIB
hybrid porins, deletions in PIB or novel PIA
molecules. FEMS Microbiol. Lett. 162:75-82.
Dillard, J.P. and Yother, J. 1994. Genetic and molecular characterization of capsular polysaccharide
biosynthesis in Streptococcus pneumoniae type
3. Mol. Microbiol. 12:959-972.
Engebrecht, J., Brent, R., and Kaderbhai, M.A.
1991. Minipreps of Plasmid DNA. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.6.11.6.10. John Wiley & Sons, Hoboken, N.J.
Genco, C.A., Chen, C.Y., Arko, R., Kapczynski,
D., and Morse, S.A. 1991. Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron
from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers. J. Gen.
Microbiol. 137:1313-1321.
Gibbs, C.P. and Meyer, T.F. 1996. Genome plasticity in Neisseria gonorrhoeae. FEMS Microbiol.
Lett. 145:173-179.
Goodman, S.D. and Scocca, J.J. 1988. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. U.S.A.
85:6982-6986.
Gunn, J.S. and Stein, D.C. 1996. Use of a nonselective transformation technique to construct a
multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae. Mol. Gen. Genet.
251:509-517.
Genetic
Manipulation of
N. gonorrhoeae
4A.2.18
Current Protocols in Microbiology
Stein, D.C. 1991. Transformation of Neisseria gonorrhoeae: Physical requirements of the transforming DNA. Can. J. Microbiol. 37:345349.
Stein, D.C., Silver, L.E., Clark, V.L., and Young,
F.E. 1983. Construction and characterization of
a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae. Gene 25:241-247.
Stern, A., Brown, M., Nickel, P., and Meyer, T.F.
1986. Opacity genes in Neisseria gonorrhoeae:
Control of phase and antigenic variation. Cell
47:61-71.
Stohl, E.A., Brockman, J.P., Burke, K.L., Morimatsu, K., Kawalczykowski, S.C., and Seifert,
H.S. 2003. Eschericihia coli RecX inhibits
RecA recombinase and coprotease activities in
vitro and in vivo. J. Biol. Chem. 278:2278-2285.
Swanson, J., Kraus, S.J., and Gotschlich, E.C. 1971.
Studies on gonococcus infection I. Pili and
zones of adhesion: Their relation to gonococccal growth patterns. J. Exp. Med. 134:886-906.
Wegener, W.S., Hebeler, B.H., and Morse, S.A.
1977. Cell envelope of Neisseria gonorrhoeae:
Relationship between autolysis in buffer and
the hydrolysis of peptidoglycan. Infect. Immun.
18:210-219.
Key Reference
Gunn, J.S. and Stein, D.C. 1996. See above.
This article describes the spot transformation
method that has greatly increased the frequency of
obtaining nonselected mutations and also reviews
the data on the many restriction systems of N. gonorrhoeae.
Beta
Proteobacteria
4A.2.19
Current Protocols in Microbiology
Interaction of Enterohemorrhagic
Escherichia coli (EHEC) with Mammalian
Cells: Cell Adhesion, Type III Secretion,
and Actin Pedestal Formation
UNIT 5A.1
The interaction of enterohemorrhagic Escherichia coli (EHEC) with host cells is critical
for colonization of the mammalian intestine. A likely first step is bacterial adhesion to
enterocytes, and Basic Protocol 1 describes the assessment of bacterial binding to cultured
mammalian cells. After initial adhesion, the pathogen alters the host cell by injection of
effectors via a type III secretion apparatus. Basic Protocol 2 describes methods to detect
type III secreted proteins in culture medium, with a method for quantitating the actin
pedestal phenotype described in the Support Protocol. Finally, one of the most notable
alterations induced by EHEC is the induction of localized actin assembly directly beneath
bound bacteria, resulting in the formation of actin pedestals that raise them above the
plane of the membrane; Basic Protocol 3 describes detection and quantitation of actin
pedestals in vitro.
CAUTION: Enterohemorrhagic Escherichia coli (EHEC) is a Biosafety Level 2 (BSL-2)
pathogen. Follow all appropriate guidelines and regulations for the use and handling of
pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidified 37 C, 5% CO2
incubator unless otherwise specified.
BASIC
PROTOCOL 1
Materials
HeLa cells (ATCC #CCL-2)
HeLa cell growth medium (see recipe)
LB broth (APPENDIX 4A) containing appropriate antibiotic (Table A.4A.2)
Enterohemorrhagic Escherichia coli (EHEC) strain of interest
DMEM/HEPES (see recipe) containing appropriate antibiotic
Phosphate-buffered saline (PBS; APPENDIX 2A)
HeLa cell infection medium (see recipe)
2.5% paraformaldehyde (see recipe)
PBS (APPENDIX 2A) containing 0.1% (v/v) Triton X-100 (store at room temperature)
Enteric Gamma
Proteobacteria
Contributed by Pamela J. Savage, Kenneth G. Campellone, and John M. Leong
Current Protocols in Microbiology (2007) 5A.1.1-5A.1.14
C 2007 by John Wiley & Sons, Inc.
Copyright
5A.1.1
Supplement 5
PBS (APPENDIX 2A) containing 1% (w/v) BSA (add 10 ml 10% w/v BSA to 90 ml
PBS)
Anti-0157 rabbit polyclonal antibody (Difco): resuspend lyophilized powder in
3 ml PBS (APPENDIX 2A), then dilute 1:500 (recommended) in PBS containing
1% BSA
200 U/ml Alexa568-phalloidin stock in methanol (Invitrogen)
1 mg/ml 4 ,6-diamidino-2-phenyindole dilactate (DAPI) stock
2 mg/ml goat anti-rabbit-Alexa488 (Invitrogen)
Mounting medium: 1:1 (v/v) PBS:glycerol or ProLong Gold Antifade Reagent
(Invitrogen)
Clear nail polish
Sterile glass coverslips, 12 mm, circular
24-well tissue culture plates
Sterile forceps
14-ml snap-cap culture tubes (e.g., Falcon)
Platform rocker
Centrifuge with plate carrier (e.g., Allegra 6 Benchtop Centrifuge; Beckman
Coulter)
Glass microscope slides
Opaque slide box
Additional reagents and equipment for epifluorescence microscopy (UNIT 2A.1)
Day 1: Culture HeLa cell monolayers and prepare bacterial cultures
1. Place sterile coverslips into a 24-well tissue culture plate (one coverslip/well) using
sterile forceps. Use three coverslips per strain to be analyzed in order to obtain
triplicate samples.
2. Seed coverslips with 104 HeLa cells per ml of HeLa cell growth medium. Incubate
at least 16 hr.
After overnight incubation, the HeLa cell monolayer should be 90% confluent. If a
different cell line is used, with different growth rate or degree of spreading, initial seeding
density should be adjusted to give this level of confluence.
3. For each bacterial strain, inoculate 1.5 ml LB broth (plus appropriate antibiotics;
Table A.4A.2) in a 14-ml snap-cap culture tube with a single colony from freshly
streaked plates. Incubate with agitation for 8 hr at 37 C until the OD600 is between
0.4 to 0.5.
The antibiotic is selected based on the bacterial antibiotic resistance marker (see
APPENDIX 4A).
It is best to inoculate the bacteria early on the same day that the mammalian cells are
seeded.
5A.1.2
Supplement 5
7. After mixing, dilute the culture in prewarmed (37 C) HeLa infection medium such
that the final concentration is 8 106 bacteria/ml medium.
Calculate the concentration of bacteria in the overnight culture based on an OD600
reading. Previous titration has revealed that an OD600 = 1.0 of wild-type EHEC strain
EDL933 reflects 3.5 108 bacteria/ml. Pilot titrations of other strains, performed by
plating cultures of different OD600 for cfu, may be required to determine the precise
correlation between OD600 and culture density for these strains.
8. Aspirate the mammalian culture medium from each well of the 24-well dish (from
step 2) into a waste vacuum flask using a Pasteur pipet. Immediately add 1 ml PBS
to each well using a pipet. Aspirate immediately and repeat wash once.
When adding PBS, the tip of the pipet should be placed against the wall of each well so
as not to disrupt the cell monolayers.
Fix cells
14. Add 0.5 ml room temperature 2.5% paraformaldehyde to each well and incubate,
without agitation, at room temperature for 15 min to fix the infected monolayers.
Incubate an additional 15 min with gentle rocking.
15. Aspirate paraformaldehyde and add 1 ml PBS per well. Rock the plate gently for
2 min. Aspirate PBS.
16. Add 0.5 ml PBS/0.1% Triton X-100 per well to permeabilize the monolayers. Rock
the plate gently for 5 min. Aspirate.
17. Immediately add 1 ml PBS per well to wash. Rock the plate gently for 2 min. Aspirate
and repeat the wash two more times.
19. Aspirate medium and wash wells with 1 ml PBS per well, each time rocking gently
for 5 min at room temperature. Remove wash medium and repeat two more times.
20. Using a total volume of 275 l in PBS/1% BSA, treat monolayers with the following:
Enteric Gamma
Proteobacteria
5A.1.3
Current Protocols in Microbiology
Supplement 5
21. Wrap plate in aluminum foil and incubate 30 min at room temperature, with gentle
rocking.
22. Aspirate medium and wash with 1 ml PBS per well, each time with gentle rocking
for 5 min. Aspirate medium and repeat two more times. Add 0.5 ml PBS to each
well.
Keep the plate wrapped in foil during the washes, because the fluorophores are sensitive
to light.
26. Use epifluorescence microscopy (UNIT 2A.1) with a 100 oil-immersion objective
(final magnification with oil equal to 1000) to visualize cells.
The outline of individual mammalian cells can be visualized using the red channel (=560
to 580 nm) to detect Alexa568- phalloidin, which stains filamentous actin (and thus actin
stress fibers). If antibody rather than DAPI was used to stain bacteria, visualize the
antibody-labeled bacteria using the green channel (=490 to 505 nm). See Critical
Parameters for further discussion. An excitation wavelength of 395 to 410 nm is then used
to visualize DAPI-stained bacteria.
27. For each slide, score the number of bacteria bound per cell for 50 cells; move the
stage in an ordered fashion to avoid counting same field twice.
The average of triplicate samples will thus encompass data from 150 cells.
BASIC
PROTOCOL 2
Materials
HeLa cell infection medium (see recipe), prewarmed
Desired primary antibody against host or bacterial protein of interest, diluted
appropriately in PBS/1% BSA
2 mg/ml Alexa488-conjugated secondary antibody against species in which
primary antibody was raised
Additional reagents and equipment for preparing EHEC-infected HeLa cell
monolayers (Basic Protocol 1, steps 1 to 11) and quantitation of actin pedestal
phenotype by fluorescence microscopy (Support Protocol)
Interaction of
EHEC with
Mammalian Cells
5A.1.4
Supplement 5
Fix cells
6. To fix the infected monolayers, add 0.5 ml room temperature 2.5% paraformaldehyde
to each well and incubate, without agitation, at room temperature for 15 min. Incubate
an additional 15 min with gentle rocking.
7. Aspirate medium and wash monolayer once for 2 min using 0.5 ml PBS/well.
8. Aspirate medium and add 0.5 ml PBS/0.1% Triton X-100 per well to permeabilize
the monolayer. Incubate 5 min at room temperature with gentle rocking.
9. Aspirate the medium and wash the monolayer three times, each time for 2 min with
0.5 ml/well PBS, at room temperature with gentle rocking.
Perform staining
10. To localize a host or bacterial protein of interest, add 275 l of desired primary
antibody (diluted appropriately in PBS/1% BSA) and incubate 30 to 45 min at room
temperature with gentle rocking.
11. Aspirate medium and wash coverslips three times for 5 min each with 1 PBS (0.5
ml/well) at room temperature with gentle rocking.
12. Using a total volume of 275 l in PBS/1%BSA, treat monolayers with the following:
13. Aspirate solution, then wash coverslips four times, each time for 5 min with
0.5 ml/well PBS at room temperature with gentle rocking. Leave final wash medium
in well.
Enteric Gamma
Proteobacteria
5A.1.5
Current Protocols in Microbiology
Supplement 5
15. Store slides in an opaque slide box at 4 C until ready to perform examination using
fluorescent microscopy (Support Protocol).
The slides can be stored at 4 C for up to 4 weeks.
SUPPORT
PROTOCOL
Materials
EHEC grown on HeLa cell monolayers and stained for actin pedestals (Basic
Protocol 2)
Additional reagents and equipment for epifluorescence microscopy (UNIT 2A.1)
1a. To identify bound DAPI-stained EHEC: Visualize cells using epifluorescence microscopy under 100 magnification with an excitation wavelength of 395 to 410 nm
(UNIT 2A.1).
1b. To identify associated actin pedestals: Examine cells using an excitation wavelength
of 560 to 580 nm to visualize Alexa568-stained phalloidin (UNIT 2A.1), which localizes
to filamentous actin.
2. For each slide, score the number of bacteria bound per cell and the number of
pedestals as follows:
a. Score the number of bacteria bound per cell for 50 cells. Move the stage in an
ordered fashion to avoid counting same field twice.
b. Simultaneously, score the number of pedestals on each of these 50 cells, and
thereby derive the percentage of adherent bacteria generating actin pedestals.
The average of triplicate samples will encompass data from 150 cells.
BASIC
PROTOCOL 3
Materials
Interaction of
EHEC with
Mammalian Cells
5A.1.6
Supplement 5
2. In another 14-ml snap-cap culture tube, dilute bacterial culture 500-fold into 5 ml
DMEM/100 mM HEPES (pH 7.4). Incubate 14 hr without agitation at 37 C in a 5%
CO2 atmosphere.
3. Dilute the DMEM culture 1:20 into 5 ml M9/glucose/bicarbonate medium or
DMEM/HEPES. Incubate 5 hr at 37 C in a 5% CO2 atmosphere.
The use of an M9-based medium compared to DMEM-based medium results in a relative
increase in the secretion of translocated effectors (i.e., proteins that are normally injected
into mammalian cells) and a reduction in translocators (i.e., secreted proteins that are
required for the translocation of effectors into mammalian cells; DeVinney et al., 1999).
For more information on this phenomenon, as well as a description of bacterial mutants
(deficient in SepL or SepD) that secrete very high levels of effectors, refer to Background
Information,identification of proteins secreted by the EHEC type III secretion system.
6. Resuspend bacterial pellet in 50 l of 1 SDS sample buffer and reserve for SDSPAGE (step 11).
7. Sterilize culture supernatant from step 4 by passing through a 0.22-m filter.
8. To the sterilized supernatant, add PMSF from 100 mM stock to a final concentration
of 1 M, and pepstatin, leupeptin, and aprotinin from 1 mg/ml stocks to a final
concentration of 10 g/ml each.
Enteric Gamma
Proteobacteria
5A.1.7
Current Protocols in Microbiology
Supplement 5
Figure 5A.1.1 Cultured bacterial strains were separated into bacterial and supernatant fractions
prior to SDS-PAGE and immunoblotting, as described in Basic Protocol 3. A Type III secretion
mutant (ETTA1) was included as a negative control and -OmpA blotting was used as a loadingvolume control. Reproduced from Campellone et al. (2004) with permission from Elsevier.
DMEM/HEPES
Dulbeccos Modified Eagle Medium (DMEM), high-glucose formulation with
L-glutamine and pyridoxine hydrochloride, without sodium pyruvate (commercially available from Invitrogen), supplemented with 100 mM HEPES (add from 1
M HEPES, pH 7.4, stock solution, also commercially available from Invitrogen).
Filter sterilize and store up to 4 weeks at 4 C.
Store both the DMEM medium and the 1 M HEPES stock at 4 C until expiration dates.
Interaction of
EHEC with
Mammalian Cells
Open all reagents, and prepare media, in laminar flow hood. Filter-sterilize and store
at 4 C. When dividing complete medium into aliquots for use, perform all procedures in
laminar flow hood.
5A.1.8
Supplement 5
Glycerol, 20%
Add 20 ml of 100% glycerol to 80 ml deionized water. Mix thoroughly and autoclave. Store indefinitely at room temperature.
M9/glucose/bicarbonate medium
To 750 ml sterile deionized H2 O (cooled to 50 C) add:
200 ml 5 M9 salts (see recipe)
2 ml 1 M MgSO4
20 ml 20% (w/v) glucose (filter sterilized)
0.1 ml 1 M CaCl2
44 ml 1 M NaHCO3
Bring volume to 1 liter with deionized H2 O
Autoclave and store indefinitely at room temperature
M9 salts, 5
Dissolve the following in a total volume of 1 liter deionized H2 O:
30 g Na2 HPO4 7H2 O
15 g KH2 PO4
2.5 g NaCl
5 g NH4 Cl
Divide salt solution into 200-ml aliquots and autoclave
Store indefinitely at room temperature
Paraformaldehyde, 2.5%
Dissolve 0.5 g of paraformaldehyde in 20 ml of PBS (APPENDIX 2A). Heat to fully
dissolve chemical but be careful not to boil solution. Make solution fresh on day of
experiment. Ensure solution is at room temperature prior to use.
Volume can be adjusted depending on needs of experiment.
5A.1.9
Current Protocols in Microbiology
Supplement 5
COMMENTARY
Background Information
Pathogenicity of EHEC
Enterohemorrhagic E. coli O157:H7
(EHEC) is a highly infectious Gram-negative
bacterium that causes an illness often associated with the consumption of fecally contaminated food. During colonization of the large
bowel, EHEC generates attaching and effacing (AE) lesions, characterized by loss of microvilli on the surface of the enterocyte, intimate bacterial attachment, and formation of
an actin pedestal. These AE lesions may both
promote colonization and contribute to compromising of the intestinal epithelial barrier.
The host cell interaction has been the subject
of extensive investigation based on its putative role in colonization, and each protocol in
this unit addresses steps required for the production of AE lesions by EHEC. Several other
likely important modifications of the host cell
are associated with type III secretion of effectors, but a full discussion of this topic is
beyond the scope of this protocol (for review,
see Garmendia and Frankel, 2005)
In addition to host modification via type III
secretion, EHEC produces a Shiga-like toxin
(Stx) that can damage the intestinal mucosa
and lead to hemorrhagic colitis. The toxins can
be absorbed systemically, and severe cases of
EHEC infection are associated with the lifethreatening complication, hemolytic uremic
syndrome (HUS), the triad of anemia, thrombocytopenia, and renal failure. Nevertheless,
in most cases, recovery from infection occurs
within 5 to 10 days with supportive treatment.
The role of antibiotics in the therapy of EHEC
infection is somewhat controversial, because
Stx is phage-encoded and some antibiotics induce the Stx prophage, resulting in high-level
production of toxin (Zhang et al., 2000).
At least two other pathogens also are capable of generating AE lesions. Citrobacter
rodentium is a natural mouse pathogen that is
often used as a model for EHEC. EPEC, mentioned extensively above, is an important cause
of infantile diarrhea in the developing world
and interacts with mammalian cells in culture with much greater efficiency than EHEC.
Hence, as discussed above, it can serve as a
useful positive control for each of the three
assays described in this unit.
Interaction of
EHEC with
Mammalian Cells
5A.1.10
Supplement 5
A variety of cell lines have been used to assay actin pedestal formation. One advantage of
using HeLa cells is that, not only are pedestals
formed efficiently on these cells, but it is often
possible to visualize most of the pedestals on
a given cell in a single plane of focus because
of their relatively flat morphology. Other cell
lines have also been used, such as Hep-2 cells
(Knutton et al., 1989), Caco-2 cells (which
are physiologically more similar to colonic
epithelium), and mouse embryonic fibroblasts
(MEFs), which can be derived from specific
knock-out cells, thereby providing a genetic
test of the role of different mammalian signaling molecules in actin pedestal formation
(Gruenheid et al., 2001).
Identification of proteins secreted by the
EHEC type III secretion system
Type III secretion is critical to the pathogenesis of an EHEC infection. A large pathogenicity island, termed the LEE (for locus of
enterocyte effacement) encodes components
of a needle-like structure that spans the inner
and outer membranes used for the secretion of
a specific set of proteins (Jarvis et al., 1995;
Abe et al., 1998; Perna et al., 1998), Some of
these proteins, such as EspA, EspB, and EspD
(where Esp denotes E. coli secreted protein),
are translocators required for delivery of other
secreted effector proteins into the host cell.
Effector proteins, such as EspF, EspFU , Tir,
and Map, modify diverse host cell functions
upon translocation (for review, see Garmendia et al., 2005). The ability to perform type
III translocation is required for EHEC to establish infection and cause disease (Jarvis and
Kaper, 1996). Therefore, the identification and
characterization of type III secreted effectors
have been essential to understanding of the
pathogenesis of EHEC.
The expression of the type III secretion apparatus and effectors depends on the growth
conditions of the bacterium. Secretion is very
sensitive to temperature, pH, growth phase,
and osmolarity (Kenny et al., 1997). Logphase growth in DMEM, but not LB, gives
rise to bacteria that are capable of type III
secretion, particularly secretion of those components, such as EspA, EspB, and EspD, that
are critical for translocation of effectors into
the host cell. Growth in M9 minimal media
reduces (but does not eliminate) the secretion
of these components, and increases the secretion of effector proteins such as Tir (DeVinney
et al., 1999) and EspFU (also known as TccP;
Campellone et al., 2004; Garmendia et al.,
Critical Parameters
Adhesion of EHEC to mammalian cells
It is important to include positive and negative controls when initially developing these
assays. A reasonable positive control is enteropathogenic E. coli (EPEC, e.g., strain
E2348/69; Levine et al., 1985), a close relative of EHEC. Unlike EHEC, EPEC possesses a bundle-forming pilus that promotes
efficient bacterial binding to cultured cells. Because EPEC binds more efficiently to cells
than EHEC, the authors routinely inoculate
monolayers with 5- to 10-fold fewer EPEC. A
nonadherent negative control, such as a derivative of E. coli K12 (e.g., MC1061), can be used
to establish a baseline for nonspecific binding. Alternatively, an EHEC mutant defective
for type III secretion also is largely defective
for binding.
The decision to stain bound bacteria with
DAPI or an EHEC-specific antibody is related
to the strains being analyzed. For example,
EPEC and E. coli K12 strains will not stain
with anti-O157 antibody, so the authors routinely use DAPI staining in experiments that
include either of these control bacteria. The
most significant disadvantage to using DAPI
is the difficulty in visualizing bacteria that
are overlying the mammalian nucleus, which
stains intensely with DAPI. However, because
scoring of DAPI-stained samples uniformly
excludes bacteria in this location, this
Enteric Gamma
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5A.1.11
Current Protocols in Microbiology
Supplement 5
Troubleshooting
Table 5A.1.1 presents some of the problems commonly encountered with adhesion of
EHEC to mammalian cells (see Basic Protocols 1 and 2).
Anticipated Results
Adhesion of EHEC to mammalian cells
Using the protocol outlined above, the authors have typically found 250 wild-type
EHEC strain EDL933 bound per 50 mammalian cells. With EPEC strains, one typically
finds 2- to 5-fold more bacteria bound per
given number of cells than with EHEC (e.g.,
Table 5A.1.1 Troubleshooting Guide for Analyzing Adhesion of EHEC to Mammalian Cells
Problem
Possible cause
Solution
No bacteria bound
5A.1.12
Supplement 5
500 to 1250 bacteria bound per 50 mammalian cells), even with the lower inoculum.
At most, one or two of the negative-control
E. coli K12 are typically bound per 50 cells.
Fluorescent staining of EHEC-induced
actin pedestals
Using the protocol outlined above (Basic
Protocol 2), one will normally see significant cell-to-cell variation in bound bacteria
and pedestals. Many mammalian cells will not
be associated with any bacteria, whereas others may harbor ten or more bound bacteria.
Normalizing pedestals to bound bacteria, the
authors have typically found 75% of bound
wild-type EHEC strain EDL933 are associated with easily detected actin pedestals. For
EPEC, this figure is usually closer to 90%.
Identification of proteins secreted by the
EHEC type III secretion system
The equivalent of 1.5 ml culture supernatant typically contains enough effector
molecule for easy detection by immunoblotting (Campellone et al., 2004), although this
may vary significantly depending upon the
specific effector.
Time Considerations
Basic Protocols 1 and 2 require multiple
days for the culturing of mammalian cell lines,
as well as a day to prepare the bacterial cultures. The infection and staining steps can be
completed in 1 day. Additionally, Basic Protocol 3 requires 2 days to complete: 1 day for
the culturing of the bacteria and an additional
day for lysate preparation and examination by
immunoblotting.
Acknowledgements
The authors would like to thank Brian Skehan and Michael Brady for their helpful discussion pertaining the methods described in
this unit.
Literature Cited
Abe, A., Heczko, U., Hegele, R.G., and Brett Finlay,
B. 1998. Two enteropathogenic Escherichia coli
type III secreted proteins, EspA and EspB, are
virulence factors. J. Exp. Med. 188:1907-1916.
Enteric Gamma
Proteobacteria
5A.1.13
Current Protocols in Microbiology
Supplement 5
Sasse, J. and Gallagher, S.R. 2003. Staining proteins in gels. Curr. Protoc. Mol. Biol. 63:10.6.110.6.35.
Interaction of
EHEC with
Mammalian Cells
5A.1.14
Supplement 5
UNIT 5A.2
BASIC
PROTOCOL
Enteric Gamma
Proteobacteria
Contributed by Pamela J. Savage, John M. Leong, and Kenan C. Murphy
Current Protocols in Microbiology (2006) 5A.2.1-5A.2.13
C 2006 by John Wiley & Sons, Inc.
Copyright
5A.2.1
Figure 5A.2.1 Map of pKM208. Positions of key genetic elements in plasmid pKM208 (clockwise
from top left): Ptac-gam-bet-exo operon, E. coli lacI repressor gene, repA, pSC101, ampicillinR encoding bla gene, and the f1 ori. A subset of unique restriction sites is shown. The lacI gene,
contained within a NotI fragment (not shown), is absent from plasmid pKM201. The pSC101 orits is
a low-copy-number replicon which allows growth of the plasmid at 30 C but not at 44 C (Hamilton
et al., 1989). While originally described as a tool for generating gene deletions in E. coli, it is used
here for the Red-producing plasmid to allow for easy curing of the plasmid following Red-promoted
gene replacement.
Materials
Linear DNA substrate containing drug marker sequence (see Critical Parameters
and Troubleshooting)
Polymerase: Taq polymerase (New England Biolabs)
dNTPs (APPENDIX 2A)
Appropriate PCR buffer (e.g., see APPENDIX 2A)
TE buffer (APPENDIX 2A)
Electrocompetent EHEC transformed with Red-producing plasmid (Support
Protocol)
Table 5A.2.1 Time Considerations for Red-Promoted Gene
Replacement in EHEC
Day
Activity
Time required
Day 1
Day 2
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
Day 3
Day 4
2-3 hr
Overnight
6-8 hr
2 hr
Overnight
15 min
Overnight
PCR analysis
2-4 hr
at 80 C.
5A.2.2
Current Protocols in Microbiology
Figure 5A.2.2 An example of two 60-oligomer primers used to generate a PCR product for Redpromoted
gene replacement (in this case, a lacZ::kan allele). The underlined region in primer 1 contains 5 to 3
sequence upstream of the aph gene (i.e., top strand reading rightward in the aph region when read as a
dsDNA sequence le; not shown)the aph gene encodes kanamycin resistance (kan). The underlined region
in primer 2 contains 5 to 3 sequence downstream from the aph gene, (i.e., bottom strand reading leftward
in the aph region when read as a dsDNA sequence le; not shown). These are the sequences that will prime
the PCR reaction. The region in italics in primer 1 contains 5 to 3 sequence from the N-terminal encoding
region of lacZ (top strand reading rightward in the sequence le of lacZ; not shown), while the region in italics
in primer 2 contains 5 to 3 sequence from the C-terminal encoding region of lacZ (bottom strand reading
leftward in the sequence le of lacZ; not shown). These are the regions that will dene the homology with the
target gene, lacZ.
2. Prepare a PCR reaction by combining linear DNA substrate containing drug marker
sequence, primers, polymerase, dNTPs, and appropriate buffer. Perform standard
PCR optimized for the sequences involved (Kramer and Coen, 2001).
3. Purify the PCR product using traditional DNA purification methods (Moore and
Downhan, 2002) or commercially available PCR product purification kits.
The purpose of this step is to remove salts from the PCR buffer that would interfere with
the electroporation step that follows. In some cases, gel purification of the PCR product
is warranted (see Critical Parameters and Troubleshooting, PCR Templates).
The PCR product contains the drug marker flanked on one end by a 40-bp sequence
from the N-terminal encoding region of the target gene, and on the other end by a 40-bp
sequence from the C-terminal encoding region of the target gene.
Enteric Gamma
Proteobacteria
5A.2.3
Current Protocols in Microbiology
7. Thoroughly (but quickly) dry the cuvette with Kimwipes. Insert it into the electroporation chamber and quickly pulse at 2200 V or voltage suggested by the electroporator
manufacturer for E. coli.
CAUTION: Maintain the cap on the cuvette during electroporation of EHEC cells to
prevent possible aerosol formation.
8. Immediately add 0.5 ml SOC medium and mix thoroughly with the cells in the
electroporation chamber. Transfer 0.5 ml of this mixture to a culture tube containing
1 to 2.5 ml SOC.
It is not necessary to remove the small amount of mixture contained between the electroporation plates (< 100 l), as this represents a small fraction of the total sample.
11. Following 60 to 90 min incubation, plate remaining SOC culture from step 9 onto
LB plates containing appropriate antibiotic. Incubate overnight at 37 C.
The appropriate antibiotic is one that selects for the marker contained on the linear DNA
substrate (i.e., PCR product; step 1). See Critical Parameters and Troubleshooting, Drug
Markers, for important details regarding antibiotic selection.
The authors typically plate 0.5 ml/plate. To conserve plates, one can concentrate the cells
by microcentrifuging 1 min at moderate speed (2/3 of maximum), 4 C, resuspending in
0.25 ml SOC, and spreading the entire mixture onto one plate.
12. Identify colonies on plates (see Critical Parameters and Troubleshooting). Using
an inoculating loop, pick and streak two to four colonies onto fresh LB antibioticselection plates. Incubate overnight at 37 C.
The authors typically streak two colonies per plate.
13. As an alternative (or in addition) to step 12, streak a larger number of colonies (40
streaks per plate) onto both LB and LB antibiotic-selection plates as follows.
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
a. Pick a colony off the transformation plate(s) (step 11) with an inoculating loop.
b. Transfer using one, small (0.5-cm) streak onto an LB antibiotic selection plate,
followed immediately (without flaming) by a streak of similar size onto an LB
plate.
c. Incubate overnight at 37 C.
d. Identify false positives as those that grow poorly on the LB antibiotic-selection
plate relative to their growth on the LB plate.
5A.2.4
Current Protocols in Microbiology
e. Select two to four candidates that grow well on both plates and streak onto LBantibiotic-selection plates (one to two colonies per plate) to obtain single colonies.
f. Incubate overnight at 37 C.
The presence and magnitude of false positives varies with bacterial strains, antibiotic
used, drug concentrations, and the amount of culture plated in step 11. Many times it
does not arise, but can be especially troublesome with the use of chloramphenicol in
EHEC (see Critical Parameters and Troubleshooting). With EHEC, false positives are
less of a problem if kanamycin is used as the drug marker.
14. Verify true chromosomal gene replacement via PCR (Kramer and Coen, 2001),
phenotypic screens, and/or Southern blot analysis (UNIT 14B.1).
The authors typically use a series of colony PCR reactions to verify gene replacement
(see Critical Parameters and Troubleshooting). Each of the two junctions of marker and
chromosomal sequence can be verified by using one primer complementary to the drug
marker and the other primer complementary to a nearby region of the chromosome not
contained in the original PCR product. Amplification across the drug marker using a
primer pair that flanks but does not include the original PCR product should give a
product (containing the marker and flanking chromosomal sequence) of the predicted
size. Finally, a primer pair specific to the putative deleted sequence should not generate
an amplicon from recombinant colonies but should result in the predicted amplicon when
the parental wild-type strain is used.
ALTERNATE
PROTOCOL
The following method is a variation of the Basic Protocol where instead of using a
drug marker in place of the gene, following the protocol first used by Ried and Collmer
(1987), a cassette containing the cat (positive selection) and sacB (negative selection)
genes is used. SacB is a levansucrase that transfers fructosyl residues from sucrose
to different cellular receptors such as alcohols, sugars, and levans (Dedonder, 1966).
Expression of sacB kills E. coli in the presence of sucrose (Gay et al., 1983). Thus,
two rounds of Red recombination are required: the first selects for chloramphenicol
resistance (CamR /SucS ), while the second inserts the precise deletion and evicts the
cassette (generating CamS /SucR colonies).
The second electroporation uses a linear DNA species, typically from a previously
constructed plasmid, that defines the precise deletion desired. The authors have used a
plasmid that contains the upstream region of the target gene fused to the downstream
region, constructed in such a way that this linear DNA can be liberated from the plamid by
a restiction enzyme digest (Murphy et al., 2000). Using PCR manipulations to construct
these plasmids, the authors usually fuse the first three condons of the target gene to the
last three codons in order to allow translational read through of the deletion once crossed
into the chromosome.
The construction of an in-frame unmarked deletion, one that contains no drug marker
or other scar, is useful when one is limited by the number or availability of antibiotic
resistance genes used for a particular pathogen. For instance, once a drug marker is
removed from the chromosome, it can then be used at a second location within the
same bacterium. Also, if one plans to test the mutant bacterial strain in animal studies,
it is best to use a strain containing no antibiotic resistant determinants to (1) rule out
polar effects of the drug marker (or its expression) on bacterial genes nearby the site of
the deletion and (2) prevent the accidental spread of antibiotic resistant determinants in
animal populations.
A potential problem with the use of sacB is the generation of spontaneous SucR colonies
due to loss or mutation of the sacB gene. The authors have found this to be more of
Enteric Gamma
Proteobacteria
5A.2.5
Current Protocols in Microbiology
Table 5A.2.2 Time Considerations for Generation of Unmarked Chromosomal Deletion in EHEC Using SacB Counterselection
Day
Activity
Time required
Day 1
Day 3
4 hr
3 daysa
Day 5
1-2 hr
Day 6
6-8 hr
3 daysa
Day 9
Overnight
Day 10
2-4 hr
a problem when sacB is on a plasmid and much less so when sacB is integrated into
the chromosome. Starting the electrocompetent culture from a single overnight colony
minimizes the presence of spontaneous sacB loss and mutation.
Table 5A.2.2 presents time considerations involved in performing this procedure.
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
5A.2.6
Current Protocols in Microbiology
The plasmid should be constructed in such a manner that a DNA fragment containing the
sequence upstream and downstream of the target gene (as well as the cat-sacB cassette
if the substrate is to be prepared on a plasmid; see step 2a) can be liberated as an intact
fragment by a restriction digest (for details, see Murphy et al., 2000).
2b. For preparation of substrate using PCR: Prepare a PCR substrate containing the
cat-sacB cassette flanked by 40 bp of DNA which contains sequence from upstream
and downstream of the region of interest (Kramer and Coen, 2001).
This PCR fragment will be larger (3.6 kb) than the PCR products described in the
Basic Protocol, which are typically <1 kb). Thus, PCR-generated sequence errors in
sacB that generate a SacB phenotype (SucR ) will have to be screened out of the pool of
transformants. Use of high fidelity polymerases will likely alleviate this problem, though
this has not yet been tested in the authors laboratory.
3. If necessary (i.e., for substrates prepared on plasmids), liberate the fragment containing the cat-sacB cassette and flanking upstream and downstream sequences using an
appropriate restriction enzyme(s). Purify the fragment by agarose gel electrophoresis
(Moore et al., 2002).
This DNA fragment must be gel purified prior to electroporation, as simple transformants
of uncut plasmid will appear as gene replacement candidates when performing Redpromoted gene replacement.
4. Using either the PCR product or the gel-purified restriction fragment, perform steps
5 to 11 of the Basic Protocol, selecting for CamR colonies.
5. Streak isolated CamR colonies from the transformation plate onto LB/Cam to verify
positive selection, LB/Suc for negative selection, and LB/Amp to verify the presence
of pKM201 or pKM208 as follows. Each set of plates can contain 40 streaks.
a. Pick a colony off the transformation plate(s) with an inoculating loop.
b. Transfer using one, small (0.5-cm) streak onto an LB/Suc plate, followed immediately
(without flaming) by a streak of similar size onto an LB/Amp plate, and again onto
an LB/Cam plate.
c. Incubate 1 day at 30 C.
d. Identify CamR /SucS /AmpR transformants. Streak onto LB/Amp plates to obtain
single colonies.
e. Incubate 1 day at 30 C.
Plasmid pKM201 and pKM208 are many times lost spontaneously following electroporation, which is advantageous if one wants to study the gene knockout in an otherwise
wild-type background. If it is not lost spontaneously, streaking out the transformation
at 42 C will promote loss of the Red plasmid. Here, however, one needs to retain the
Red plasmid for the subsequent gene eviction step. Therefore, incubation is performed at
30 C. (Red-producing plasmids contain temperature-sensitive origins of replication; see
Fig. 5A.2.1.)
It should be evident here why the recombinant fragment from the second plasmid
described in step 1a must be gel purifiedi.e., transformation by plasmid would also
yield CamR /SucS /AmpR transformants. Minilysates can be prepared from the candidates
to verify the absence of the substrate plasmid and the presence of Red-producing plasmid.
Enteric Gamma
Proteobacteria
5A.2.7
Current Protocols in Microbiology
Also, a PCR test, or a phenotypic test (if available), for the gene knockout can be used at
this step to verify the absence of the target gene.
For an unknown reason, selection for SucS mutants is more readily achieved using LB
plates without sodium chloride.
6. Prepare a CamR /SucS /AmpR transformant from this second LB/Amp plate for electrocompetence and hyper-recombinogenicity as described in steps 2 to 12 of the
Support Protocol.
7. Using the technique described in steps 5 to 8 of the Basic Protocol, electroporate a
restriction digest of the plasmid containing the precise (unmarked) deletion of the
target gene (or region of interest) constructed in step 1.
Note that the recombining fragment need not be gel purified, since the selection in this step
(SucR ) involves loss of a chromosomal marker rather than the acquisition of an antibiotic
marker carried by a potentially contaminating plasmid.
9. If the culture has been grown for 5 hr, plate onto LB/Suc plates at 0.5 ml/plate.
Alternatively, if the culture has been grown overnight to saturation, prepare four
10-fold dilutions in LB medium and plate 0.1 ml of each onto LB/Suc.
10. Incubate overnight at 37 C. Select for SucR colonies.
11. Streak individual SucR colonies onto LB and LB/Cam plates to identify CamS
transformants.
12. Simultaneously, screen for AmpS candidates as well to identify cells that have
spontaneously lost the Red plasmid. If no AmpS transformants are found, streak at
42 C to cure the cell of the Red plasmid.
13. Verify deletion by PCR analysis (Kramer and Coen, 2001).
See Basic Protocol, step 14, and Critical Parameters and Troubleshooting for more
information.
SUPPORT
PROTOCOL
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
5A.2.8
Current Protocols in Microbiology
shock of electroporation, resulting in higher rates of survival, and thus increased numbers
of transformants. This effect can vary from 2- to 50-fold; the mechanism of this effect,
and its variability, is not understood. In this description, a 20-ml starting culture is used
for the prepration of electrocompetent cells, which is enough for two electroporations.
The procedure can be scaled up as needed.
Materials
LB plates (APPENDIX 4A) containing 100 g/ml ampicillin (LB/AMP)
EHEC containing pKM201 or pKM208 (Murphy and Campellone, 2003)
SOC medium (APPENDIX 4A) containing 100 g/ml ampicillin, 30 C
100 mM IPTG (see recipe)
20% (w/v) glycerol, 4 C
30 and 42 C shaking water baths
1. Streak EHEC containing pKM201 or pKM208 onto LB plates containing 100 g/ml
ampicillin. Incubate 24 hr at 30 C.
Lower drug concentrations are sometimes required for selecting plasmid transformants.
However, once established, the transformants grow well at higher drug concentrations.
The authors have observed this with pKM208 in EHEC. Thus, for transformation of
EHEC with pKM208, one should try low levels of ampicillin (50 g/ml or lower), but
once established, the transformant should be maintained at 100 g/ml. Also see Critical
Parameters and Troubleshooting.
2. Inoculate 20 ml warm SOC medium containing 100 g/ml ampicillin with one to
four purified colonies.
LB medium (APPENDIX 4A) can be used in place of SOC medium.
3. Incubate culture in a 30 C shaking water bath until the OD600 reaches 0.3 (cell
density 107 cells/ml).
4. Induce the culture by adding 100 mM IPTG to a final concentration of 1 mM.
In the case of pKM201, IPTG is used to inactivate endogenous wild-type repressor.
6. Chill the culture in an ice-water bath for 10 min with slow constant swirling.
7. Transfer cells to appropriate bottles and centrifuge 10 min at 5000 g, 4 C. Discard
the supernatant.
8. Resuspend the pellet in 1 ml of ice-cold 20% glycerol.
The purpose of the glycerol is to protect the membranes from the shock of electroporation
and to act as a cryoprotectant if the cells are to be frozen for future use.
The use of 1 mm MOPS has been identified as an optimal component in a survey of
electroporation buffers for transformation of P. aeruginosa (Farinha and Kropinski, 1990)
and has been adapted for use in some of the authors experiments, both here and in
step 11.
Enteric Gamma
Proteobacteria
5A.2.9
Current Protocols in Microbiology
12. Use bacteria within 1 hr or freeze at 80 C for future use (up to 3 to 6 months).
IPTG, 100 mM
Dissolve 238 mg IPTG powder (Sigma) into 10 ml deionized H2 O
Pass through a 0.2-m filter (Acrodisc) to sterilize
Store up to 6 months at 20 C
COMMENTARY
Background Information
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
5A.2.10
Current Protocols in Microbiology
Enteric Gamma
Proteobacteria
5A.2.11
Current Protocols in Microbiology
Rapid Allelic
Exchange in
EHEC and
Other E. coli
Using Red
Recombination
No transformants
Follow the guidelines and alternate protocols discussed above. If no transformants are
obtained, examine cell survival after electroporation by plating on solid LB medium. In
the final culture following electroporation, a
survival count of 107 to 108 /ml is desired.
Cell survivals <106 indicate excessive killing.
Since frequencies of gene replacement are typically in the range of 104 to 105 /survivor,
excess killing will prevent successful recovery of transformants. If this happens, search
for possible salt contamination in the electrocompetent cell prep or DNA substrate, lower
the electroporation voltage and/or decrease or
eliminate heat shock exposure.
Anticipated Results
Farinha, M.A. and Kropinski, A.M. 1990. High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions. F.E.M.S.
Microbiol. Lett. 70:221-226.
Time Considerations
Table 5A.2.1 and Table 5A.2.2 present time
considerations for the protocols described in
this unit.
Literature Cited
Campellone, K.G., Giese, A., Tipper, D.J., and
Leong, J.M. 2002. A tyrosine-phosphorylated
12-amino-acid sequence of enteropathogenic
Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to
actin pedestals. Mol. Microbiol. 43:1227-1241.
Campellone, K., Robbin, D., and Leong, JM. 2004.
EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nckindependent actin assembly. Dev. Cell 7:217228.
5A.2.12
Current Protocols in Microbiology
Enteric Gamma
Proteobacteria
5A.2.13
Current Protocols in Microbiology
UNIT 6A.5
Until the late 1970s and early 1980s, Vibrio cholerae, the causative agent of cholera,
was viewed as host adapted, i.e., resident in the human gut. Numerous studies conducted
during the last three decades, however, have provided clear evidence establishing the
microorganism as an autochthonous (i.e., native) member of the aquatic environment
(Colwell and Spira, 1992). These organisms can be detected in marine, estuarine, and
fresh-water environments throughout the year if appropriately sensitive methods are
employed (Colwell et al., 1981; Huq et al., 1990; Louis et al., 2000, 2003). However, V.
cholerae may not be detected for several reasonse.g., if it is present in low numbers,
if it exists in a state that does not facilitate growth, or if it is simply overlooked. When
the bacteria are viable (determined by direct-detection methods) but do not grow on
conventional culture media or in broth, they are termed viable but not culturable (VBNC;
Xu et al., 1982; Roszak and Colwell, 1987). V. cholerae, along with a host of other, mostly
Gram-negative bacteria, often occur as VBNC cells in the environment, an indication that
this state may be an important strategy for persistence and survival of non-spore-forming
microorganisms (Colwell et al., 1985; Roszak and Colwell, 1987).
Currently, many molecular-biological tools are used to study bacteria, including VBNC
cells, in clinical, environmental, or industrial samples (Knight, 2000). With the availability of these tools and a renewed interest in V. cholerae, the presence of these bacteria in
various geographical locations has been reported with increasing frequency. However,
standard methods for detection and enumeration have not been universally established.
The choice of method for detection and enumeration of V. cholerae is largely dependent
on the type of sample and the desired end purposei.e., simple detection or isolation.
Often, combinations of different methods are used for a specific type of sample. Therefore, commonly used bacteriological, molecular, and immunological methods targeted at
microbiological analysis of environmental water samples will be discussed in this unit.
Several methods will be described, and investigators will have to determine the most
appropriate method consistent with their purpose and needs and with the availability
of resources. For the readers convenience, Table 6A.5.1 provides an overview of the
methods provided in this unit.
CAUTION: V. cholerae is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate
guidelines and regulations for the use and handling of pathogenic microorganisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
STRATEGIC PLANNING
There are numerous methods and protocols available in the literature for detecting and
isolating V. cholerae. A number of them are described in this unit; however, all may not
be required in one study. In addition, availability of resources in a particular laboratory
will be a critical predetermining factor in making decisions on the choice of methods
to include; therefore, stepwise selection cannot be standardized. In other words, it is
extremely important to contrive a strategic plan to determine the choice of methods at
the beginning of the study. A general method for specimen collection and transportation
is provided (Basic Protocol 1). The traditional culture method (Basic Protocol 2) is the
so-called gold standard in bacteriology, especially when an isolate is needed for the
study. In the conventional culture method, selective plates are often used to reduce the
Contributed by Anwar Huq, Christopher Grim, Rita R. Colwell, and G. Balakrish Nair
Current Protocols in Microbiology (2006) 6A.5.1-6A.5.38
C 2006 by John Wiley & Sons, Inc.
Copyright
Nonenteric
Gamma
Proteobacteria
6A.5.1
Supplement 2
Table 6A.5.1 Overview of Methods Presented in This Unit for Isolation and Detection of V.
cholerae
Protocols
Included methodologies
Basic Protocol 2
Basic Protocol 3
Serogroup determination
Basic Protocol 5
Direct PCR
Alternate Protocol 1
Basic Protocol 6
Alternate Protocol 2
Support Protocol
Basic Protocol 8
volume of work and to identify a specific organism or variety of organisms. Once this
culture method has been used and an organism isolated, a battery of biochemical tests
is employed for characterization and identification. If such colonies are picked from
selective plates, the number of biochemical tests needed may decline. Therefore, the
number of tests may vary depending on the source of the strain. However, as more
tests are performed, more convincing and accurate results will be obtained. Serogroup
determination for V. cholerae is described in Basic Protocol 3.
Although conventional culture methods, including biochemical tests, are routinely used,
they can be time consuming as well as labor intensive. The authors have included two
abbreviated alternative methods that use fewer tests for a rapid presumptive identification.
Colony blot hybridization (Basic Protocol 6 and Alternate Protocol 2; probe labeling is
described in Support Protocol) can be used for rapid detection of Vibrio cholerae, and
in cases involving large numbers of samples, this method may be preferred. However, it
too is dependent on the culturability of the cells.
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.2
Supplement 2
Molecular approaches target specific genes that are unique to the organism in question.
Usually, species-specific genes are targeted. This approach is extremely valuable for
monitoring the presence of the organism. A single probe, designed carefully, is very
specific and will be amenable to screening a larger number of strains at one time. Likewise,
a series of such probes can be used for extensive characterization of an organism. These
methods can be based on both culturable and nonculturable cells. Community DNA can
be used to determine the presence of an organism without culture; however, results will
be dependent on the quality and quantity of the extracted nucleic acids.
Strategically, if resources are available, it is appropriate to start with the conventional
culture method and the DFA-DVC method. Those samples exhibiting positive results by
the DFA-DVC method can be confirmed by PCR (Basic Protocol 4). If one must avoid
conventional culture, one may wish to use direct PCR (Basic Protocol 5 or Alternate
Protocol 1) and DFA-DVC (Basic Protocol 7), which should provide fairly convincing
results. Use of PCR alone for environmental samples may not be reliable, as background
noise often poses a significant obstacle in the interpretation of results.
BASIC
PROTOCOL 1
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6A.5.3
Current Protocols in Microbiology
Supplement 2
Processing of samples should begin soon after collection (on site or in situ) or within
1 hr of collection. If processing is delayed, the sample should be stored in a cool box
at a temperature of 10 to 15 C until processing begins (not to exceed 8 hr; Clesceri
et al., 1998). This is a critical parameter if enumeration of V. cholerae from the sample
will be performed. A recent study suggests that transporting samples at ambient air
temperature prior to processing can increase the number of V. cholerae strains isolated
(Alam et al., 2006). Based on the type of examination, samples may require treatment,
e.g., addition of direct viable count (DVC) reagents, before proceeding with further
examination and testing. It is recommended that some basic physiochemical parameters
e.g., temperature, salinity, pH, dissolved oxygen, conductivityof the water source be
measured at the time of collection, for later analysis. Results of environmental studies
conducted in Bangladesh have revealed a direct correlation of water temperature with
clinical outbreaks of cholera. Furthermore, significant correlation of water depth, rainfall,
conductivity, and copepod density with cholera outbreaks has been found, with lag periods
from 0 to 8 weeks from optimum environmental conditions to cholera outbreaks (Huq
et al., 2005). These parameters can be measured on site using portable meters.
Materials
500-ml plastic containers with caps (Nalgene) for water sampling, sterile
Portable meter(s) that measure temperature, dissolved oxygen and pH (dissolved
oxygen and pH meter Model 210A from Orion Laboratories), and salinity
(Model CO150 conductivity meter from Hach Chemical Company)
Simple plankton net, 64-m mesh size, or nets of different mesh sizes for size
filtration of plankton (Aquatic Research Instruments,
http://www.aquaticresearch.com; Fig. 6A.5.1)
Net-mounted flow meter (General Oceanics; http://www.generaloceanics.com/)
Bucket of known volume (e.g., 5 liters; optional)
240-ml glass containers for plankton collection (Qorpak), sterile
To collect water sample
1a. Measure temperature, dissolved oxygen, pH, and salinity using portable meters.
2a. Uncap presterilized 500-ml plastic bottle and submerge to fill.
3a. Remove sample bottle from water. Cap bottle, leaving enough air inside for agitation
and mixing.
4a. Transport samples to the laboratory for processing in a cold box (Styrofoam box with
ice, 10 to 15 C), or, preferably, begin processing on site within 1 hr of collection
(Basic Protocol 2).
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
Figure 6A.5.1 Simple plankton net (Aquatic Research Instruments). The net consists of a metal
ring and bridle, a heavy-duty nylon net of variable mesh size, and a PVC cod-end or collecting
bucket, which can be removed for easy sample collection. A ow meter may be mounted in the
mouth of the net on the metal ring to measure volume when the net is towed.
6A.5.4
Supplement 2
A recent study (Alam et al., 2006) found that transportation or maintenance at ambient air
temperature (22 to 25 C) after collection for up to 24 hr may enhance recovery of Vibrio
species. This can be a useful alternative to increasing sample volume for geographical
areas and/or seasons when the density of V. cholerae is low. This aspect of the protocol
may need to be optimized for the water source and environmental conditions.
To collect plankton
1b. Rinse plankton net and cod-end of the net in the body of water to be sampled.
2b. Filter 10 to 40 liters water through the plankton net by towing; use a calibrated,
net-mounted flow meter to determine volume. Alternatively, pour known volumes
through the net with a small bucket.
3b. Remove cod-end from plankton net and decrease volume to 100 ml by continuing to
filter.
4b. Measure (with graduated cylinder) and decant plankton sample into sterile 240-ml
glass container.
5b. Transport samples to the laboratory in a cold box (Styrofoam box with ice, 10 to
15 C) for processing or, preferably, begin processing on site within 1 hr of collection
(Basic Protocol 2).
See annotation to step 4a for additional considerations.
BASIC
PROTOCOL 2
Overnight enrichment is performed using alkaline peptone water (APW), pH 8.6. Some
investigators recommend two successive enrichments. Surface aliquots are streaked
onto selective bacteriological media. The two favored selective media used for V.
cholerae isolation are thiosulfate citrate bile salts sucrose (TCBS) agar and tellurite
taurocholate gelatin agar (TTGA), also known as Monsur medium (Monsur, 1961). V.
cholerae produces translucent, flat, yellow sucrose-fermenting colonies with elevated
centers on TCBS (Fig. 6A.5.2A). The organism produces colorless colonies on TTGA
(Fig. 6A.5.2B), often with a characteristic dark center after 2 days growth, surrounded
by a halo, which appears due to the hydrolysis of gelatin. Both media have advantages
as well as disadvantages.
TCBS, or cholera medium, is highly selective, inhibiting growth of Gram-positive and
typical intestinal flora, which cannot grow under alkaline conditions. However, TCBS
can inhibit some members of the genus Vibrio, including V. cholerae itself, as evidenced
by comparing simultaneous growth of V. cholerae on TCBS versus nonselective media.
In addition, TCBS can support the growth of several closely related bacteria, most notably Aeromonas and Proteus. One of the major advantages of this medium is that it
Nonenteric
Gamma
Proteobacteria
6A.5.5
Current Protocols in Microbiology
Supplement 2
Figure 6A.5.2 Growth of V. cholerae O1 on (A) TCBS and (B) TTGA. Courtesy of Dr. Munir Alam, International Center for Diarrheal Disease Research, Bangladesh. For the color version of this gure go to http://www.
currentprotocols.com.
is commercially available and does not require an autoclave to prepare plates, thereby
making it possible to use the medium under field conditions, without a laboratory or
autoclave. TTGA medium is generally considered less selective and less toxic to V.
cholerae than TCBS, so recovery of V. cholerae colonies may be enhanced as compared to that obtained with TCBS; however, these plates preferably should be incubated
longer (48 hr versus 24 hr for TCBS) to observe the characteristic dark center. Also,
the formulation is not commercially available and the medium must be autoclaved after
preparation. Once presumptive strains are purified on a nonselective medium such as
gelatin agar or modified nutrient agar, they are identified and confirmed by biochemical
tests and serogrouped using simple slide agglutination with polyclonal V. cholerae O1
and monoclonal O139 antiserum.
Materials
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.6
Supplement 2
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Proteobacteria
6A.5.7
Current Protocols in Microbiology
Supplement 2
Table 6A.5.2 Selected Biochemical Tests Used for the Identication of V. cholerae
Test
Medium
Methyl Red
Reagent to add
Positive result indicates
prior to reading
Temperature
Duration
MR-VP medium
37 C
48 hr
5-6 drops
methyl red
indicatora
VogesProskauer
MR-VP medium
37 C
48 hr
3 ml Barritts
reagent Ab and
1 ml Barritts
reagent Bc
Production of acetoin,
formed from pyruvic
acid during glucose
fermentation
Arginine
dihydrolase
37 C
24 hr
None
Lysine
Moeller decarboxylase broth
decarboxylased base (see recipe) containing
1% (w/v) L-lysine
37 C
24 hr
None
37 C
24 hr
None
37 C
24 hr
None
Utilization of the
carbohydrate arabinose
by production of acid
end products
Mannitol, acid
production
37 C
24 hr
None
Utilization of the
carbohydrate mannitol
by production of acid
end products
Oxidase
37 C
Esculin
hydrolysis
37 C
3 days
None
Hydrolysis of esculin to
glucose and esculetin
Growth in
nutrient broth
with 0% NaCl
37 C
5 days
None
Growth in
nutrient broth
with 6% NaCl
37 C
5 days
None
Organism is tolerant of
moderate salt
concentration
Growth in
nutrient broth
with 8% NaCl
37 C
5 days
None
Organism is tolerant of
high salt concentration
Presence of cytochrome
oxidase
a Dissolve 0.1 g methyl red in 300 ml of 95% ethanol. Adjust volume to 500 ml with water. Store up to 6 months at 2 to 8 C.
b Dissolve 5 g -naphthol in 100 ml absolute ethanol. Store up to 6 months at 2 to 8 C.
c Dissolve 40 g potassium hydroxide in 100 ml distilled water. Store up to 6 months at 2 to 8 C.
d For lysine and ornithine decarboxylase, it is useful to inoculate a tube of decarboxylase broth base without the addition of either amino acid for
comparison as a negative control. All decarboxylase test tubes must be overlaid with mineral oil.
e Nutrient broth must not contain any sodium chloride.
6A.5.8
Supplement 2
Results
Test
% Positive
Positive
Negative
Methyl Red
99
Red
Yellow
Voges-Proskauer
75
Red
Copper
Arginine dihydrolase
Pink
Orange
Lysine decarboxylase
99
Dark purple
Yellow
Ornithine decarboxylase
99
Dark purple
Yellow
Yellow
Purple
99
Yellow
Purple
Oxidase
100
Purple
Esculin hydrolysis
Clear
b
No fluorescence
Fluorescence
100
Turbid
Clear
53
Turbid
Clear
Turbid
Clear
a Traditional biochemical characterization of presumptive colonies (also see Table 6A.5.2). Adapted from (Lennette
et al., 1985).
b Loss of fluorescence is used since many V. cholerae strains produce melanin, which interferes with the normal
7a. If a positive result is achieved using the oxidase reagent test, inoculate one tube
of each of the other tests outlined in Table 6A.5.2 (eleven total after oxidase test
has been performed) for each suspected colony. Inoculate a second tube of each
test with a known positive control. Include a tube of decarboxylase base medium
(without the amino acid supplement) as a negative control for the lysine and ornithine
decarboxylase tests. Refer to Table 6A.5.3 for interpretation of results.
8a. Overlay arginine, lysine, and ornithine tubes (and decarboxylase broth base used as
negative control) with sterile mineral oil. Incubate all tubes at 37 C for the duration
indicated in Table 6A.5.2.
9a. Proceed to serogroup determination (Basic Protocol 3) with presumptive V. cholerae
colonies.
Nonenteric
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6A.5.9
Current Protocols in Microbiology
Supplement 2
7c. Perform the oxidase test (step 6a) on colonies giving an alkaline slant over acid butt
(purple slant and yellow butt) with no gas production or blackening of the butt due
to H2 S production.
8c. Proceed to serogroup determination (Basic Protocol 3) with presumptive V. cholerae
colonies.
BASIC
PROTOCOL 3
Serogroup Determination
Over 210 serogroups of V. cholerae have been described to date based on antigenic
properties of cell surface polysaccharides, of which serogroup O1 and O139 have been
implicated in epidemics of cholera, while serogroup O37 has been held responsible for
localized outbreaks of cholera in Czechoslovakia and Sudan. The remaining serogroups
collectively and commonly termed non-O1/non-O139 are predominant among the
strains of V. cholerae isolated from the aquatic environment (Sack et al., 2003). Although
reported mostly in O1 and O139 serogroups, the cholera toxin gene has been reported
in non-O1/non-O139 strains, also from the aquatic environment, in India (Chakraborty
et al., 2000). However, because of the epidemic potential, the method to determine O1
and O139 serogroup is described below. Strains other than O1 or O139 serogroup need
not be serogrouped unless there is a special need, in which case those strains should be
sent to a Reference Center for serotyping, since antisera for serogroups other than O1
and O139 are not commercially available.
Materials
Phosphate buffered saline (PBS; APPENDIX 2A)
V. cholerae colonies (6- to 16-hr subculture on nonselective modified nutrient agar,
e.g., Basic Protocol 2)
Polyvalent antisera for serogroup O1 and O139 V. cholerae (BD Biosciences)
Glass microscope slides
1. Add two separate drops of PBS to a microscope slide.
2. Add a loopful of fresh growth from a 6 to 16 hr subculture of V. cholerae on
nonselective medium to each drop, and mix.
3. Add an equal-sized drop of group O1 polyvalent antiserum to one of the drops.
4. Mix the antiserum-culture suspension by tilting the slide back and forth. Determine
if the reaction clumps (i.e., agglutinates) within 0.5 to 1 min, indicating a positive
result.
Autoagglutination, i.e., clumping in the saline solution without antiserum, is indicative
of a rough morphotype which cannot be typed by antisera. For typing of colonies with
rough morphotype, see Alternate Protocol 1.
5. Test non-O1 serogroup colonies using O139 antisera, repeating steps 1 to 4, but
substituting anti-O139 antiserum for anti-O1 antiserum.
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
Following the introduction of molecular methods into bacteriology, there has been an
influx of newer techniques aimed at understanding the mechanism of infection and at the
detection of pathogens, or organisms with virulent properties, in clinical or environmental
samples. Soon after the V. cholerae genome sequence was published in 2000 (Heidelberg
et al., 2000), PCR-based simple molecular probes became an important tool for detecting
V. cholerae and its antigens. Direct-detection methods have proven to be important when
conventional culture methods are recognized as ineffective because of the viable but not
culturable (VBNC) organisms present in samples collected for analysis.
6A.5.10
Supplement 2
BASIC
PROTOCOL 4
Materials
Overnight culture of putative V. cholerae in liquid medium or on agar plates (Basic
Protocol 2)
H2 O, sterile
1 PCR amplification buffer (APPENDIX 2A) containing 15 mM MgCl2 and 0.1%
(w/v) gelatin
25 mM dNTP stock: dATP, dCTP, dGTP, dTTP (APPENDIX 2A)
20 M PCR primers for ITS (Table 6A.5.4)
Taq DNA polymerase
1.5% agarose gel (Voytas, 2000)
TAE buffer (APPENDIX 2A)
Molecular weight ladder (e.g., Hyperladder IV, Bioline)
1 g/ml ethidium bromide (APPENDIX 2A)
2-ml microcentrifuge tubes, sterile
Boiling water bath
PCR tubes
Thermal cycler (MJ Research)
UV transilluminator, handheld UV lamp, or digital gel documentation system
Additional reagents and equipment for agarose gel electrophoresis and ethidium
bromide staining of gels (Voytas, 2000)
Prepare crude template
1a. From liquid medium: Centrifuge a 1-ml culture (overnight growth at 37 C) and
resuspend in 1 ml sterile water.
1b. From agar plates: Resuspend a loopful of pure culture (50 to 100 colonies) of
suspected or presumptive V. cholerae into 300 l sterile water.
Plates should contain a fresh overnight subculture.
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6A.5.11
Current Protocols in Microbiology
Supplement 2
Table 6A.5.4 PCR Primers for Genes Associated with Virulence of Toxigenic V. cholerae O1/O139 and Expected
Amplicon Size
Target
Primer
Amplicon
size
Reference
ITS
pVC-F2
TTAAGCSTTTTCRCTGAGAATG
295-310
PVCM-R1
AGTCACTTAACCATACAACCCG
PCTA-94F
CGGGCAGATTCTAGACCTCCTG
563
PCTA-614R
CGATGATCTTGGAGCATTCCCAC
pToxR-101F
CCTTCGATCCCCTAAGCAATAC
778
pToxR-837R
AGGGTTAGCAACCGATGCGTAAG
pTcpA-72F
CACGATAAGAAAACCGGTCAAGAG
452-621
pTcpAET-477R
CGAAAGCACCTTCTTTCACGTTG
pTcpACL-647R
TTACCAAATGCAACGCCGAATG
PZot-225F
TCGCTTAACGATGGCGCGTTTT
946
PZot-1129R
AACCCCGTTTCACTTCTACCCA
pOmpU-80F
ACGCTGACGGAATCAACCAAAG
868
pOmpU-906R
GCGGAAGTTTGGCTTGAAGTAG
VCT1
ACAGAGTGAGTACTTTGACC
308
VCT2
ATACCATCCATATATTTGGGAG
O1F2-1
GTTTCACTGAACAGATGGG
192
O1R2-2
GGTCATCTGTAAGTACAAC
O139F2
AGCCTCTTTATTACGGGTGG
449
O139R2
GTCAAACCCGATCGTAAAGG
ompW-F
CACCAAGAAGGTGACTTTATTGTG
588
ompW-R
GAACTTATAACCACCCGCG
CtxA-F
CTCAGACGGGATTTGTTAGGCACG
302
CtxA-R
TCTATCTCTGTAGCCCCTATTACG
16S-F
CAGCMGCCGCGGTAATWC
888
16S-R
ACGGGCGGTGTGTRC
ctxA
toxR
tcpA
Zot
ompU
ctxA
O1-rfb
O139-rfb
ompW
ctxA
16S
rDNAb
a Currently, there are no published 23S rDNA PCR primers specific for V. cholerae.
3. Place the microcentrifuge tube containing the resuspended culture into a boiling
water bath for 10 min.
4. Cool tube to room temperature by allowing tube to sit on bench (30 min).
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.12
Supplement 2
6. Amplify the V. cholerae-specific ITS target with the following cycling conditions:
1 cycle:
30 cycles:
1 cycle:
1 min
1 min
1 min
1 min
10 min
94 C
94 C
60 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
Refer to Kraemer and Coen (2001) for information about optimizing PCR.
Figure 6A.5.3 Results (gel) of PCR assays used to detect and characterize V. cholerae. Lane
1, Hyperladder IV (Bioline); lane 2, V. cholerae-specic ITS; lane 3, ctxA (pCTA); lane 4, tcpA of
V. cholerae O1 Classical; lane 5, tcpA of V. cholerae O1 El Tor; lane 6, tcpA of V. cholerae O139;
lane 7, toxR; lane 8, zot; lane 9, ompU; lane 10, O1-O139/ctxA multiplex of V. cholerae O1 and
O139. See Table 6A.5.4 for amplicon sizes.
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6A.5.13
Current Protocols in Microbiology
Supplement 2
BASIC
PROTOCOL 5
Materials
Water or plankton sample (Basic Protocol 1) or sediment sample
TE buffer, pH 8.0 (APPENDIX 2A)
10% (w/v) SDS (APPENDIX 2A)
20 mg/ml (2%) proteinase K
5 M NaCl
CTAB/NaCl solution (see recipe)
25:24:1 phenol/chloroform/isoamyl alcohol (APPENDIX 2A)
24:1 chloroform/isoamyl alcohol
Isopropanol
70% ethanol
1 PCR amplification buffer (APPENDIX 2A) containing 15 mM MgCl2 and 0.1%
(w/v) gelatin
25 mM dNTP stock: dATP, dCTP, dGTP, dTTP (APPENDIX 2A)
20 M forward and reverse PCR primers (Table 6A.5.4) for ompW (ompW-F, R)
and ctxA (ctxA-F, -R)
Taq DNA polymerase
1.5% agarose gel (Voytas, 2000)
TAE buffer (APPENDIX 2A)
Molecular weight ladder (e.g., Hyperladder IV, Bioline)
1 g/ml ethidium bromide (APPENDIX 2A)
37 and 65 C water baths
Vacuum desiccator or lyophilizer
Thermal cycler (MJ Research)
UV transilluminator, handheld UV lamp, or digital gel documentation system
Additional reagents and equipment for sample collection/transportation (Basic
Protocol 1) and enrichment (Basic Protocol 2), PCR (Kramer and Coen, 2001),
agarose gel electrophoresis and ethidium bromide staining of gels (Voytas,
2000), and isolation of V. cholerae by traditional culture method (Basic Protocol
2; optional)
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.14
Supplement 2
Extract DNA
3. Microcentrifuge a 1-ml aliquot from the upper surface (i.e., top 1 to 2 mm) of the
APW enrichment culture for 5 min at 12,000 g, room temperature.
4. Resuspend the cell pellet in 567 l TE buffer, pH 8.0.
5. Add 30 l of 10% SDS, then 3 l of 20 mg/ml proteinase K. Incubate the suspension
1 hr at 37 C.
6. Add 100 l of 5 M NaCl, then add 80 l of CTAB/NaCl solution. Incubate mixture
10 min at 65 C.
7. Add 800 l of 25:24:1 phenol/chloroform/isoamyl alcohol, vortex, and centrifuge 5
min at 12,000 g, room temperature.
8. Transfer aqueous (upper) phase to a new microcentrifuge tube. Add 800 l of
chloroform/isoamyl alcohol (24:1), vortex, and centrifuge 5 min at 12,000 g,
room temperature.
9. Transfer aqueous (upper) phase to a new microcentrifuge tube. Precipitate DNA by
adding an equal volume of isopropanol.
10. Microcentrifuge 5 min at 12,000 g, room temperature, and remove the supernatant.
Wash DNA by adding 1 ml of 70% ethanol and microcentrifuging 5 min at 12,000
g, room temperature.
11. Dry pellet in vacuum desiccator or lyophilizer and resuspend in 100 l TE buffer,
pH 8.0.
Perform multiplex PCR assay for detection of ompW (V. cholerae-specific) and ctxA
(toxigenicity) (Nandi et al., 2000)
12. Set up ompW-ctxA multiplex PCR in a PCR tube for a total reaction volume of
25 l containing the following:
10 to 20 ng extracted genomic DNA (step 11)
1 PCR amplification buffer
250 M each dNTP
1.2 pmol/l forward and reverse primers for ompW (ompW-F, -R)
0.25 pmol/l forward and reverse primers for ctxA (ctxA-F, -R)
0.625 U Taq DNA polymerase.
This protocol may be modified for the analysis of cultured organisms by using crude
template DNA (see Basic Protocol 4, step 4) in this mixture instead of extracted genomic
DNA.
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1 cycle:
5 min
30 sec
30 sec
30 sec
7 min
94 C
94 C
64 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
18. Screen samples giving a positive result for isolation of V. cholerae using the traditional
culture method as described in Basic Protocol 2, if desired.
ALTERNATE
PROTOCOL 1
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.16
Supplement 2
Final extension:
5 min
1 min
1 min
1 min
7 min
94 C
94 C
55 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
4. Run PCR product out on a 2.0% agarose gel in 1 TAE for 1 to 2 hr at 5 V/cm
(Voytas, 2000), along with a molecular weight ladder (e.g., Hyperladder).
5. Stain the gel by incubating in 1 g/ml ethidium bromide staining solution for 15 min
at room temperature.
6. Destain the gel by incubating in distilled water for 15 min at room temperature.
7. Visualize the products by viewing the gel using a handheld UV lamp, transilluminator, or digital gel documentation system.
The O1-rfb, O139-rfb, and ctxA amplicons are 192, 449, and 308-bp in length, respectively
(see Fig 6A.5.3).
8. Screen samples giving a positive result for isolation of V. cholerae using the traditional
culture method as described in Basic Protocol 2, if desired.
BASIC
PROTOCOL 6
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Materials
Enrichment flask with Vibrio cholerae culture (see Basic Protocol 2) (for detection)
or unenriched sample (for enumeration)
Alkaline peptone water (APW; see recipe)
LB plates (APPENDIX 4A) or modified nutrient agar plates (see recipe)
10% (w/v) SDS (APPENDIX 2A)
3 SSC (APPENDIX 2A)
Prewashing solution (see recipe), 60 C
DEPC-treated H2 O (APPENDIX 2A)
RNA colony blot hybridization solution (see recipe)
25 ng/l fluorescein-labeled Vchomim1276 probe
(5 -ACTTTGTGAGATTCGCTCCACCTCG-3 , with 5 fluorescein label; Sigma
Genosys)
Washing solution for colony blot hybridization with fluorescein-labeled probes (see
recipe), 60 C
85-mm sterile nitrocellulose membranes, 0.22-m (GE Water and Process
Technologies)
65 C incubator
Pyrex dishes
Whatman no. 3 filter paper cut slightly larger than the nitrocellulose membranes
70 C oven
60 C water bath
Hybridization oven
Typhoon Scanner (GE Healthcare) or Dark Reader (Clare Chemical)
NOTE: All solutions should be DNase and RNase-free. For RNA colony blot hybridization, use DEPC-treated water (see APPENDIX 2A) to make hybridization solutions.
6. Replica plate the membrane onto a fresh modified nutrient agar plate, transferring
the orientation markings. Maintain master and replica plate at 10 to 15 C, sealed
with Parafilm.
Perform this step only when subsequent analysis of a positive colony is necessary.
6A.5.18
Supplement 2
7. For each plate, prepare two Pyrex dishes by placing a piece of Whatman no. 3 filter
paper cut slightly larger than the nitrocellulose membrane into each. Prewet the filter
paper in one dish with 10% SDS and the other with 3 SSC, pouring or pipetting
the liquid onto the filter paper, letting it soak briefly, removing the air bubbles, then
pouring off the excess. Ensure that there is no pooled liquid on the filter paper prior
to placing membrane.
It is important to use filter paper prewetted but not saturated with the appropriate solutions, to prevent colonies from over-swelling and losing their circular shape.
8. Preheat 10% SDS, 3 SSC, and the Pyrex dishes with the prewetted filter paper to
65 C.
9. Place membrane colony-side-up on the SDS-prewetted filter paper in the corresponding dish, cover the membrane with Saran Wrap or equivalent plastic wrap to prevent
filter paper from drying out, and incubate 5 min at 65 C.
10. Transfer the membrane colony-side-up to the filter paper prewetted with 3 SSC,
cover membrane with Saran wrap or equivalent plastic wrap to prevent the filter
paper from drying out, and incubate 5 min at 65 C.
11. Transfer membranes to dry filter paper and allow to air dry 10 min at room temperature.
12. Bake membranes 15 min in a 70 C oven.
6A.5.19
Current Protocols in Microbiology
Supplement 2
ALTERNATE
PROTOCOL 2
4. Overlay the corresponding membrane onto each of the incubated spread plates,
starting from the center to ensure there are no air bubbles.
5. Allow at least 15 min for transfer.
6A.5.20
Supplement 2
6. Transfer the blot onto a new LB plate keeping colony side up and incubate 3 hr at
37 C. Wrap the master plate with Parafilm and keep at 10 to 15 C.
Master plates can be maintained in this manner for up to 2 weeks.
7. Place membranes, colony-side-up, onto Whatman no. 3 filter paper prewetted with
lysis buffer in a Pyrex dish (see Basic Protocol 6, step 7, for additional considerations). Incubate 10 min at room temperature.
It is important to use filter paper prewetted but not saturated with the appropriate solutions, to prevent colonies from over-swelling and losing their circular shape.
8. Remove the membrane from lysis buffer and place onto Whatman no. 3 filter paper
prewetted with neutralization solution in a Pyrex dish. Incubate 5 min at room
temperature.
9. Transfer membrane to a fresh filter paper prewetted with neutralization solution and
incubate 5 min at room temperature.
10. Remove membrane from neutralization solution, place onto dry no. 3 Whatman
filter paper and allow to air dry (15 min) at room temperature, but not to complete
dryness.
11. Immobilize colonies onto the membrane using a UV cross-linker or transilluminator.
Damp membranes should be cross-linked at an output intensity of 120-mJ/cm2 , which
corresponds to the optimal or auto-cross-link setting on commercially available machines. Transilluminators or hand-held UV lamps can be used, if calibrated; however, the
following times should be sufficient: 1 min for 254-nm lamps or 3 min for 302-nm lamps.
12. Rinse the blot twice in 1 SSC buffer and air dry (30 min).
13. Treat membranes 30 min at 42 C with 100 ml of 40 g/ml proteinase K solution
using gentle shaking.
14. Rinse filters three times in 1 SSC in a shaking water bath at room temperature,
each time for 10 min. Allow to air dry (30 min) at room temperature.
21. Wash membrane twice in stringency wash solution I for 5 min at room temperature
under constant agitation.
22. Wash membrane twice in stringency wash solution II for 15 min at 65 C under
constant agitation.
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31. From master plates (step 6), subculture colonies that were positive by blot hybridization for further analysis.
SUPPORT
PROTOCOL
Materials
DNA extracted from toxigenic (ctxA+ ) V. cholerae reference strain (ATCC)
Forward and reverse pCTA primers for amplifying ctxA (Table 6A.5.4)
Plasmid pKTN901 (available from Dr. James Kaper, jkaper@umaryland.edu) or
other source of ctxA probe
EcoRI restriction endonuclease (or other restriction enzyme depending on source
of ctxA probe) and restriction buffer
5 DIG High Prime labeling mixture (Roche)
0.2 M tetrasodium EDTA, pH 8.0
Boiling water bath
65 C water bath
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
Additional reagents and equipment for purifying DNA fragments (Moore et al.,
2002)
6A.5.22
Supplement 2
BASIC
PROTOCOL 7
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6A.5.23
Current Protocols in Microbiology
Supplement 2
Figure 6A.5.4 DFA staining of V. cholerae O1 using the Cholera DFA Kit (New Horizon Diagnostics). (A) Fresh culture; (B) VBNC cells; (C) and (D), DVC-incubated cells. For the color version of
this gure go to http://www.currentprotocols.com.
Materials
Concentrated water or homogenized plankton sample (Basic Protocol 2)
2.5% (w/v) yeast extract in distilled water
0.2% (w/v) nalidixic acid in distilled water
37% to 40% formaldehyde solution or fresh 4% formaldehyde prepared from
paraformaldehyde
Absolute methanol
Cholera DFA and/or Bengal DFA kit (New Horizon Diagnostics) containing:
FITC-conjugated DFA reagent
Positive and negative control
Fluorescent mounting medium
Phosphate-buffered saline (PBS; APPENDIX 2A)
Humidified chamber: 50-ml conical centrifuge tube containing 1 to 2 strips of
water-saturated filter paper
Multiwell slides and coverslips
Epifluorescent microscope with FITC filter set
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All solutions should be filter-sterilized through a 0.1-m filter, as VBNC cells of
V. cholerae appear as small coccoid cells in a size range of 0.1 to 0.8 m.
The water sample can be concentrated by filtration through a 0.22-m filter followed by
resuspension or by centrifugation for 10 min at 12,000 g, room temperature, followed
by removal of the supernatant and resuspension.
Freeze a parallel 1-ml sample for PCR confirmation (see Basic Protocol 5).
6A.5.24
Supplement 2
BASIC
PROTOCOL 8
Materials
Concentrated water or homogenized plankton sample (Basic Protocol 2)
95% ethanol
Phosphate-buffered saline (PBS; APPENDIX 2A)
FA Rhodamine counterstain (Becton Dickinson)
Polyvalent V. cholerae O1-specific antiserum (BD Bioscience)
FITC-conjugated anti-rabbit globulin goat serum (Sigma)
Low-fluorescence antiquenching mounting medium: e.g., FA (BD Biosciences) or
Citifluor AF1/AF3 (Electron Microscopy Sciences)
Multiwell, Teflon-coated slides
35 and 55 C incubators
Humidified chamber: 50-ml conical centrifuge tube containing 1 to 2 strips of
water-saturated filter paper
Glass coverslips
Epifluorescent microscope with FITC bandwidth filter
Prepare and fix samples
1. Add an appropriate amount (dependent on concentration of sample and well size) of
each sample to be tested to a Teflon-coated multiwell slide. Air dry 15 to 20 min at
room temperature.
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2. Fix the sample by adding 95% ethanol to each sample-containing well. Air dry 5 to
10 min at room temperature.
3. Heat slide 10 min in a 55 C incubator.
Slides may be stored up to 1 month at 70 C at this point.
Stain samples
4. Rinse the slide(s) with 50 ml PBS and air dry 15 to 20 min.
5. While slide is drying, equilibrate humidified chamber in 35 C incubator 15 min.
6. To each dry sample well, add 1 to 2 drops of a 1:20 dilution of FA Rhodamine in
distilled water. Incubate in humidified chamber 30 min at 35 C.
Minimize exposure to light from this step forward.
7. Rinse slide by gently flooding with 50-ml PBS, then soak in PBS 10 min at room
temperature. Remove slide from PBS and rinse again briefly in PBS.
8. Allow slide to air dry 15 to 20 min.
9. Add 5 to 10 l of V. cholerae O1-specific antiserum. Incubate in humid chamber
30 min at 35 C.
10. Repeat steps 7 and 8 using fresh PBS for washing.
11. Add 1 to 2 drops undiluted FITC-conjugated anti-rabbit globulin goat serum and
incubate in humidified chamber 30 min at 35 C.
12. Repeat washing steps 7 and 8 using fresh PBS.
6A.5.26
Supplement 2
Gelatin agar
4 g neopeptone
1 g yeast extract
5 g sodium chloride
15 g gelatin
15 g agar
Adjust volume to 1 liter with water
Autoclave to sterilize
Store up to 1 month at 2 to 8 C
Kligler iron agar
Combine the following:
20 g polypeptone
10 g lactose
1 g glucose
5 g sodium chloride
0.5 g ferric ammonium citrate
0.5 g sodium thiosulfate
0.025 g phenol red
15 g agar
Adjust volume to 1 liter with water
Boil to dissolve agar completely
Dispense 5-ml portions of the medium into 16 125mm tubes (other size tubes
can be used, adjust dispensed media volume to about 1/3 volume of test tube).
Autoclave 15 min at 121 C. Allow tubes to solidify in a slanted position to give a
generous butt (underneath a 20 to 40 slant). Store up to 6 months at 2 to 8 C.
This medium is also available commercially as a dehydrated powder from Difco (BD
Biosciences).
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6A.5.27
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Oxidase reagent
Dissolve 0.05 g N,N,N N -Tetramethyl-p-phenylenediamine dihydrochloride into
5 ml water. Prepare fresh
Final concentration is 1% (w/v).
Prewashing solution
Prepare the following in DEPC-treated water (APPENDIX 2A)
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.28
Supplement 2
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6A.5.29
Current Protocols in Microbiology
Supplement 2
COMMENTARY
Background Information
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.30
Supplement 2
DVC-DFA (Basic Protocol 7) and IFA (Basic Protocol 8) of V. cholerae are relatively
rapid protocols compared to the other methods
presented. One pitfall of these immunofluorescence methods is the confounding observation
of autofluorescing constituents found in environmental water samples. All positive samples should be documented by digital or film
photography; confirmation by PCR of a parallel sample (frozen, but not fixed) is advisable.
Table 6A.5.5 outlines some of the more
common problems that may be experienced
in performing the basic and alternate protocols from this unit. This is not an exhaustive
list; others may be encountered. Consult the
troubleshooting sections from the referenced
units of Current Protocols in Molecular Biology (see Literature Cited) for further advice.
Anticipated Results
V. cholerae can be easily isolated from
estuarine environments during the warm
summer monthseven in nonepidemic areas.
The chance of successful isolation decreases
during the colder winter months, even from
samples that are positive by PCR or DFA.
Figure 6A.5.2 shows the typical growth of V.
cholerae on TCBS and TTGA media. Growth
on TCBS by V. cholerae appears as small
(1- to 3-mm diameter) flat, yellow colonies
(Fig. 6A.5.2A). V. cholerae appear as medium
to large (2- to 5-mm in diameter) flat,
translucent colonies on TTGA (Fig. 6A.5.2B).
There will be a zone of clearing (or halo)
surrounding V. cholerae colonies due to
hydrolysis of gelatin. A dark center usually
develops after 24 hr. All strains of V. cholerae
can exhibit various colony morphologies,
including a rugose form (White, 1940). The
strain in Figure 6A.5.2A exhibits two colony
morphologies, translucent and opaque. For
the sake of comparison, several different
positive-control V. cholerae strains should
be subcultured at the time of subculturing
from APW to selective medium. The selective
media TCBS and TTGA still allow the
growth of other Vibrio species and related
bacteria. For example, V. parahaemolyticus
will appear as blue-green colonies, V. mimicus will appear as green colonies, and V.
alginolyticus as larger, yellow colonies on
TCBS. Therefore, colonies growing on these
media are not necessarily all V. cholerae and
are labeled presumptive. The goal of each
step in the biochemical identification method
is to reduce the number of suspected or presumptive colonies, which will include other
Vibrio species and related Vibrionaceae, until
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Problem
Solution
Basic Protocol 1
Little or no plankton in cod-end
collecting bucket
Basic Protocol 2
Failure of positive control to give correct Use multiple positive controls
results for biochemical tests
Prepare multiple replicates for each positive control
Results of biochemical tests difficult to
interpret
Basic Protocol 3
Autoagglutination or clumping in saline
without antisera
Basic Protocol 4
No PCR product with positive control
Basic Protocol 5
No PCR product with positive control
Alternate Protocol 1
No PCR product with positive control
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.32
Supplement 2
Table 6A.5.5 Troubleshooting Guide for V. cholerae Isolation and Detection, Contined
Problem
Solution
Basic Protocol 6
Positive control is negative
Alternate Protocol 2
Positive control is negative or weak
Support Protocol
Positive control from Alternate Protocol Check efficiency of probe labeling reaction
2 is negative or weak
Increase probe concentration (>25 ng/ml)
Basic Protocol 7
Positive control is negative
Basic Protocol 8
Positive control signal is weak
and El Tor (ET). The two biotypes are distinguished from each other on the basis of their
differences in several phenotypic properties
(WHO, 1987), including hemolysis (sheep red
blood cells; CL , ET+/ ), hemagglutination
(chicken red blood cell; CL , ET+ ; Finkelstein
and Mukerjee, 1963), Voges-Proskauer reaction (CL , ET+ ), susceptibility to polymyxin
B (CL+ , ET ; Han and Khie, 1963), and sensitivity to biotype-specific phages (Nair et al.,
2002). More recent studies have identified
genes unique to the classical and seventh pandemic El Tor strains.
Figure 6A.5.3 shows a gel separation of
the expected amplicons for several of the PCR
protocols listed in the sections above. Table
6A.5.4 lists the amplicon size for all of the PCR
primers mentioned in this chapter. This is not
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6A.5.33
Current Protocols in Microbiology
Supplement 2
Figure 6A.5.5 Growth of V. cholerae in a Kligler iron agar (KIA) slant. Notice the alkaline butt
and acid slant, with no gas production or blackening due to hydrogen sulde production. For the
color version of this gure go to http://www.currentprotocols.com.
Time Considerations
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
Acknowledgements
This work was partially supported by Grant
No. 01200622 from the National Science
Foundation and Grant No. 1RO1A139129-01
from the National Institutes of Health.
Literature Cited
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N.A., Nair, G.B., Siddique, A.K., Sack, D.A.,
Ahsan, S., Huq, A., Sack, R.B., and Colwell,
R.R. 2006. Effect of transport at ambient temperature on detection and isolation of Vibrio
cholerae from environmental samples. Appl. Environ. Microbiol. 72:2185-2190.
Amann, R., Ludweg, W., and Schleifer, K.H. 1995.
Phylogenetic identification and in situ detection
of individual microbial cells without cultivation.
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Bergey, J. Holt, G., Krieg, N.R., Bergey, D.H., and
Sneath, P.H.A. 1994. Bergeys Manual of Determinative Bacteriology. Lippincott, Williams &
Wilkins, Baltimore, Md.
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J., Bass, S., Qin, H., Dragoi, I., Sellers, P., McDonald, L., Utterback, T., Fleishmann, R.D.,
Nierman, W.C., White, O., Salzberg, S.L.,
Smith, H.O., Colwell, R.R., Mekalanos, J.J.,
Venter, J.C., and Fraser, C.M. 2000. DNA sequence of both chromosomes of the cholera
pathogen Vibrio cholerae. Nature 406:477483.
Heidelberg, J.K., Heidelberg, K.B., and Colwell,
R.R. 2002. Seasonality of Chesapeake Bay bacterioplankton species. Appl. Environ. Microbiol.
68:5488-5497.
Hood, M.A., Ness, G.E., and Rodrick, G.E. 1981.
Isolation of Vibrio cholerae serotype O1 from
the eastern oyster, Crassostrea virginica. Appl.
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Hoshino, K., Yamasaki, S., Mukhopadhyay, A.K.,
Chakraborty, S., Basu, A., Bhattacharya, S.K.,
Nair, G.B., Shimada, T., and Takeda, Y. 1998.
Development and evaluation of a multitest PCR
assay for rapid detection of toxigenic Vibrio
cholerae O1 and O139. FEMS Immunol. Med.
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Huq, A., Colwell, R.R., Rahman, R., Ali, A.,
Chowdhury, M.A., Parveen, S., Sack, D.A.,
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Huq, A., Rivera, I.N.G., and Colwell, R.R. 2000.
Epidemiological Significance of viable but
nonculturable microorganisms. In Nonculturable Microorganisms in the Environment. (R.
Colwell and D. Grimes, eds.) pp. 301-323. ASM
Press, Washington, D.C.
Huq, A., Sack, R.B., Nizam, A., Longini, I.M.,
Nair, G.B., Ali, A., Morris, J.G., Khan, M.N.H.,
Siddique, A.K., Yunus, M., Albert, M.J., Sack.
D.A., and Colwell, R.R. 2005. Critical factors
influencing the occurrence of Vibrio cholerae in
the environment of Bangladesh. Appl. Environ.
Microbiol. 71:4645-4654.
Islam, M., Hasan, N.K., Miah, M.A., Qadri, F.,
Yunus, M., Sack, R.B., and Albert, M.J. 1993.
Isolation of Vibrio cholerae O139 Bengal from
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Islam, M.S, Rahman, M.Z., S.I. Khan, Mahmud,
Z.H., Ramamurthy, T., Nair, G.B., Sack, R.B.,
and Sack, D.A. 2005. Organization of the CTX
prophage in environmental isolates of Vibrio
mimicus. Microbiol. Immunol. 49:779-784.
Kaper, J., Lockman, H., Colwell, R.R., and Joseph,
S.W. 1979. Ecology, serology, and enterotoxin
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Knight, I. 2000. Molecular genetic methods for detection and identification of viable but nonculturable microorganisms. In Nonculturable Microorganisms in the Environment (R. Colwell
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Minami, A., Hashimoto, S., Abe, H., Arita, M.,
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Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
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Morris, G.K., Merson, M.H., Huq, I., Kibrya, A.K.,
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retention of Vibrio cholerae O1 in the eastern
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Current Protocols in Microbiology
Supplement 2
Detection,
Isolation, and
Identication of V.
cholerae from the
Environment
6A.5.38
Supplement 2
UNIT 7B.1
Bdellovibrio and like organisms (BALOs) are commonly retrieved from natural and
man-made habitats. As they are obligate predators of other Gram-negative bacteria,
they should be cultivated with prey cells in two-member cultures. Their growth cycle
includes two spatially separated phases: a free-swimming attack phase and an intracellular
phase during which the predatory bacterium establishes, elongates, and grows within the
periplasm of its host at the expense of the latters cell content. BALO-infected prey are
called bdelloplasts. Isolation from environmental samples requires separation of the small
BALOs from the majority of the other bacteria to ensure that plaques are not overgrown
by contaminants.
This unit describes ways to isolate BALOs from the environment (see Basic Protocol 1 and
Alternate Protocols 1 to 3), store BALOs (see Basic Protocol 2), obtain host-independent
derivatives (see Basic Protocol 3), synchronize BALOS (see Basic Protocols 4 and 5),
separate the various phases of growth (see Basic Protocol 6), and classify BALOs (see
Basic Protocol 7). BALOs are isolated in double-layered agar plates as plaques growing
in top agar containing prey cells. When found at very low levels, samples containing
BALOs can be enriched by incubation with potential prey prior to isolation. Hostindependent (HI) mutants are readily isolated from wild-type BALOs grown in pure and
dense cultures, and are grown in rich medium. The different phases of the growth cycle
of both wild-type BALOs and HI mutants can be separated to obtain defined fractions.
Moreover, wild-type as well as HI-mutant cultures can be synchronized to obtain a
majority of cells at the same stage of the growth cycle. Classification is based on the
analysis of the 16S rRNA gene with the support of specific primers.
CAUTION: Bdellovibrio and like organisms are Biosafety Level 1 (BSL-1) microbes.
Such organisms are not known to consistently cause disease in healthy adult humans, and
are of minimal potential hazard to laboratory personnel and the environment. Standard
microbiological practices should be followed when working with these organisms. Prey
cells are selected by the experimenter and may therefore vary in biosafety level(s). This
should be checked prior to starting work. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
IMPORTANT NOTE: When it is important to remove prey cells from a BALO culture
(i.e., DNA, RNA, or protein analysis), lytic suspensions should be passed through a 0.45m filter at least once. Also, filtration may have to be performed to remove contaminants
carried over during the isolation of BALOs.
IMPORTANT NOTE: When working with bacteria-domain-directed primers, exercise
extra care to prevent contamination of PCR reagents.
BASIC
PROTOCOL 1
BALO cells are small (0.25 to 0.5 0.75 to 1.25 m); therefore, they can be separated
from the major fraction of the bacterial population. This is achieved using differential
centrifugation and filtration.
Delta
Proteobacteria
Contributed by Edouard Jurkevitch
Current Protocols in Microbiology (2005) 7B.1.1-7B.1.17
C 2005 by John Wiley & Sons, Inc.
Copyright
7B.1.1
Materials
Soil or water sample suspected of containing BALOs
H2 O, sterile (for soil samples)
HM buffer, 4 C (see recipe)
Potential prey (Support Protocol)
HM top agar, molten (see recipe)
HM plates (see recipe)
1.2-m Nucleopore filter
28 C incubator
0.45-m syringe filters (Supor Acrodisc, Pall Corp.; Minisart, Sartorius)
1a. For soil or rhizosphere samples: Prepare a slurry containing 10 to 50 g soil using a
1:2 to 1:10 soil/sterile water ratio according to soil texture (i.e., less for heavy clay
soils and more for light sandy soils). Shake 30 min at room temperature using a
platform shaker at 150 rpm or an orbital shaker at 200 to 250 rpm.
1b. For aquatic samples: Collect 50 to 250 ml sample.
2. Centrifuge 5 min at 500 g, 4 C. Discard the pellet.
3. Centrifuge supernatant 20 min at 27,000 g, 4 C. Decant supernatant and dissolve
resulting pellet in a few milliliters of cold HM buffer.
HM buffer is used in place of a richer medium to limit the growth of prey or potential
environmental contaminants. This is especially important when isolating BALOs from the
environment.
4. Pass through a 1.2-m Nucleopore filter. Maintain at 4 C (up to a few days); process
as soon as possible.
If a high level of contaminants (carried over from isolation) grow on the HM top agar,
blurring the BALO plaques, an extra filtration through a 0.45-m filter may be necessary.
Such a step may, however, further reduce the yield of BALOs.
To deal with clogged filters, see Alternate Protocol 1 and the Troubleshooting section.
7. Allow top agar to solidify at room temperature (60 min). Seal each plate with
Parafilm.
8. Incubate sealed plates upside-down for 3 to 8 days at 28 C. Check for the growth of
regular lytic plaques on the background of the prey lawn.
Isolation and
Classication of
Bdellovibrio and
Like Organisms
7B.1.2
Current Protocols in Microbiology
9. Cut 1-ml pipettor tips with a sterile scalpel. Gently aspirate plaques into the tip.
Resuspend individual plaques in 0.5 ml HM buffer, room temperature. Let stand for
a few minutes, then vortex.
10. Place a drop of the suspension on a clean glass slide and cover with a cover slip.
Check for minute, fast moving cells using phase-contrast microscopy.
11. Pass through a 0.45-m syringe filter. Maintain at 4 C; process as soon as possible.
12. Repeat steps 5 to 11 twice more to obtain pure cultures.
13. Grow and store as described in Basic Protocol 2.
ALTERNATE
PROTOCOL 1
ALTERNATE
PROTOCOL 2
Delta
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7B.1.3
Current Protocols in Microbiology
7. Allow top agar to solidify at room temperature (<60 min). Wrap plates in Parafilm.
8. Incubate at 25 C for at least one week.
9. Check for the growth of regular lytic plaques on the background of the prey lawn
(up to 8 days). When apparent, treat plaques as in the Basic Protocol 1, steps 9 to 13,
substituting Pp medium for HM buffer.
ALTERNATE
PROTOCOL 3
Materials
Pure culture of BALO in double-layered agar plate (Basic Protocol 1 and Alternate
Protocols 1 to 3)
Diluted nutrient broth (DNB; see recipe)
Potential prey (Support Protocol; optional)
80% glycerol, sterile
28 C incubator
Isolation and
Classication of
Bdellovibrio and
Like Organisms
1. Cut 1-ml pipettor tips with a sterile scalpel. Gently aspirate a BALO plaque from a
double-layered agar plate, including some surrounding agar containing prey. Transfer
to a 125-ml Erlenmeyer flask containing 5 ml DNB.
7B.1.4
Current Protocols in Microbiology
BASIC
PROTOCOL 3
Note that HI strains lose their predatory behavior when repetitively grown in the absence
of prey and also are pleiotropic. HI strains vary in growth rates and other phenotypic
characteristics.
Materials
Wild-type, host-dependent BALO strain
Streptomycin-resistant (Smr ) and -sensitive (Sms ) prey (Support Protocol)
50 mg/ml streptomycin
Dilute nutrient broth (DNB; see recipe) with and without 50 g/ml streptomycin
PYE medium (see recipe) containing 50 g/ml streptomycin
PYE plates with and without host extract (see recipe)
HM buffer (see recipe; optional)
Glycerol, sterile
28 C incubator
0.45-m syringe filter (Supor Acrodisc, Pall Corp.; Minisart, Sartorius)
Additional reagents and equipment for preparing double-layered HM plates (see
Basic Protocol 1; optional)
1. Grow wild-type, host-dependent BALO strain on Smr prey (109 cells/ml) 24 hr at
28 C. Add 50 mg/ml streptomycin to a final concentration of 50 g/ml. Incubate an
additional 12 hr at 28 C for a total of 36 hr.
Streptomycin is used to select for SmR BALOs.
2. Inoculate 20 ml DNB containing 109 cells/ml Smr prey and 50 g/ml streptomycin
with the 36-hr culture to a final concentration of 15% (v/v). Incubate at 28 C until
the prey is consumed (2 hr).
Delta
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7B.1.5
Current Protocols in Microbiology
7. Streak onto a PYE plate. Incubate at 28 C until colonies develop (up to 3 to 5 days).
Repeat this step twice to obtain pure cultures.
8. Optional: To retain predatory efficiency in HI mutants, plate HI strains in doublelayered HM plates (e.g., Basic Protocol 1, steps 6 to 10) or grow in HM buffer.
Include Sms prey every second or third transfer.
Do not subculture HI strains too many times. Start from stock after 3 passages.
10. To revive an HI culture, thaw and spread 100 l on a PYE plate supplemented with
7% (v/v) host extract.
BASIC
PROTOCOL 4
Materials
Prey (Support Protocol)
HM buffer (see recipe), 4 C
Pure BALOs (Basic Protocol 1 or Alternate Protocols 1 to 3)
0.45-m syringe filter (Supor Acrodisc; Pall Corp.; Minisort, Sartorius)
28 C incubator
1. Grow 50 ml prey cells to the end of exponential or early stationary phase.
2. Centrifuge 10 min at 5,000 g, 4 C. Decant the supernatant and add 5 vol cold HM
buffer. Repeat centrifugation and resuspend in cold HM buffer such that the final
concentration is at least 5 109 cells/ml.
3. Prepare a fresh 100-ml lytic culture of BALOs on the same prey.
Isolation and
Classication of
Bdellovibrio and
Like Organisms
4. Centrifuge the BALO culture 20 min at 10,000 g, 4 C. Decant the supernatant and
add 5 vol cold HM buffer. Repeat centrifugation and resuspend in cold HM buffer
such that the final concentration is at least 2 1010 cells/ml.
7B.1.6
Current Protocols in Microbiology
7. Combine predator (step 6) and prey (step 2) at a ratio of 2:1, at final concentrations
of 1010 and 5 109 cell/ml, respectively.
8. Incubate with strong shaking (250 rpm) at 28 C.
Prey cells are penetrated within 30 min and a full cycle completed within 2.5 to 4 hr,
depending on the strains and conditions. After the first cycle, the prey culture is essentially
consumed and no prey cells remain to start a second cycle.
BASIC
PROTOCOL 5
Materials
Host-independent (HI) mutant (Basic Protocol 3)
PYE medium with 7% (v/v) host extract (see recipe)
28 C incubator
1. Grow HI mutant in PYE medium with host extract at 28 C overnight.
2. Centrifuge 10 min at 10,000 g, 4 C.
3. Resuspend in fresh PYE supplemented with 7% (v/v) host cell adjusted to 5 mg
protein/ml to obtain a final volume 10 larger that of the overnight culture (step 1).
4. Grow at 28 C and check for culture homogeneity according to needs by phasecontrast microscopy.
Synchrony is maintained until stationary phase (8 to 12 hr).
Growth can be tracked by spectrophotometry (OD600 ).
BASIC
PROTOCOL 6
Materials
Synchronous BALO culture (Basic Protocols 4 and 5)
0.05 M potassium phosphate buffer, pH 7.2 (APPENDIX 2A)
Percoll
0.25 M sucrose (85.5 g/liter)
14.5 mM NaCl
70-ml ultracentrifuge tube
Sorvall Centricon T-1170 ultracentrifuge and A-641 rotor (or equivalent)
1. Centrifuge a 100-ml synchronous BALO culture 10 min at 20,000 g, 4 C. Wash
with 5 to 10 ml of 0.05 M potassium phosphate buffer and resuspend in a few
milliliters of the same.
Delta
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7B.1.7
Current Protocols in Microbiology
2. Mix five parts Percoll with four parts 0.25 M sucrose. Transfer 66 ml into a 70-ml
ultracentrifuge tube.
3. Layer BALOs onto the top of the Percoll-filled tube and ultracentrifuge 30 min at
50,000 g, 4 C.
4. Using gentle pipetting, first remove the floating fluffy white material, then harvest
prey cell remains or bdelloplasts (depending on the growth stage) from a ring at the
top of the tube.
The floating fluffy white material is composed of a mixture of growth stages.
5. Harvest the attack phase cells from an opaque band located 1.5 cm from the bottom
of the tube, also using gentle pipetting.
6. To remove Percoll, wash the retrieved fractions by diluting in 5 vol of 14.5 mM
NaCl. Centrifuge 10 min at 20,000 g, 4 C.
7. Resuspend in a solution appropriate for further work, such as the extraction of cellular
constituents.
SUPPORT
PROTOCOL
BASIC
PROTOCOL 7
PREY PREPARATION
Prey organisms should be prepared ahead of working with BALOs. In order to accomplish
this, first grow potential Gram-negative prey in an appropriate medium (e.g., see APPENDIX
2C). Collect cells at the end of exponential to early stationary phase. Centrifuge 10 min
at 5,000 g, 4 C. Wash once with HM buffer. Resuspend in HM buffer (see recipe) to a
final concentration of 5 109 to 1010 cfu/ml. For routine growth, prey stock can be used
10 to 14 days if kept at 4 C. The most commonly used prey include E. coli ML35 and
various strains of Pseudomonas fluorescens and P. putida.
CLASSIFICATION OF BALOS
The classification of BALOs is based on the analysis of the 16S rRNA gene. BALOs
belong to two recognized families, Bdellovibrionaceae and Bacteriovoracaceae, forming
the order Bdellovibrionales within the -Proteobacteria. Bdellovibrio bacteriovorus is
the only recognized species within the Bdellovibrionaceae, while Bacteriovorax stolpii,
Bacteriovorax marinus, Bacteriovorax littoralis, and Peridibacter starrii belong to the
Bacteriovoracaceae. The G+C content of the Bdellovibrionaceae is 50%, and that of
the Bacteriovoracaceae is between 37% to 44%. Amplified rDNA restriction analysis
(ARDRA) of an 800-bp fragment enables a first classification of BALOs. Precise
phylogenetic affiliation is performed using the (almost) whole 16S rRNA gene sequence
length.
Materials
Isolation and
Classication of
Bdellovibrio and
Like Organisms
7B.1.8
Current Protocols in Microbiology
Primer
Sequence
5 -CGWCACTGAAGGGGTCAA- 3
5 -CAGGCCTAACACATGCAAGTC-3
5 -CCCCGTCAATTCCTTTGAGTTT-3
a Any general Bacteria (i.e., Eubacteria or Archaea) domain targeted primer near the 3 end can be utilized.
3 mM MgCl2
20 M (each) dNTPs
1 PCR reaction buffer
1.25 U Taq DNA polymerase
1 M each 63F and 842R primer
12.5 g BSA
5. Amplify using the following program:
Initial step:
35 cycles:
Final step:
4 min
1 min
1 min
1 min
5 min
94 C
94 C
50 C
72 C
72 C
(denaturation)
(denaturation)
(annealing)
(extension)
(extension).
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7B.1.9
Current Protocols in Microbiology
Figure 7B.1.1 Restriction patterns of amplied 16S rRNA gene fragments of Bdellovibrio and
like organisms after digestion with HaeIII, HinfI, and RsaI. Lane designated MW was loaded with
a 100-bp molecular marker. The combination of the patterns denes the different ARDRA groups.
(Reproduced with permission from Davidov and Jurkevitch, 2004).
ARDRA
7. Purify amplification products by standard techniques (e.g., Moore and Downhan,
2002).
8. Prepare separate restriction digestions according to manufacturers protocols utilizing HaeIII, HinfI, or RsaI, and 15 l PCR product.
See Bloch and Grossman (1995) for general information about performing restriction
digestion analysis.
10. Compare restriction patterns to Figure 7B.1.1, find the corresponding group in
Table 7B.1.2, and the phylogenetic affiliation in Figure 7B.1.2.
11. If required (i.e., organism belongs to Group C or D; Table 7B.1.2), prepare a new
restriction digestion utilizing EcoRI to enhance resolution.
7B.1.10
Current Protocols in Microbiology
HaeIII
HinfI
RsaI
10
11
10
8d
Rf
12d
9d
Sf
13d
10d
Tf
11d
Ue
Delta
Proteobacteria
7B.1.11
Current Protocols in Microbiology
Figure 7B.1.2 Phylogenetic 16S rRNA tree of the (A) Bdellovibrionaceae (B) Bacteriovoracaceae based on
maximum-likelihood analysis. The ARDRA groups shown correspond to the patterns shown in Figure 7B.1.1 and to
the key in Table 7B.1.2 (Reproduced with permission from Davidov and Jurkevitch, 2004.)
Isolation and
Classication of
Bdellovibrio and
Like Organisms
7B.1.12
Current Protocols in Microbiology
HM top agar
Prepare 25 mM N-[2-hydroxyethyl]piperazine-N -[2-ethanesulfonic acid]
(HEPES). Add agar to 0.7% final. Adjust to pH 7.4 with 10 N sodium hydroxide.
Sterilize by autoclaving and cool to 60 C. Add 6 ml of 0.5 M CaCl2 2H2 O (3 mM
final) and 3.33 ml of 0.6 M MgCl2 7H2 O (2 mM), passing each solution through a
0.20-m syringe filter as it is added to the medium. Rapidly prepare 4-ml aliquots
in sterile test tubes before the agar solidifies. Prior to use, melt in an 80 C water
bath, then maintain at 42 C to keep molten.
A concentration of 0.7% can be obtained by adding 7 g agar per liter medium.
Solidified tubes can be stored for 1 to 2 weeks but should be wrapped to prevent evaporation.
Host extract
Centrifuge one liter of an overnight E. coli culture prepared in nutrient broth (e.g.,
LB or NB; APPENDIX 4A). Centrifuge 10 min at 10,000 g, 4 C. Resuspend in 10
ml HM buffer (see recipe), autoclave, and remove debris by centrifuging 10 min
at 10,000 g, 4 C. Retain the supernatant. Determine protein concentration using
Bradfords reagent (APPENDIX 3A) so that the addition of extract to growth medium
is standardized.
Ocean water, sterile
Obtain ocean water or prepare from sea salts such as Instant Ocean (Aquarium
Systems). Autoclave and pass through a 0.2-m filter. Store up to 2 months at room
temperature.
Polypeptone 20 (Pp) medium
Dissolve 1 g polypeptone 20 (Pp) in one liter ocean water (see recipe for sterile
ocean water). If necessary, add HCl to obtain a pH of 7.7 to 7.8. Autoclave and pass
through a 0.2-m filter. Store up to 3 months at room temperature.
Delta
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7B.1.13
Current Protocols in Microbiology
Pp plates
Autoclave polypeptone 20 (Pp) medium (see recipe) containing 1.5% (w/v) agar.
Cool to 60 C. Pour into 15 150mm plates using 60 to 65 ml per plate. Let
solidify and dry. Seal in bags and store up to 2 months at 4 C.
A concentration of 1.5% can be obtained by adding 15 g agar per liter medium.
Pp top agar
Autoclave polypeptone 20 (Pp) medium (see recipe) containing 1.95% agar. Prepare
3.3-ml aliquots in sterile test tubes. Before use, melt, then maintain at 42 C to keep
molten.
A concentration of 1.95% can be obtained by adding 19.5 g agar per liter medium.
PYE medium
Add 3 g yeast extract and 10 g Bacto peptone to 1 liter water. Sterilize by autoclaving.
Cool to room temperature and add 6 ml of 0.5 M CaCl2 2H2 O (3 mM final) and
3.33 ml of 0.6 M MgCl2 7H2 O (2 mM), passing each through a 0.20-m syringe
filter as it is added to the medium. Store up to 3 months at room temperature.
PYE plates
Add 3 g yeast extract, 10 g Bacto peptone, and 15 g agar to 1 liter water. Cool to
60 C. If desired, add 70 ml (7% v/v final) host extract (see recipe). Add 6 ml of 0.5
M CaCl2 2H2 O (3 mM final) and 3.33 ml of 0.6 M MgCl2 7H2 O (2 mM), passing
each through a 0.20-m syringe filter as it is added to the medium. Pour into 15-mm
plates using 20 to 25 ml per plate. Seal in bags and store up to 2 months at 4 C.
Salt solution
0.75 g KCl
23.4 g NaCl
6.9 g MgCl2 7H2 O
Adjust pH to 7.2 with 1 N NaOH
Store indefinitely at room temperature
SWYE plates
Dissolve 10 g proteose peptone, 3 g yeast extract, and 20 g agar in 1 liter salt
solution (see recipe). Sterilize by autoclaving. Cool to 60 C. Pour into standard
1.5 90mm plates using 20 to 25 ml per plate. Seal in bags and store up to 2
months at 4 C.
COMMENTARY
Background Information
Isolation and
Classication of
Bdellovibrio and
Like Organisms
7B.1.14
Current Protocols in Microbiology
Critical Parameters
Amplification of the 16S rRNA gene (Basic Protocol 7) or part thereof with the
polymerase-chain reaction should be performed with great care when using general
primers targeting the Bacteria domain. In that
case, BALO suspensions should be thoroughly
filtered and washed to remove traces of the
prey.
Troubleshooting
Table 7B.1.3 presents some common problems encountered when performing the protocols described in this unit, as well as their
common causes and potential solutions.
Anticipated Results
Isolation from environmental samples
BALOs plaques should almost always be
obtained from environmental samples. The
number of plaques may vary greatly between
habitats, seasons and the prey chosen, among
other parameters. Usually tens to tens of thousands of plaques are formed per milliliter or
gram sample.
Isolation of HI mutants
The procedure described has enabled the
isolation of HI derivatives from all the strains
tested so far (Seidler and Starr, 1969).
Classification
At least the family affiliation (Bdellovibrionaceae, Bacteriovoracaceae, or as yet undescribed groups) of the analyzed isolates
should be obtained. Most strains originating
from mesophilic, but not extreme terrestrial
and aquatic habitats, may belong to one of the
described ARDRA groups.
Time Consideration
Isolation from environmental samples
Plaques become apparent 3 to 8 days after
inoculation. Pure cultures, obtained by plating the plaques repeatedly, should be obtained
within two weeks.
Growth and storage of pure BALO cultures
This procedure should take 2 to 3 days.
Isolation of HI mutants
Isolating host-independent mutants can
take up to two weeks.
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Proteobacteria
7B.1.15
Current Protocols in Microbiology
Table 7B.1.3 Troubleshooting Guide for Isolation and Classication of Bdellovibrio and Like
Organisms
Problem
Possible Cause
Solution
Change prey
Overgrowth of contaminants
No amplification product
Classification
Literature Cited
Moore, D. and Downhan, D. 2002. Purification and
concentration of DNA from aqueous solutions
In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. 2.1.1-2.1.10. John Wiley & Sons, Hoboken,
N.J.
Isolation and
Classication of
Bdellovibrio and
Like Organisms
sification of Bdellovibrio stolpii and Bdellovibrio starrii into a new genus, Bacteriovorax
stolpii comb. nov. and Bacteriovorax starrii
comb. nov., respectively. Int. J. Syst. Environ.
Microbiol. 50:219-224.
Barel, G. and Jurkevitch, E. 2001. Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J. Arch.
Microbiol. 176:211-216.
Bloch, K.D. and Grossman, B. 1995. Digestion of
DNA with restriction endonucleases. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.1.13.1.21. John Wiley & Sons, Hoboken, N.J.
Davidov, Y. and Jurkevitch, E. 2004. Diversity
and evolution of Bdellovibrio-and-like organisms (BALOs), reclassification of Bacteriovorax
starrii as Peredibacter starrii gen. nov., comb.
nov., and description of the BacteriovoraxPeredibacter clade as Bacteriovoracaceae fam. nov.
Int. J. Syst. Evol. Microbiol. 54:1439-1452.
7B.1.16
Current Protocols in Microbiology
Key References
Davidov and Jurkevitch, 2004. See above.
A comprehensive presentation of BALO classification.
Martin, 2002. See above.
A review of various predatory bacteria and of
their modes of action. The first in-depth report on
Bdellovibrio. A classic.
Jurkevitch, E. 2005. The genus Bdellovibrio. In
The Prokaryotes, Release 3.19 (M. Dworkin,
S. Flakow, E. Rosenberg, K.H. Schleifer, and
E. Stackebrandt, eds.). Springer-Verlag, New
York. (available online at http://141.150.157.
117:8080/prokPUB/index.htm.)
A review on BALOs.
Kelley, J.I., Turng, B.F., Williams, H.N., and Baer,
M.L. 1997. Effects of temperature, salinity, and
substrate on the colonization of surfaces in situ
by aquatic bdellovibrios. Appl. Environ. Microbiol. 63:84-90.
Describes the tracking of BALO populations in the
marine environment.
Stolp, H., and Starr, M.P. 1963. Bdellovibrio bacteriovorus gen. et sp., a predatory, ectoparasitic, and bacteriolytic microorganism. Anton.
van Leeuwen. 29:217-248.
The first independent report on Bdellovibrio: a
classic.
Delta
Proteobacteria
7B.1.17
Current Protocols in Microbiology
UNIT 7B.2
ABSTRACT
Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys upon other Gramnegative bacteria. It does this by penetrating the outer membrane and peptidoglycan to
establish itself in the periplasm where it grows at the expense of the contents of the
prey cell before ultimately lysing the prey. Wild-type Bdellovibrio are prey-(or host-)
dependent and the protocols described in this unit deal with the techniques required to
grow this bacterium on its prey cells. Protocols are also presented to generate and culture
host-independent mutants as well as novel protocols to evaluate Bdellovibrio predation.
C 2008 by John Wiley & Sons, Inc.
Curr. Protoc. Microbiol. 9:7B.2.1-7B.2.13.
Keywords: Bdellovibrio bacteriovorus r predator-prey interactions r
luminescence assay r plaque assay r host-independent growth r storage of cultures
INTRODUCTION
Bdellovibrio are small, predatory, delta-Proteobacteria. They collide with, attach to, and
invade Gram-negative bacterial prey by lysing a small temporary pore into the outer
layers of the prey through which they enter and establish growth in the prey periplasm in
a structure known as the bdelloplast (Tudor et al., 1990). Bdellovibrio growth is achieved
by attaching to the cytoplasmic membrane of prey and hydrolyzing and taking up prey
biomolecules for Bdellovibrio cell synthesis. Bdellovibrio grow in a filamentous form
within their prey, and when nutrients are exhausted, they septate into individual cells,
which synthesize flagella and swim within the bdelloplast. Ultimately, the prey outer
membrane is lysed to release fresh, motile progeny Bdellovibrio. Their ability to prey
upon many disease-causing bacteria raises the interesting possibility that they may be
developed for use as biocontrol or therapeutic agents.
Routine laboratory maintenance of Bdellovibrio is carried out by growth on suitable
Gram-negative prey cells such as E. coli by either the double-layer overlay technique
(Rittenberg, 1982) or as prey lysates in buffer (Koval and Hynes, 1991). Alternatively,
a proportion of the cells of any given population of Bdellovibrio can grow axenically in
the absence of hosts (Seidler and Starr, 1969a). These are most commonly referred to as
host-independent (HI) strains and often have many properties differing from wild-type,
such as morphology (Fig. 7B.2.1) and they may or may not retain the ability to form
plaques on prey lawns.
Recently, molecular genetic techniques have been developed for the manipulation of
Bdellovibrio, and since the sequencing of the genome of B. bacteriovorus HD100, genes
can now be targeted for insertional inactivation or deletion (Lambert et al., 2003). Basic
Protocol 1 describes the standard methods of growing Bdellovibrio in suspensions of
prey E. coli. Basic Protocol 2 explains further the standard procedure for rescuing
Bdellovibrio from long-term storage. Basic Protocol 3 details growth of Bdellovibrio
as single plaques for enumeration, which is a technique used in many experiments to
determine the number of Bdellovibrio in a preparation. Basic Protocol 4 describes the
differential filtration technique to isolate HI derivatives. Basic Protocol 5 shows how
Delta
Proteobacteria
Current Protocols in Microbiology 7B.2.1-7B.2.13, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc07b02s9
C 2008 John Wiley & Sons, Inc.
Copyright
7B.2.1
Supplement 9
such strains are grown. Basic Protocol 6 gives a robust protocol for reliable storage of
Bdellovibrio. Basic Protocol 7 is a novel assay for observing phenotypic differences
between strains.
CAUTION: Bdellovibrio bacteriovorus and E. coli are Biosafety Level 1 (BSL-1) organisms. Such organisms are not known to consistently cause disease in healthy adult
humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these
organisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: Some prey cells are Biosafety Level 2 (BSL-2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms.
See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
BASIC
PROTOCOL 1
Materials
Laboratory
Maintenance of
Bdellovibrio
Prey cells (e.g., E. coli K12; refer to APPENDIX 4A for methods of culturing E. coli
K12)
1% YT agar plates (see recipe for YT agar)
YT broth (see recipe)
HEPES buffer supplemented with CaCl2 (see recipe)
Overnight culture of prey lysate
7B.2.2
Supplement 9
37 C static incubator
250-ml conical flasks or sterile universal tubes (Sterilin)
29 and 37 C shaking incubator
Phase contrast microscope with a 100 oil immersion lens
1. Streak prey cells out to produce single colonies on 1% YT agar plates and incubate
inverted overnight at 37 C.
These plates can be stored up to 2 weeks at 4 C.
4. Check by phase contrast microscopy after 24 hr, preferably with a 100 oil immersion lens, for the presence of bdellovibrios (usually highly motile) and the absence
of prey cells and bdelloplasts (indicating successful lysis of prey).
Prey lysates of this nature retain Bdellovibrio viability for up to 2 weeks if stored at 4 C.
BASIC
PROTOCOL 2
Materials
Prey cells (e.g., E. coli K12; refer to APPENDIX 4A for methods of culturing E. coli
K12)
1% YT agar plates (see recipe for YT broth)
YT broth (see recipe)
1% YPSC bottom agar (see recipe for YPSC broth)
25 g/liter CaCl2 2H2 O solution, sterile
0.6% YPSC top agar (see recipe for YPSC broth)
Bdellovibrio frozen stock (made from a previous prey lysate, see Basic Protocol 6)
HEPES buffer supplemented with CaCl2 (see recipe)
29 and 37 C static incubators
37 C shaking incubator
Test tubes
55 C water bath
Phase contrast microscope
Grow E. coli prey cells
1. Streak E. coli prey cells onto 1% YT agar plates and incubate inverted overnight at
37 C to produce single colonies.
These plates can be stored up to 2 weeks at 4 C.
Current Protocols in Microbiology
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4. To a test tube, add 100 l of the overnight culture of prey cells and 5 ml of 0.6%
YPSC top agar incubated at 55 C, with CaCl2 added. Quickly pour this mixture onto
the bottom plate and leave undisturbed for 5 min at room temperature to set.
The agar has to be at 55 C to ensure that it is completely molten so that the prey can be
adequately mixed in a room temperature tube before pouring. Therefore, the prey must be
able to withstand addition to molten agar at 55 C (as is the case for E. coli prey).
5. Spot 50 l of a Bdellovibrio frozen stock onto the center of this double layer plate
and incubate upright overnight at 29 C until a lawn of prey cells is observed.
6. Turn plate upside down and incubate for an additional 1 to 5 days at 29 C until
clearing of the prey lawn where the Bdellovibrio were spotted is observed.
Higher concentrations cause quicker clearing of the prey lawn.
7. Pick up some of the area of clearing from the soft agar layer using a 1000-l pipet
set to 250 l. Aseptically transfer this into a mini lysate containing 2 ml HEPES
buffer supplemented with CaCl2 and 150 l of overnight prey culture.
8. Incubate 1 to 2 days at 29 C with shaking at 200 rpm until lysate appears clear and
phase contrast microscopy reveals the presence of bdellovibrios and lysis of prey
cells.
If the Bdellovibrio are pipetted from an area of plate that is thoroughly cleared, then the
mini lysate should clear after an overnight (16 hr) incubation. If the plate has only partial
clearing, then the lysate may take longer to clear.
YPSC overlay plates can be stored up to 2 weeks at room temperature and the Bdellovibrio
within remain viable, more so than if stored at 4 C.
BASIC
PROTOCOL 3
Materials
Laboratory
Maintenance of
Bdellovibrio
Prey cells (e.g., E. coli K12; refer to APPENDIX 4A for methods of culturing E. coli
K12)
1% YT agar plates (see recipe for YT broth)
YT broth (see recipe)
1% YPSC bottom agar (see recipe for YPSC broth)
25 g/liter CaCl2 2H2 O solution, sterile
HEPES buffer supplemented with CaCl2 (see recipe)
7B.2.4
Supplement 9
Sample to be enumerated
0.6% YPSC top agar (see recipe for YPSC broth)
29 and 37 C static incubators
37 C shaking incubator
Test tubes
55 C water bath
Grow E. coli prey cells
1. Streak prey cells out onto 1% YT agar plates and incubate inverted overnight at 37 C
to produce single colonies.
These plates can be stored up to 2 weeks at 4 C.
4. Prepare ten-fold serial dilutions in HEPES buffer supplemented with CaCl2 of the
sample to be enumerated.
5. Add 100 l of these serial dilutions to separate test tubes with 100 l of an overnight
prey culture and then add 5 ml of 0.6% top agar YPSC incubated at 55 C, with
CaCl2 added to a final concentration of 0.25 g/liter. Quickly pour this mixture onto
the bottom plate and leave undisturbed for 5 min at room temperature to set.
6. Incubate plates inverted 3 to 10 days at 29 C until clear plaques appear in the lawn
of prey cells.
Typically, useful dilutions are 104 to 106 for a prey lysate that has completely cleared.
BASIC
PROTOCOL 4
Materials
Overnight prey lysate of host-dependent Bdellovibrio
PY agar (see recipe for PY broth)
PY broth (see recipe)
Phase contrast microscope
Petri dishes
0.45-m syringe filters
10-ml syringe
15-ml tubes (Falcon)
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Current Protocols in Microbiology
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Benchtop centrifuge
Plate spreaders
29 C static incubator
Bijou universal tubes (7-ml screw-top plastic tubes; Sterilin cat. no. 129B)
29 C shaking incubator
1. Grow a 50-ml prey lysate overnight (as in Basic Protocol 1) and check by phase
contrast microscopy for the presence of Bdellovibrio and complete prey lysis, i.e.,
the absence of prey cells and bdelloplasts.
2. Pour plates with 40 ml of 1% PY agar per petri dish and leave to set 10 min at
room temperature.
3. Filter 10 ml of prey lysate through a 0.45-m syringe filter attached to a 10-ml
syringe into a 15-ml tube.
4. Balance the tubes and centrifuge 10 min at 5000 g, room temperature, in a
benchtop centrifuge.
5. Pour off the supernatant and resuspend pellet in 100 l of PY broth.
6. Spread entire 100 l onto a PY plate and dry briefly by standing at room temperature
with lids slightly off (preferably in a laminar flow cabinet). Incubate inverted 3 to 10
days at 29 C.
7. Check on the following day for E. coli growth (imperfections in filters can sometimes
allow a few cells through).
Tiny yellow colonies will start to appear, which grow slowly over several days.
8. When colonies are >1 mm, pick into 0.5 ml PY broth in a bijou universal tube and
incubate at 29 C with shaking at 200 rpm.
Growth in broth can be achieved by scraping an area containing many small colonies
but such a culture would not be clonal and may have a number of plaquing (and other)
phenotypes.
9. In 1 to 3 days, as the culture grows (OD600 > 0.5), add more PY broth, doubling the
volume, then incubate overnight at 29 C with shaking at 200 rpm.
This process can be continued until the required culture volume has been reached for an
experiment. The culture should be incubated in a vessel at least five times the volume of
the liquid.
PY plates of HI cultures can be stored for up to 2 weeks at room temperature.
BASIC
PROTOCOL 5
Materials
PY agar (see recipe for PY broth)
Frozen stock of HI strain
PY broth (see recipe)
Laboratory
Maintenance of
Bdellovibrio
Petri dishes
29 C static incubator
Bijou universal tubes
7B.2.6
Supplement 9
Conical flasks
29 C shaking incubator
Grow from frozen stocks
1. Pour a petri plate with 50 ml 1% PY agar and leave to set 10 min at room
temperature.
2. Pipet 50 l of a thawed (5 min at room temperature) stock of HI strain onto the plate.
3. Streak repeatedly across the plate successively to obtain single colonies.
4. Incubate upright overnight at 29 C.
5. Incubate inverted for an additional 2 to 8 days at 29 C.
A large area of yellow growth will be observed within 2 to 4 days, and later single colonies
will be observed.
Grow in broth
6. When colonies are >1 mm, pick and place into 0.5 ml PY broth in a bijou universal
tube and incubate at 29 C with shaking at 200 rpm.
7. In 1 to 3 days as the culture grows (OD600 > 0.5), add more PY medium, doubling
the volume, then incubate overnight at 29 C with shaking at 200 rpm.
This process can be continued until the required volume has been reached for an experiment. The culture should be incubated in a vessel at least five times the volume of the
liquid.
BASIC
PROTOCOL 6
Materials
Culture of host-dependent Bdellovibrio
Culture of host-independent Bdellovibrio
Sterile 80% glycerol
Liquid nitrogen in suitable Dewar container
Phase contrast microscope
1.5-ml plastic freezer tubes (Nunc)
Perforated ladle/slotted spoon
80 C freezer containing freezer box
1. Check culture of host-dependent Bdellovibrio by phase contrast microscopy for
presence of bdellovibrios and absence of prey cells and bdelloplasts. Check hostindependent Bdellovibrio culture for presence of bdellovibrios and absence of contaminants.
2. Pipet 700 l of culture into 1.5-ml freezer tubes.
3. Add 150 l of 80% glycerol and cap tube. Mix well.
4. Drop into liquid nitrogen and leave until frozen (1 min). Remove tubes with
perforated ladle or slotted spoon.
5. Place in a precooled freezer box in a 80 C freezer.
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Current Protocols in Microbiology
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The stock can withstand several freeze-thaw cycles, but it is advisable to have many stocks
and not to access one stock more than three times to ensure growth.
It is important to prepare many frozen stocks of recently isolated HI strains, as upon
repeated subculturing they have been demonstrated to lose their ability to infect prey cells.
BASIC
PROTOCOL 7
Materials
Bdellovibrio mutant strains to be tested (usually kanamycin resistant due to
insertional inactivation of a gene under study)
109JK control strain
Luminescent E. coli S17-1 (pCL100, kanamycin resistant) (Lambert et al., 2003)
E. coli S17-1 (pZMR100) (Rogers et al., 1986)
YT agar (see recipe for YT broth)
YPSC agar (see recipes for both top and bottom agar)
HEPES buffer supplemented with CaCl2 (see recipe)
YT broth (see recipe)
PY broth (see recipe)
50 mg/ml kanamycin sulfate
Petri dishes
55 C and boiling water baths
1.5-ml microcentrifuge tubes
Multichannel pipettor
96-well microtiter plates for luminometer
Photoluminometer capable of reading a kinetic assay of luminescence over time in
a 96-well microtiter well and with temperature control and shaking (e.g., Anthos
Lucy, Perkin-Elmer Instruments Victor, BMG Labtechnology Fluostar Optima,
Tecan Genios)
Grow bacterial strains
1. Grow Bdellovibrio mutant strains to be tested and the 109JK control strain for 3 days
on luminescent E. coli S17-1 (pCL100) (Lambert et al., 2003), subculturing at the
same time each day. On the final day, also grow E. coli S17-1 (pZMR100) (Rogers
et al., 1986) to use to enumerate the Bdellovibrio.
Prepare plates and bacteria
2. Pour three 1% YT agar plates and three YPSC bottom agar plates supplemented with
50 g/ml kanamycin sulfate per Bdellovibrio strain tested and melt the appropriate
amount (5 ml per plate) of YPSC top agar, maintain melted agar at 55 C.
3. Prepare controls by dispensing 1 ml of each Bdellovibrio strain to be tested into
1.5-ml microcentrifuge tubes and boil for 5 min, then leave to cool to room temperature for at least 10 min.
Laboratory
Maintenance of
Bdellovibrio
7B.2.8
Supplement 9
Enumerate bacteria
9. Immediately after setting the plate for incubation, enumerate samples of each of the
Bdellovibrio strains used in the assay plate by plating at dilutions between 104 and
106 onto YPSC overlays, incubate 3 to 10 days at 30 C and then count plaques.
10. Enumerate the E. coli S17-1 (pCL100) in the PY broth and HEPES supplemented
with CaCl2 mix by plating dilutions between 104 and 106 onto YT plates and
incubate overnight at 37 C.
Analyze data
11. Analyze data by plotting log10 luminescence against time.
From this plot, the time taken to reduce to half of the maximum log10 luminescence value
for each amount of Bdellovibrio is calculated and this is plotted against the amount of
Bdellovibrio initially added as calculated by the plaque overlay enumeration (Fig. 7B.2.3).
If a mutant strain is reduced in its efficiency of predation relative to the 109JK control,
then this should be shown on this plot as the line of best fit is shifted (Fig. 7B.2.3).
Figure 7B.2.2 Plot showing luminescence (in relative luminescent units) against time (in steps of
30 min; i.e., 1 =30 min, 2 = 60 min, etc.) for an infection of Bdellovibrio on luminescent E. coli. After
an initial rise in luminescence, more bdellovibrios added results in faster diminution of light. The
suspension added contains 1 105 viable bdellovibrios as determined by plaque enumeration.
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Current Protocols in Microbiology
Supplement 9
Figure 7B.2.3 Plot showing log10 numbers of bdellovibrios initially added against time to half
log10 luminescence values comparing two strains; a wild type 109J and a mutant MCP strain
(Lambert et al., 2003).
Laboratory
Maintenance of
Bdellovibrio
7B.2.10
Supplement 9
YT broth
5 g/liter sodium chloride
5 g/liter peptone
8 g/liter tryptone
Adjust to pH 7.5 using 2 M NaOH
Sterilize by autoclaving
Store up to 6 months at room temperature
For YT agar, add 10 g/liter agar
COMMENTARY
Background Information
Bdellovibrio bacteriovorus is a highly
motile delta-proteobacterium that preys on
other Gram-negative bacteria (Stolp and Starr,
1963). Bdellovibrio are ubiquitous in nature,
having been discovered in a wide variety of
environments that include both aquatic and
terrestrial habitats as well as mammalian intestines (Varon and Shilo, 1980). Their prey
includes plant, animal, and human pathogens
(Martin, 2002). Thus, studying the molecular
mechanisms of Bdellovibrio predation offers
cues for the design of antibacterial agents.
Critical Parameters
One important consideration when setting up experiments with Bdellovibrio is the
physiological state of the prey lysate. As
Bdellovibrio cycle through attack phase and
intraperiplasmic growth, and then release into
attack phase again, they undergo many different developmental and metabolic states and
this could have a drastic effect on the outcome
of any given experiment. Similarly, newly released attack phase cells are very different
from cells from older lysates (more rapidly
motile and often smaller). To avoid fluctuations between experiments, it is essential to
subculture at exactly specified times over a
period of several days (e.g., every 24 hr for 3
days). It may also be vital to subculture for several rounds on a new prey, or under new conditions, before using the Bdellovibrio progeny
for an experiment.
Host-independent Bdellovibrio appear to
have an as yet unquantified reliance upon cell
density, for example, small colonies seem to
appear on plates mainly in high density and a
relatively high inoculum per volume is necessary for consistent growth.
For culturing, all media and solutions
should be sterilized by autoclaving and it is
important to work with good aseptic technique. If possible, work should be carried
out in a Class II biohazard fume hood to
Troubleshooting
Table 7B.2.1 presents commonly encountered difficulties in maintaining Bdellovibrio,
as well as the potential causes and possible
solutions.
Anticipated Results
For the culture of host-dependent
Bdellovibrio, overlay plates should develop a
large region of clearing where the Bdellovibrio
has been spotted onto a cloudy lawn of prey
cells, or individual plaques where dilutions
have been used. These plaques start out as
small and cloudy (2 to 4 days) and become
clearer and larger over several days (3 to 10
days) incubation.
Culture of Bdellovibrio in prey lysates
should result in the clearing of the cloudy prey
lysate over 1 to 2 days and phase contrast
microscopy should reveal prey cells turning
into bdelloplasts (rounded prey cells containing Bdellovibrio within), then lysing to release
small, highly motile attack phase Bdellovibrio
cells.
Culture and generation of HI Bdellovibrio
should produce initially (2 to 4 days) very
small, yellow-orange colonies on PY plates,
which grow slowly over time (3 to 10 days).
Similarly, growth in broth produces yelloworange coloration. Phase contrast microscopy
should show, in addition to some small, motile
attack phase Bdellovibrio, a very heterogeneous mix of cell morphology and size. A
much smaller proportion of HI cells tend to
be motile and it is not unusual to see no swimming cells at all (Fig. 7B.2.1).
Data from the predation assay on luminescent prey should show light emissions initially
increasing as the prey cells grow in the nutrients, followed by the levels slowly lowering in
the control with no live Bdellovibrio. For each
well containing Bdellovibrio, the light levels
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Current Protocols in Microbiology
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Laboratory
Maintenance of
Bdellovibrio
Problem
Cause
Solution
Environmental/skin bacteria
Frozen stock has been through Use fresh frozen stocks with
too many freeze/thaw cycles fewer than four freeze-thaw
cycles
CaCl2 missing
Unknown
Literature Cited
Time Considerations
7B.2.12
Supplement 9
Seidler, R.J. and Starr, M.P. 1969a. Isolation and characterisation of host-independent
bdellovibrios. J. Bacteriol. 100:769-785.
Seidler, R.J. and Starr, M.P. 1969b. Factors affecting the growth of Bdellovibrio bacteriovorus
developing within E. coli. J. Bacteriol. 97:912923.
Stolp, H. and Starr, M.P. 1963. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic,
and bacteriolytic microorganism. Antonie Van
Leeuwenhoek 29:217-248.
Tudor, J.J., McCann, M.P., and Acrich, I.A. 1990.
A new model for the penetration of prey cells by
bdellovibrios. J. Bacteriol. 172:2421-2426.
Varon, M. and Shilo, M. 1980. Ecology of aquatic
bdellovibrios. In Advances in Aquatic Microbiology, vol. 2. (M.R. Droop and H.W. Jannesch,
eds.) pp. 1-41. Academic Press, London.
Key References
Beck, S., Schwudke, D., Strauch, E., Appel, B.,
and Linscheid, M. 2004. Bdellovibrio bacteriovorus strains produce a novel major outer
membrane protein during predacious growth in
the periplasm of prey bacteria. J. Bacteriol.
186:2766-2773.
Key paper that refutes earlier work on outermembrane protein transfer from predator to prey.
Cotter, T.W. and Thomashow, M.F. 1992. Identification of a Bdellovibrio bacteriovorus genetic
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UNIT 8A.1
ABSTRACT
Campylobacter jejuni is a fastidious organism, growing in microaerophilic conditions
with a temperature range between 37 and 42 C. Multiple types of media can be used to
cultivate it; however, Mueller Hinton broth and agar support the best C. jejuni growth.
Optimum atmosphere for C. jejuni is 85% N2 , 10% CO2 , and 5% O2 . Curr. Protoc.
C 2008 by John Wiley & Sons, Inc.
Microbiol. 10:8A.1.1-8A.1.7.
Keywords: campylobacteriosis r microaerophilic growth r Mueller Hinton
INTRODUCTION
Campylobacter jejuni is one of the major causes of bacterial gastroenteritis worldwide and
is primarily acquired through the ingestion of contaminated poultry products. Research
on C. jejuni has been greatly impaired due to poor culturing techniques and genetic
tools. C. jejuni was successfully isolated in the early 1970s, utilizing specialty agar and
microaerophilic atmospheric conditions. Since its isolation, it has been connected with
bacterial gastroenteritis as well as the neurological disorders Guillen-Barre Syndrome
and Fisher Syndrome in humans. It is now one of the leading causes of gastroenteritis in
both the developed and developing worlds. (Young et al., 2007).
C. jejuni is a fastidious organism, requiring modified atmospheric conditions, a longer
growing time, a narrow temperature range, and specialized media when compared to
such bacteria as E. coli. All these factors must be taken into consideration when working
with C. jejuni.
STRATEGIC PLANNING
Atmosphere
Campylobacter jejuni requires microaerophilic conditions for growth. Optimum growth
is maintained in a tri-gas incubator (e.g., Thermo Forma Series II Water-Jacketed CO2
Incubator), with 85% N2 , 10% CO2 , and 5% O2 . If a tri-gas incubator is unavailable,
specific gas packs (i.e., BBL Campy Pak Plus) or formulated compressed air can be
applied with sealable gas chambers or plastic bags, respectively. Gas packs are expensive,
and growth in plastic bags is suboptimal compared to that obtained by using a tri-gas
incubator. Notes concerning the differences are in the specific protocols outlined. C.
jejuni is also able to grow in anaerobic conditions.
Temperature
C. jejuni has a narrow temperature range of growth, with optimum growth occurring
between 37 and 42 C. The bacteria are able to survive at 7 C and perform vital cellular
processes such as protein synthesis; however, growth and recovery at low temperatures
Epsilon
Proteobacteria
Current Protocols in Microbiology 8A.1.1-8A.1.7, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc08a01s10
C 2008 John Wiley & Sons, Inc.
Copyright
8A.1.1
Supplement 10
is difficult (Hazeleger et al., 1998). Significant loss of viable bacteria occurs when
the bacteria are left at room temperature and atmosphere for only 10 min. Therefore,
when working with C. jejuni, the amount of time the bacteria are out of incubators and
microaerophilic atmosphere must be limited.
Media
Many types of media have been used to culture C. jejuni. Mueller-Hinton medium, blood
agar, Columbia blood agar, and BBL medium are commonly used. However, Mueller
Hinton (MH) has the highest recovery rate and is recommended in this unit (Ng et al.,
1985). Campylobacter defined medium was developed by Leach and colleagues (Leach,
1997). Although a minimal medium has not been developed that supports C. jejuni
growth, the defined medium can be used to adjust components based on experimental
needs.
Growth Conditions
C. jejuni can grow in both static and shaking broth cultures. The stringent growth
conditions do not lend themselves well to a shaking apparatus. Some tri-gas incubators
are equipped to have an internal shaker; however, most are not, and the gas packs in
bags or jars do not always allow shaking conditions. C. jejuni grown in shaking cultures
grow faster than those in static culture. Aggregates form when C. jejuni is grown in static
broth (Joshua et al., 2006). This aggregation phenotype has been studied extensively, and
several genes involved in this have been identified. Based on availability, the protocols
suggested in this unit use static growth as the standard.
Strain Selection
Several isolates of C. jejuni have been used in previous research (Table 8A.1.1). Strain
selection is important, and depends upon the proposed research. Strains have been isolated from chicken, human, and environmental sources. Phenotypic variation among
strains from varying sources has been observed for many traits including invasion, cytolethal distending toxin (cdt) production, chick colonization, lipooligosaccharide (LOS)
Table 8A.1.1 Most Commonly Used C. jejuni Strainsa
Sources
Sequences
260.94
Human
480
Human
811681116
Human
Lab strain
11168
Human
Gaynor (2004)
81-176
Human
BTI
Chicken
CG8245
Human
F38011
Human
Environment
Human
M1
RM1221
Notes
a The most commonly used isolates of C. jejuni are listed here, including their source of isolation and whether their
Laboratory
Maintenance of
C. jejuni
genome has been sequenced to date. Strains can be acquired from individual labs working on the particular isolate.
b GBS, Guillain-Barre Syndrome.
8A.1.2
Supplement 10
production, and natural transformation. The ability of C. jejuni to undergo natural transformation has led to a high degree of horizontal gene transfer between C. jejuni strains,
leading to high genetic diversity among C. jejuni strains. C. jejuni strains 81-176 and
11168 are well characterized strains, and the most commonly used in pathogenesis
studies.
Growth Curves
Mueller Hinton agar is the recommended medium for standard growth curves of C. jejuni.
Biphasic MH medium, prepared in tissue culture flasks (Fig. 8A.1.1), is the classic way
to perform growth curves. The biphasic medium is made using 50% MH agar and 50%
MH (poured after the agar solidifies). Note that MH agar should never be microwaved
for C. jejuni agar plates and biphasic media.
Biphasic medium is recommended for growing conditions outside of a tri-gas incubator,
such as in bags or sealed containers. If a tri-gas incubator is used, growth curves can be
performed in MH broth alone. However, no volume below 1 ml should be used, due to
poor recovery and inconsistent growth.
Each time point should be an individual sample. Static growth of C. jejuni results in
aggregative bacterial raft formation, and disruption of this growth has led to variable and
inconsistent growth. Therefore, each time-point should be separate and undisturbed. Furthermore, each time-point should be done in triplicate to obtain a statistically significant
average. Growth curves are normally performed for 36 or 48 hr.
The inoculum for a growth curve is 1 106 cfu/ml. From 16- to 18-hr plates, C. jejuni
should be resuspended to an OD600 of 0.4. Dilute this suspension 1:10. Inoculate 80 l
of this dilution into 20 ml of medium. A standard growth curve, measured in cfu/ml over
48 hr, is shown in Figure 8A.1.2.
CAUTION: Campylobacter jejuni is a Biosafety Level 2 (BSL-2) pathogen. Follow
appropriate guidelines and regulations for the handling of pathogenic microorganisms.
Proper hand washing is essential, as C. jejuni has been shown to cause gastroenteritis at
a small dose (10 to 100 organisms). See UNIT 1A.1 and other pertinent resources for more
information.
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Current Protocols in Microbiology
Supplement 10
1011
1010
109
cfu/ml
108
107
81-176
mutant
106
105
104
103
102
0
Figure 8A.1.2
BASIC
PROTOCOL 1
12
18
24
30
Time (hr)
36
42
48
Materials
C. jejuni frozen stock (Basic Protocol 2)
100 15mm Mueller Hinton (MH) agar plates (BD Biosciences, cat. no. 22520;
plates are poured in lab) containing 10 g/ml trimethoprim (antibiotics added in
lab)
Mueller Hinton (MH) broth (BD Biosciences, cat. no. 275730) containing
10 g/ml trimethoprim
Equipment for maintaining Campylobacter-specific microaerophilic atmosphere
(see Strategic Planning)
Additional reagents and equipment for streaking bacteria (APPENDIX 4A)
1. From frozen stock (in MH broth plus 20% glycerol; see Basic Protocol 2), heavily
streak out C. jejuni strain on 100 15mm Mueller Hinton agar plates containing
10 g/ml trimethoprim. Grow for 16 to 20 hr in microaerophilic conditions (see
Strategic Planning).
Growth in a tri-gas incubator allows more rapid growth, while bag plating may require
up to 48 hr of growth. See Strategic Planning for additional discussion of atmospheric
conditions required for growing C. jejuni.
Laboratory
Maintenance of
C. jejuni
8A.1.4
Supplement 10
Antibiotic
Concentration
Kanamycin
50 g/ml
Chloramphenicol
15-20 g/ml
Streptomycin
Trimethoprim
10 g/ml
Cefoperazone
20 g/ml
Nalidixic acid
30 g/ml
Tetracycline
12.5 g/ml
Figure 8A.1.3 Growth of C. jejuni 81-176 on an Mueller Hinton agar plate containing 10 g/ml
trimethoprim after 24 hr in a tri-gas incubator.
2. Restreak C. jejuni onto a new Mueller Hinton plate containing 10 g/ml trimethoprim. Grow for 16 to 20 hr in microaerophilic conditions.
Single colonies will not be observed until culture has been incubated >24 hr. Scrape up
a large amount of cells from the lawn (see Figure 8A.1.3).
After this incubation, C. jejuni is ready to be used.
For single colonies, streak for isolated colonies on plates and grow for 48 hr or until
individual colonies are observed.
3. Optional: If large amounts are needed, restreak a heavy inoculum onto several
Mueller Hinton plates containing 10 g/ml trimethoprim or inoculate into Mueller
Hinton broth containing 10 g/ml trimethoprim.
4. Optional: Passage C. jejuni an additional two to three times.
Further passages from frozen stock are not recommended.
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Current Protocols in Microbiology
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BASIC
PROTOCOL 2
PRESERVATION OF C. JEJUNI
Stocks of C. jejuni should be kept in 20% glycerol stocks, stored at 80 C. Incubations
can be performed in a tri-gas incubator or in the other recommended conditions at 37 C.
Materials
C. jejuni organisms (cannot be purchased from ATCC or similar sources; must be
obtained from individual labs)
100 15mm Mueller Hinton (MH) agar plates (BD Biosciences, cat. no. 22520;
plates are poured in lab) containing 10 g/ml trimethoprim (antibiotics added in
lab)
Mueller Hinton (MH) broth (BD Biosciences, cat. no. 275730) containing 20%
(v/v) glycerol
Sterile cotton swabs
2-ml cryotubes
1. Heavily streak out C. jejuni strain on an MH agar plate containing 10 g/ml trimethoprim. Grow for 18 to 20 hr.
After making a mutant (see UNIT 8A.2), streak for single colonies, which takes 36 to 48 hr.
Pick a single colony and streak out. Finally, restreak it heavily onto a MH agar plate and
carry out the following steps to preserve.
4. Never thaw frozen stocks. Instead, scrape off an ice chip from the frozen stock using
a sterile implement and plate directly onto MH agar plates containing 10 g/ml
trimethoprim.
COMMENTARY
Background Information
Laboratory
Maintenance of
C. jejuni
8A.1.6
Supplement 10
Anticipated Results
Literature Cited
Time Considerations
Following the protocol, large amounts of
viable C. jejuni will be available after 36 to 48
hr in a tri-gas incubator. If gas jars are used,
growth will be slower, with usable growth appearing after 40 to 48 hr, depending on the
amount being streaked.
Freezing a newly constructed C. jejuni
strain may take 4 to 5 days. Streak from a
single colony. Growth will be visible after
48 hr. Heavily restreak the growth onto a new
MH agar plate and incubate for another 24 to
36 hr.
Epsilon
Proteobacteria
8A.1.7
Current Protocols in Microbiology
Supplement 10
UNIT 8A.2
ABSTRACT
Molecular manipulation has been a limiting factor in C. jejuni research for many years.
Recent advances in molecular techniques adapted for C. jejuni have furthered our understanding of the organism. This unit is dedicated to common molecular tools in bacterial
research specifically tailored for C. jejuni. These include colony PCR, DNA isolation,
and RNA isolation. The unit also reviews techniques for genetic manipulation, such as
the use of plasmids, natural transformation, electroporation, conjugation, and transposition. In addition, a reporter system, the arylsulfatase assay, can be used to study gene
C 2008 by John Wiley & Sons,
expression. Curr. Protoc. Microbiol. 10:8A.2.1-8A.2.17.
Inc.
Keywords: natural transformation r arylsulfatase r genomic DNA r RNA isolation
INTRODUCTION
Molecular biology has been a limiting factor in C. jejuni research. Indigenous plasmids
are commonly used for cloning and expression vectors. C. jejuni is most efficiently
transformed by plasmid DNA from its own species, thereby limiting the ease of genetic
manipulation. Mutations are produced in C. jejuni using allelic replacement via suicide
plasmids. Because these methods are described elsewhere (see Hendrixson et al., 2001,
for a strategy that works well for C. jejuni), we will not discuss them in this unit, which
will focus on moving plasmids into C. jejuni by conjugation (Basic Protocol 1), electroporation (Basic Protocol 2), and natural transformation (Basic Protocol 3). Numerous
plasmids have been developed for C. jejuni research and may be used in lieu of specific
plasmids described here. However, those discussed (see Table 8A.2.1) are commonly
used and have proven successful in many studies. An inducible expression plasmid has
yet to be developed for C. jejuni.
Table 8A.2.1 Commonly Used Plasmids
Stable in
Source
C. jejuni
Notes
Antibiotic
resistance
Ampicillin
Mobilizable with
pRK212.1?
Source
pUC19
E. coli
pRY108
Kanamycin
pRY112
Chloramphenicol
pECO102
pRY112
Chloramphenicol
Wiesner et al.
(2003)
Replicating plasmid
Epsilon
Proteobacteria
Current Protocols in Microbiology 8A.2.1-8A.2.17, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc08a02s10
C 2008 John Wiley & Sons, Inc.
Copyright
8A.2.1
Supplement 10
This unit also outlines several common molecular techniques for bacterial studies that
have been adapted for C. jejuni. Common techniques include transposition (Basic Protocol 4), colony PCR (Basic Protocol 5), isolation of DNA (Basic Protocol 6) and RNA
(Basic Protocol 7 and Alternate Protocol), and an arylsulfatase assay used as a reporter
for C. jejuni gene expression studies (Basic Protocol 8).
The sequencing of genomes of several C. jejuni strains, as well as the development of
a variety of genetic tools for use in C. jejuni, have provided the opportunity for rapid
expansion in the knowledge of the biology of this organism. The protocols outlined in
this unit, combined with genome sequence data, can be used to make and complement
in-frame deletions, create libraries of transposon mutants, and study gene expression
using gene fusions and techniques such as microarrays and qRT-PCR.
CAUTION: Campylobacter jejuni is a Biosafety Level 2 (BSL-2) pathogen. Follow
appropriate guidelines and regulation for the handling of pathogenic microorganisms.
Proper hand washing is essential because C. jejuni has been shown to cause gastroenteritis
at a small dose (10 to 100 organisms). See UNIT 1A.1 and other pertinent resources for
more information.
NOTE: Incubations are performed at 37 C in a tri-gas incubator, as recommended in
UNIT 8A.1.
BASIC
PROTOCOL 1
Materials
DNA fragment of interest
C. jejuni plasmid (e.g., pRY108 or pECO101; also see Table 8A.2.1)
Donor strain: E. coli DH5 [pRK212.1]
LB agar plates with appropriate selective antibiotics (APPENDIX 4A)
Recipient strain: C. jejuni (UNIT 8A.1)
Mueller Hinton (MH) agar plates, with and without appropriate selective
antibiotics (BD Biosciences)
LB liquid medium with appropriate selective antibiotics (APPENDIX 4A)
Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
Additional reagents and equipment for cloning of DNA (Struhl, 2000), introduction
of plasmid DNA into E. coli (Seidman et al., 1997), growth of E. coli on solid
medium (Elbing and Brent, 2002a) and in liquid medium (Elbing and Brent,
2002b), and growth of C. jejuni (UNIT 8A.1)
Construct E. coli donor strain
1. Clone fragment to be maintained in C. jejuni plasmid (i.e., pRY108, pECO101).
Struhl (2000) contains protocols for cloning of DNA fragments.
Seidman et al. (1997) describe methods for introducing plasmid DNA into E. coli cells.
8A.2.2
Supplement 10
3. Grow the transformed E. coli (Elbing and Brent, 2002a) on LB plates containing
selection antibiotics: ampicillin (100 g/ml), tetracycline (12.5 g/ml), and plasmidspecific antibiotic (e.g., kanamycin, chloramphenicol).
Chloramphenicol would be used with pECO102.
5. Streak C. jejuni recipient strain onto MH plate with appropriate antibiotics for 16 to
20 hr at 37 C in microaerophilic conditions (UNIT 8A.2). Start an overnight culture of
the E. coli donor strain in LB liquid medium with appropriate antibiotics.
6. Inoculate 5 ml LB liquid medium containing selective antibiotic for the plasmid with
375 l of the overnight culture of the donor strain prepared in step 5. Incubate at
37 C while shaking until OD600 = 0.45 to 0.5.
7. Resuspend recipient C. jejuni strain from the 16- to 20-hr plates (step 5) in 1.8 ml
MH broth without antibiotics to an OD of 1.0.
8. Wash 0.5 ml of the donor strain culture from step 6 once by microcentrifuging
5 min at 10,000 g, 4 C, removing the supernatant, adding 10 ml MH broth
without antibiotics, then centrifuging again as before and removing the supernatant
to eliminate any antibiotics. Resuspend donor pellet with 1 ml of C. jejuni recipient
strain suspension.
9. Spin bacterial mixture for 2 min at full speed in microcentrifuge, and remove supernatant.
10. Resuspend cells in 100 l MH broth without antibiotics. Spot onto an MH plate
without any antibiotics.
11. Incubate plate organism-side-up at 37 C in microaerophilic conditions (UNIT 8A.1) for
5 hr.
12. Resuspend bacteria from plate in 1.8 ml MH broth. Pellet bacteria by centrifuging
5 min at 10,000 g, 4 C, and removing the supernatant.
13. Spread bacteria on MH agar with 10 g/ml trimethoprim (TMP), 20 g/ml cefoperazone, 2 mg/ml streptomycin, and an antibiotic for selection of plasmid.
14. Incubate for 4 to 5 days at 37 C in microaerophilic conditions (UNIT 8A.1).
ELECTROPORATION OF C. JEJUNI
Electroporation of bacteria involves the application of electrical current to the bacterial
cells, which permeabilizes the membrane, allowing uptake of DNA from the environment.
This technique is primarily used for the incorporation of plasmid DNA. This technique
was first described for C. jejuni by Miller et al. (1988). Electroporation of C. jejuni
is primarily done to incorporate DNA from suicide plasmids into the chromosome of
C. jejuni. Electroporation is useful for this because plasmids derived from E. coli will
not transform well into C. jejuni by natural transformation.
BASIC
PROTOCOL 2
Epsilon
Proteobacteria
8A.2.3
Current Protocols in Microbiology
Supplement 10
Materials
C. jejuni frozen stock (UNIT 8A.1)
Mueller Hinton (MH) agar plates with appropriate selective antibiotics (BD
Biosciences)
Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
Wash buffer: 15% (v/v) glycerol/9% (w/v) sucrose
DNA of interest, to be electroporated into C. jejuni (15 g DNA/reaction)
SOC medium (see recipe)
Nitrocellulose membrane (0.025-m pore size VSWP (Millipore, cat. no.
VSWP04700)
Electroporation cuvettes (BioRad Gene Pulser Cuvettes, 0.2-cm electrode; cat. no.
165-2006)
Electroporator (BioRad E. coli Pulser)
Additional reagents and equipment for streaking bacteria (APPENDIX 4A) and growing
C. jejuni (UNIT 8A.1)
Grow C. jejuni
1. Streak strain (APPENDIX 4A) from frozen stock onto one MH agar plate containing
10 g/ml trimethoprim (TMP). Grow for 24 hr at 37 C under microaerophilic conditions (UNIT 8A.1).
2. Restreak strain onto one MH agar plate containing 10 g/ml TMP, using a heavy
inoculum. Grow for 16 hr at 37 C under microaerophilic conditions (UNIT 8A.1).
Perform electroporation
9. For large volumes (>5 l) of DNA that have been prepared in TE buffer, dialyze
DNA on top of a nitrocellulose membrane floating in 20 ml distilled deionized water
for 20 min.
10. Cool electroporation cuvettes on ice.
11. In a microcentrifuge tube, combine 50 l electrocompetent bacteria (from step 8)
and 15 g DNA. Keep on ice.
12. Transfer bacteria-DNA mix to an electroporation cuvette. Electroporate the sample
at 2.5 kV, 200 , and 25 F.
The time constant is usually 5 msec.
13. Flush cuvette with 100 l SOC medium. Spread bacteria onto MH agar plate containing 10 g/ml TMP.
Genetic
Manipulation of
C. jejuni
8A.2.4
Supplement 10
15. Harvest bacteria by resuspending in 1.8 ml of MH broth without antibiotics. Microcentrifuge bacteria for 2 min at maximum speed and remove supernatant. Resuspend
bacteria in 100 l MH broth. Plate onto MH agar containing the appropriate selective
antibiotics.
16. Incubate for 2 to 4 days at 37 C in microaerophilic atmosphere (UNIT 8A.1).
BASIC
PROTOCOL 3
Materials
C. jejuni frozen stock (UNIT 8A.1)
Mueller Hinton (MH) agar plates with and without appropriate selective antibiotics
(BD Biosciences)
Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
DNA of interest, to be transformed into C. jejuni (C. jejuni genomic DNA prepared
as in Basic Protocol 6)
Additional reagents and equipment for streaking bacteria (APPENDIX 4A) and growing
C. jejuni (UNIT 8A.1)
Grow C. jejuni
1. From frozen stock, streak strain (APPENDIX 4A) onto MH agar containing 10 g/ml
trimethoprim (TMP). Grow for 16 to 24 hr at 37 C under microaerophilic conditions
(UNIT 8A.1).
2. From the 16 to 24 hr growth plate, streak strain onto MH agar containing 10 g/ml
TMP, using a heavy inoculum. Streak plates 16 hr before the start of the transformation experiment.
3. Make fresh MH agar (without antibiotics) and pipet 1-ml aliquots into plastic tubes.
4. Resuspend the entire growth from the 16-hr plate (from step 2) in 1 ml MH broth
(without antibiotics).
Perform transformation
5. Dilute the bacteria to an OD600 of 0.5 (1 109 cfu/ml) with MH broth (without
antibiotics).
6. Add 0.5 ml resuspended bacteria to each tube (from step 3) containing 1 ml of
solidified MH agar. Incubate tubes for 3 hr at 37 C in microaerophilic conditions
(UNIT 8A.1).
7. Gently mix tubes.
8. Add 5 to 10 g of the DNA to be transformed to each tube and pipet up and down.
9. Incubate for 4 hr at 37 C in microaerophilic conditions (UNIT 8A.1).
10. Transfer bacteria into a microcentrifuge tube. Microcentrifuge for 2 min at maximum
speed, room temperature.
11. Resuspend bacteria and plate onto MH agar containing 10 g/ml TMP and appropriate selection antibiotics.
12. Incubate plates at 37 C in microaerophilic environment (UNIT 8A.1) for 2 to 4 days.
Epsilon
Proteobacteria
8A.2.5
Current Protocols in Microbiology
Supplement 10
BASIC
PROTOCOL 4
Materials
5 salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA)
100 mM dithiothreitol (DTT)
Donor DNA (containing transposon)
Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA
prepared as in Basic Protocol 6)
Transposase: (available from Dr. David Lampe (Lampe et al., 1999)
TE buffer, pH 8.0 (APPENDIX 2A)
dNTP mix: 1.25 mM each dNTP (APPENDIX 2A)
T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen)
T4 ligase and T4 ligase buffer (Invitrogen)
30 and 70 C water baths and 11 and 16 C recirculating water baths (or thermal
cycler)
Nitrocellulose membrane (0.025 m pore size VSWP (Millipore, cat. no.
VSWP04700)
Additional reagents and equipment for phenol:chloroform extraction and ethanol
precipitation of DNA (Moore and Dowhan, 2002)
Perform transposition reaction
1. Prepare transposition reaction by combining the following:
16.0 l 5 salt buffer
2.0 l 10 mg/ml BSA
1.6 l 100 mM DTT
1.0 g donor DNA
2.0 g recipient DNA
500 ng transposase
Distilled deionized H2 O to 80 l
Incubate reactions at 30 C for 4 hr.
Table 8A.2.2 Transposons
Plasmid
Transposon
Transposase
HImar1
Himar1
picard
Himar1
pFalcon
Solo
Himar1
pOTHM
Mariner
Himar1
pFD1
pEnterprise
Genetic
Manipulation of
C. jejuni
Reference
8A.2.6
Supplement 10
Purify DNA
2. Extract DNA once with phenol:chloroform, then ethanol precipitate DNA (Moore
and Dowhan, 2002).
3. Microcentrifuge DNA 30 min at maximum speed, 4 C.
4. Remove supernatant and resuspend pellet in 40 l TE buffer, pH 8.0.
BASIC
PROTOCOL 5
Materials
Proteinase K
K buffer (see recipe)
100 C water bath
Additional reagents and equipment for PCR (Kramer and Coen, 2001)
1. Add proteinase K to a final concentration of 100 l/ml in K buffer immediately
before use.
Epsilon
Proteobacteria
8A.2.7
Current Protocols in Microbiology
Supplement 10
2. Resuspend one or two isolated colonies in this 100 l of K buffer containing proteinase K, using a toothpick or a similar implement.
Spot the individual colonies onto a fresh plate of MH agar containing 10 g/ml TMP
before resuspending in K buffer and keep in microaerophilic conditions at 37 C to preserve
the colonies. Individual colonies will take 3 to 5 days to grow on MH agar containing
10 g/ml TMP in a tri-gas incubator. See UNIT 8A.1 for further information.
Materials
C. jejuni frozen stock (UNIT 8A.1)
Mueller Hinton (MH) agar plates (BD Biosciences) with 10 g/ml trimethoprim
(TMP)
Phosphate-buffered saline (PBS; APPENDIX 2A)
10 mM TrisCl, pH 8.0 (APPENDIX 2A)/0.1 mM EDTA
10 mM TrisCl, pH 8.0 (APPENDIX 2A)/0.1 mM EDTA containing 1 mg/ml lysozyme
Proteinase K
20% (w/v) SDS (APPENDIX 2A)
Buffered phenol (APPENDIX 2A)
24:1 chloroform:isoamyl alcohol
7.5 M ammonium acetate
Isopropanol
70% (v/v) ethanol
1 mM TrisCl, pH 7.5 (APPENDIX 2A)/0.1 mM EDTA
Filter paper (Whatman)
Additional reagents and equipment for growing C. jejuni (UNIT 8A.1) and
phenol/chloroform extraction of DNA (Moore and Dowhan, 2002)
Days 1 and 2: Grow C. jejuni
1. From a frozen stock, streak strain onto MH with 10 g/ml trimethoprim (TMP) agar.
Grow for 16 to 24 hr at 37 C under microaerophilic conditions (UNIT 8A.1).
2. From the 16- to 24-hr growth plate incubated in step 1, restreak strain onto one plate
of MH agar containing 10 g/ml TMP, using a heavy inoculum. Incubate 18 hr at
37 C under microaerophilic conditions (UNIT 8A.1).
8A.2.8
Supplement 10
Materials
C. jejuni frozen stock (UNIT 8A.1)
Mueller Hinton (MH) agar plates (BD Biosciences) with 10 g/ml trimethoprim
(TMP)
Mueller Hinton (MH) broth (BD Biosciences) with 10 g/ml trimethoprim (TMP)
RNA Protect Bacteria Reagent (Qiagen)
TE buffer, pH 8.0 (APPENDIX 2A) containing 1 mg/ml lysozyme
RNeasy kit (Qiagen) including:
Buffer RLT
Buffer RW1
Buffer RPE
RNA Mini Spin columns
2-ml collection tubes
RNase-free H2 O
2-mercaptoethanol (2-ME)
100% ethanol, ice cold
10 DNase buffer
10 U/l DNase (Invitrogen)
Buffered phenol (APPENDIX 2A)
BASIC
PROTOCOL 7
Epsilon
Proteobacteria
8A.2.9
Current Protocols in Microbiology
Supplement 10
Chloroform
3 M sodium acetate, pH 5.2 (APPENDIX 2A)
70% (v/v) ethanol
75-cm2 tissue culture flask
15-ml conical centrifuge tubes
Centrifuge
Spectrophotometer
Additional reagents and equipment for growing C. jejuni (UNIT 8A.1)
Grow C. jejuni
1. Grow a full lawn of C. jejuni from frozen stock on one MH agar plate with 10 g/ml
trimethoprim for 16 to 20 hr.
2. Prepare a biphasic 75-cm2 tissue culture flask (UNIT 8A.1; see Fig. 8A.1.1) with 20 ml
MH agar and 20 ml MH broth with 10 g/ml trimethoprim (TMP).
3. Resuspend the bacteria from the prepared overnight plate (step 1) to an OD600 of 0.4
in MH broth with 10 g/ml trimethoprim (TMP).
4. Dilute the bacterial suspension 1:10 with MH broth containing 10 g/ml trimethoprim (TMP) and inoculate 80 l into the biphasic flask.
5. Incubate for 48 hr at 37 C in microaerophilic atmosphere (UNIT 8A.1).
6. Transfer 4-ml aliquots of the MH broth containing the 48-hr bacterial growth into
15 ml conical tubes.
Due to the limitations of the RNeasy Spin Column, better yield is achieved when 4 ml of
the culture is used per spin column. Multiple samples can be performed and combined
prior to DNase treatment, for higher yields.
Lyse bacteria
7. Add 8 ml RNA Protect Bacteria Reagent per 4 ml of the 48 hr culture in the 15-ml
conical tube. Vortex for 5 sec and incubate for 5 min at room temperature.
8. Centrifuge tubes at 10 min at 10,000 g, 4 C. Decant supernatant and dab on paper
towel.
9. Add 400 l TE buffer, pH 8.0, with 1 mg/ml lysozyme to each tube and resuspend
the bacteria by pipetting.
10. Incubate for 10 min at room temperature, vortexing every 2 min.
16. Add 500 l Buffer RPE onto the RNeasy spin column. Microcentrifuge 2 min at
10,000 rpm and discard the flowthrough.
8A.2.10
Supplement 10
17. Place column into fresh 2-ml collection tube and microcentrifuge for an additional
1 min at 12,000 rpm, to eliminate any remaining buffer.
18. Transfer RNeasy column into a fresh 1.5-ml microcentrifuge tube.
19. Add 50 l RNase-free water to the RNeasy membrane in the column and centrifuge
for 1 min at 10,000 rpm.
20. Combine any multiple samples done from the original 48 hr overnight culture (see
step 6).
21. Dilute sample to 200 l with RNase-free water and add 20 l of 10 DNase buffer.
22. Add 1 l of 10 U/l DNase to each tube. Incubate at room temperature for 1 hr.
23. Add 220 l of buffered phenol and vortex. Let sit for 5 min at room temperature.
24. Microcentrifuge 15 min at maximum speed, 4 C.
25. Save top layer and transfer to a new microcentrifuge tube.
26. Add 1 volume chloroform, vortex, and let sit for 5 min.
27. Microcentrifuge 15 min at maximum speed, 4 C.
28. Save top layer and transfer to a new microcentrifuge tube.
29. Precipitate RNA by adding 0.1 vol of 3 M sodium acetate pH 5.2, and 2.5 vol of
ice-cold 100% ethanol.
30. Incubate at 20 C overnight (or 2 to 3 hr, minimum).
31. Spin down RNA by microcentrifuging 15 min at maximum speed, 4 C.
32. Remove supernatant, then wash pellet by adding 1 ml of 70% ethanol and microcentrifuging 7 min at maximum speed, 4 C.
33. Remove supernatant and dry pellet completely.
34. Resuspend pellet in 25 to 50 l RNase-free water.
35. Quantitate RNA by measuring OD260 in a spectrophotometer and determine purification of RNA by comparing OD260 /OD280 (should be >1.8 to 2.0).
OD260 = 1 is equivalent to 40 g/ml RNA.
ALTERNATE
PROTOCOL
Epsilon
Proteobacteria
8A.2.11
Current Protocols in Microbiology
Supplement 10
Harvest bacteria
1. Prepare a 48-hr liquid culture of C. jejuni as described in Basic Protocol 7, steps 1
to 5.
2. Harvest from liquid culture by centrifuging 10 min at 10,000 g, 4 C, removing
the supernatant, and resuspending the pellet in 1 stop solution at 1/10 the original
volume.
3. Invert several times to mix.
4. Microcentrifuge 5 to 10 min at 11,000 g, 4 C. Aspirate supernatant.
5. Optional: Wash by adding 1 ml of 1 stop solution, centrifuging again as in step 4,
and aspirating the supernatant.
This optional wash removes any left-over medium and helps ensure a clean isolation.
6. Freeze in liquid nitrogen in 1ml aliquots in 1.5-ml microcentrifuge tubes and store
at 80 C until ready to isolate RNA.
This is a good stopping point if taking multiple samples over a period of time.
8A.2.12
Supplement 10
ARYLSULFATASE ASSAY
Similar to the lacZ reporter system in E. coli, the arylsulfatase assay, described in Hendrixson and DiRita (2003), quantifies the expression of a gene of interest. Arylsulfatase
cleaves sulfates from substrates, and is encoded by the astA gene in C. jejuni. The assay
measures arylsulfatase activity colorimetrically, using nitrophenyl sulfate as a substrate.
Arylsulfatase liberates the sulfate from nitrophenyl sulfate (a colorless compound),
forming nitrophenol. Nitrophenol is yellow in color, and by monitoring a colorimetric
change, this assay can be used to determine the amount of arylsulfatase activity. Briefly,
a promoterless astA gene is cloned into the gene of interest on the chromosome of C.
jejuni, creating an astA transcriptional fusion. Expression of the gene of interest can
then be monitored through the arylsulfatase assay, described below.
BASIC
PROTOCOL 8
Materials
p-Nitrophenol (Sigma, cat. no 104-8)
Arylsulfatase buffer 1 (AB1): 0.1 M TrisCl, pH 7.2 (APPENDIX 2A)
C. jejuni (UNIT 8A.1)
Mueller Hinton (MH) agar plates with antibiotics (BD Biosciences)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Arylsulfatase buffer 2 (AB2): 2 mM tyramine in 0.1 M TrisCl, pH 7.2
Arylsulfatase buffer 3 (AB3): 20 mM nitrophenyl sulfate (potassium 4-nitrophenyl
sulfate; Sigma, cat. no. N3877) in 0.1 M TrisCl, pH 7.2 (APPENDIX 2A)
0.2 N NaOH
Spectrophotometer
Additional reagents and equipment for growing C. jejuni (UNIT 8A.1)
Prepare standard curve
1. Prepare standard curve using p-nitrophenol:
a. Prepare 200 M nitrophenol in buffer AB1 by dissolving 27.82 mg nitrophenol
in 10 ml AB1, then diluting this solution 1:100 with buffer AB1 for a 200 M
solution.
b. Serially dilute the 200 M nitrophenol 1:2 with buffer AB1 for six dilutions.
c. Read OD420 for the dilutions of nitrophenol. Create a standard curve of OD420
values versus M nitrophenol.
Prepare bacteria
2. Streak C. jejuni onto MH agar containing 10 g/ml trimethoprim and grow at 37 C
for 24 hr under microaerophilic conditions (UNIT 8A.1).
3. Restreak heavily onto one MH agar plate. Grow at 37 C for 18 hr.
4. Resuspend the growth in PBS to an OD600 of 1.0.
5. Divide each sample into two 1-ml aliquots.
6. Wash one aliquot with AB1 by microcentrifuging 5 min at maximum speed, room
temperature, and removing the supernatant. Add 1 ml of buffer AB1, repeat microcentrifugation, and remove supernatant. Resuspend in 1 ml AB1.
7. Wash the second aliquot in AB2 using the technique described in step 6. Resuspend
in 1 ml AB2.
Current Protocols in Microbiology
Epsilon
Proteobacteria
8A.2.13
Supplement 10
Perform assay
8. Add 200 l of each sample to 200 l of freshly prepared AB3 in microcentrifuge
tubes.
9. Incubate for 1 hr at 37 C.
10. Stop reaction by adding 800 l of 0.2 N NaOH.
11. Read OD420 .
Samples in AB1 are blanks for AB2 samples.
DNase buffer, 10
200 mM sodium acetate, pH 4.5
100 mM MgCl2
100 mM NaCl
Store up to 1 year at 20 C
K buffer
50 mM KCl
10 mM TrisCl, pH 8.0 (APPENDIX 2A)
2.5 mM MgCl2
0.5% (v/v) Tween 20
Filter sterilize
Store up to 1 year at room temperature
Salt buffer, 5
6.25 ml 80% (w/v) glycerol (50% final)
1.25 ml 1 M HEPES, pH 7.9 (125 mM final)
1.25 ml 4 M NaCl (500 mM final)
0.25 ml 1 M MgCl2 (25 mM final)
1 ml H2 O
Store up to 1 year at room temperature
SOB medium
2 g Bacto tryptone (BD Biosciences)
0.5 g yeast extract (BD Biosciences)
0.05 g NaCl
10 ml 250 mM KCl
Adjust pH to 7.0 using 1 M HCl
Store up to 1 year at room temperature
SOC medium
Genetic
Manipulation of
C. jejuni
8A.2.14
Supplement 10
COMMENTARY
Background Information
Since its discovery, work on Campylobacter species has been limited due to challenging
culture requirements and limited genetic tools.
However, work in recent years has contributed
much to our understanding of Campylobacter
jejuni, as well as the tools used to study it.
Epsilon
Proteobacteria
8A.2.15
Current Protocols in Microbiology
Supplement 10
Protocol
Description
Time required
Basic Protocol 1
Conjugation
7-8 daysa
Basic Protocol 2
Electroporation
5-7 daysa
Basic Protocol 3
Natural transformation
5-7 daysa
Basic Protocol 4
In vitro transposition
2 daysb
Basic Protocol 5
<1 dayc
Basic Protocol 6
3-4 daysa
Basic Protocol 7 or
Alternate Protocol
RNA isolation
4-5 daysa
Basic Protocol 8
Arylsulfatase assay
3 daysa
a Time indicated includes time from streaking C. jejuni strain out from freezer stocks to end of
protocol as written. For making new strains via introduction of foreign DNA, this includes time
until single colonies will appear on selective media, but not the time required to verify the identity
of the new strain, isolate single colonies, and freeze down the new strain.
b Time does not include purification of transposase or natural transformation of transposition reaction
into C. jejuni.
c Time indicated does not include time required to grow C. jejuni strain before beginning the protocol.
procedure does not always efficiently eliminate any contaminating DNA. If DNA contamination is a problem, perform DNase treatment as indicated in Basic Protocol 7. DNase
treatment can be repeated multiple times, followed by phenol and chloroform extractions
and RNA precipitation. RNA can be tested for
contaminating DNA by a PCR reaction (without prior reverse transcription) and DNase
treatment can be repeated until there is no PCR
product.
Arylsulfatase assay
The substrate for the arylsulfatase assays
(Basic Protocol 8), nitrophenyl sulfate in solution, can be unstable and degrade after
long-term storage at 20 C, resulting in high
background readings. If this occurs, a fresh
stock of nitrophenyl sulfate should correct the
problem.
Time Considerations
Table 8A.2.3 provides time considerations
for the protocols in this unit. It should be noted
that for protocols that aim to produce new
strains of C. jejuni, the time indicated does not
include the time to verify the identity of the
new strain, colony purify the strain, and freeze
the strain down into new glycerol stocks.
Literature Cited
Elbing, K. and Brent, R. 2002a. Growth on solid
media. Curr. Protoc. Mol. Biol. 59:1.3.1-1.3.6.
Genetic
Manipulation of
C. jejuni
8A.2.16
Supplement 10
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Proteobacteria
8A.2.17
Current Protocols in Microbiology
Supplement 10
UNIT 8B.1
In vitro, Helicobacter species grow quite slowly and only under conditions of reduced (but
not absent) oxygen (i.e., microaerophilic conditions) and rich media. Primary cultures
may take up to 5 to 7 days to achieve optimal growth, although subsequent passages take
only a few days. Identification of H. pylori can be made by confirming the presence of
urease, catalase, and oxidase enzymes, and by observing microscopic evidence of high
motility and a spiral or curved morphology (Windsor and ORourke, 2000). Colonies are
small and round, and appear translucent against the blood agar background. H. pylori
is resistant to sulfonamides, vancomycin, trimethoprim, and cephalothin, which allows
for the creation of a very effective selective media when isolating bacteria from clinical
biopsies. It is sensitive, however, to many common antibiotics including tetracycline,
gentamicin, kanamycin, and penicillin. These antibiotics may therefore be used to kill
extracellular H. pylori when performing co-culture experiments with phagocytes, or as
selection markers when introducing gene cassettes into the H. pylori genome.
In the present unit, protocols for growing Helicobacter organisms on plates (Basic Protocol 1) or in liquid cultures (Basic Protocol 2) are presented first, followed by Basic
Protocol 3 for culturing H. pylori from clinical biopsy specimens. Three support protocols are presented that describe the preparation of solid media for growth on plates
(Support Protocol 1), a method for quantifying numbers of Helicobacter organisms in
an actively growing culture (Support Protocol 2), and techniques for quantifying H.
felis in vitro (Support Protocol 3). Additionally, a method for liquid culture of
Helicobacter has been provided that does not require the use of a tissue culture incubator
(see Alternate Protocol). Finally, methods are provided for confirming growth of H. pylori
(see Support Protocol 4).
CAUTION: H. pylori and H. mustelae are Biosafety Level 2 (BSL-2) pathogens; H. felis is
a BSL-1 pathogen. Adult acquisition of H. pylori is thought to be relatively rare, and most
infected individuals remain asymptomatic and healthy for life. Follow all appropriate
guidelines and regulations for the use and handling of pathogenic microorganisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
STRATEGIC PLANNING
Several different Helicobacter selective media supplements have been described (see
Table 8B.1.1). Skirrows recipe, available commercially from Difco, BBL, and Oxoid,
was originally developed for isolation of Campylobacter species. Although Skirrows
supplement (10 g/ml vancomycin, 5 g/ml trimethoprim, and 2.5 IU/ml polymyxin B)
plus amphotericin B has been used for isolation and culture of Helicobacter species, the
authors have found the recipes in Table 8B.1.1 to be more effective. In particular, the
so-called Glaxo selective supplement A (GSSA) is useful for the isolation of H. pylori
from mice when attempting to adapt a new H. pylori strain to mice (Lee et al., 1997;
McColm, 1997; McColm et al., 1995). Note, however, that use of GSSA could induce
a bias into the selection/adaptation process, as some H. pylori isolates have been shown
to be sensitive to nalidixic acid. One or the other of the supplements must be used when
attempting to isolate H. pylori or one of the other Helicobacter species from a human or
animal biopsy. This is particularly important for isolating bacteria from mice since they
are coprophagic (they consume their own feces). For pure cultures of laboratory strains,
selective medium supplementation is less critical, but in order to simplify preparation
Epsilon
Proteobacteria
Contributed by Thomas G. Blanchard and John G. Nedrud
Current Protocols in Microbiology (2006) 8B.1.1-8B.1.13
C 2006 by John Wiley & Sons, Inc.
Copyright
8B.1.1
Antibiotic
H. pylori
H. pylori
GSSAa
H. felis
H. mustelae
Amphotericin B
2.5 g/ml
2.5 g/ml
2.5 g/ml
2.5 g/ml
Bacitracin
Cefsulodin
20 g/ml
16 g/mlb
16 g/ml
Nalidixic acid
10.7 g/ml
Polymyxin B
3.3 g/ml
Trimethoprim
20 g/ml
Vancomycin
6 g/ml
10 g/ml
0.125 g/ml
20 g/ml
20 g/ml
6 g/ml
6 g/ml
of bacterial growth plates, it will do no harm to always use plates prepared with the
indicated supplements.
BASIC
PROTOCOL 1
Materials
Freshly prepared blood agar plates containing appropriate antibiotic
(see Support Protocol 1)
Plates containing viable Helicobacter colonies, liquid culture of Helicobacter
(Basic Protocol 2), or frozen stock of Helicobacter
Disposable bacterial spreaders (PGC Scientific), laboratory-made glass spreaders,
or inoculating loops
Anaerobic jar(s) with sealable lid(s) (BBL model no. 100 uses one GasPak and
holds 12 plates; model 150 uses three GasPak envelopes and holds 36 plates.)
GasPak: BBL CampyPak Plus Microaerophilic System Envelopes with Palladium
Catalyst (Becton Dickinson) or equivalent
1. Inoculate freshly prepared blood agar plates with several colonies from a viable plate
using an inoculating loop; alternatively inoculate 50 to 100 l of thawed material
from a frozen stock or liquid culture.
Laboratory
Maintenance of
Helicobacter
Species
2. Use a bacterial spreader or inoculating loop to spread the material back and forth
across the entire plate from top to bottom. Turn the plate 90 and repeat.
8B.1.2
Current Protocols in Microbiology
4. Cut the corner off of a GasPak envelope, separate the edges, and place inside the
anaerobic jar with the catalyst facing the outside. Add 10 ml water to each envelope, then seal the top of the anaerobic jar. Place the jar in a 37 C incubator or
environmental control room.
There are several different types of gas-generating GasPaks available. Be sure to select
the correct one or the Helicobacter organisms may not grow.
If an incubator is available in which three gases can be controlled, or if custom-blended
gas cylinders can be obtained, these may be used in lieu of anaerobic jars and GasPaks.
The incubator should be set to deliver 5% to 7% O2 /10% CO2 with the balance N2 . If the
incubator allows control of two gases plus air, set it at 20% to 35% air (which yields 5%
to 7% oxygen), 10% CO2 , and the balance N2 . Some trial and error may be required to
determine the optimum gas mixture for a particular incubator.
5. After 3 to 4 days open the jar and examine the plates for growth. If no growth or
only low growth is observed, plates can be returned to the jar with a fresh GasPak
for another 2 to 3 days.
H. pylori and H. mustelae will form distinct pinhead-sized colonies which gradually
become larger and easier to identify. Colonies on densely cultured plates may merge into
a confluent lawn that displays a distinctive sheen when the plate is viewed at an angle.
Thus, if individual H. pylori or H. mustelae colonies are to be counted, timing is important
and it may be necessary to examine the plates at two or three different time points. H.
felis tends to form confluent sheets within several days of culture. Early examination may
allow for enumeration of individual colonies, but these colonies may be very small and
difficult to detect.
SUPPORT
PROTOCOL 1
Successful culture of Helicobacter organisms requires the use of fresh nutrient blood
agar plates. Commercially prepared plates may work, but the freshness of these plates
cannot be controlled and they can often be too old or too dry. Additionally, they will
lack the appropriate selective antibiotics. Unless one has access to a reliable bacteriology
laboratory that can prepare fresh plates to specifications, it is better to prepare them in
ones own laboratory. Any one of several nutrient agars recommended for growth of
fastidious organisms will work, including Columbia Agar Base, Mueller-Hinton Agar
Base, and Blood Agar Base (available from BBL, a trademark of Becton Dickinson).
The important thing is that the plates be relatively fresh and moist. When preparing the
plates, let the agar harden only until it solidifies (not overnight). If the plates are not to be
used immediately, store in sealed plastic sleeves at 4 C for no longer than 2 to 3 weeks.
Materials
Nutrient agar base: Columbia Agar Base, Mueller-Hinton Agar Base, or Blood
Agar Base (available from BBL; also see APPENDIX 2C)
Stock solutions of antibiotics (see Table 8B.1.1)
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8B.1.3
Current Protocols in Microbiology
2. Loosely cover vessels and autoclave under standard conditions for liquid reagents
(18 psi/120 C) for 20 min.
3. Remove containers from autoclave and place in a 56 C water bath.
This allows the medium to cool enough for addition of the blood and antibiotics without
denaturing their activity but still being warm enough to prevent the agar from solidifying.
4. Add the appropriate amount of the antibiotics required for the particular Helicobacter
species (see Table 8B.1.1).
5. Add 35 ml defibrinated whole blood, previously warmed to room temperature.
Alternatively, laked whole blood, which has been frozen and thawed to produce
hemolysis, may be used; however, the authors have not found this to be necessary.
6. Remove the flask from the water bath and gently mix until the blood is distributed
uniformly. Keep the formation of bubbles to a minimum.
This is best accomplished by rolling the flask at an angle on the counter top.
7. Immediately dispense the medium into 100-mm petri dishes and remove bubbles by
flaming. Stack the warm plates into groups of four or five and allow to cool at room
temperature (only until agar has hardened).
Stacking will allow the agar to cool more slowly and minimize the amount of condensation
that forms on the lids (except for the top plate). However, unstacked plates will solidify
more quickly.
8. Following solidification of the agar, place plates in a plastic sleeve and store at 4 C
until needed.
BASIC
PROTOCOL 2
Materials
Laboratory
Maintenance of
Helicobacter
Species
Brucella broth or other suitable liquid medium supplemented with 10% (v/v)
heat-inactivated FBS
Agar plate containing a heavy growth of viable H. pylori (Basic Protocol 1)
Disposable bacterial spreaders (PGC Scientific) or laboratory-made glass spreaders
25-cm2 polystyrene tissue culture flasks
37 C, 5% CO2 incubator
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Current Protocols in Microbiology
4. Attach the cap of the flask so that it is loose enough to allow gas exchange but not
loose enough to fall off.
Alternatively, flasks with vented caps may by used and screwed on tightly.
6. Allow culture to grow overnight. Check for Helicobacter growth by placing the flask
horizontally on an inverted microscope at 40 magnification. When the agitation of
the culture has stabilized, look for highly motile bacteria by focusing just above the
bottom of the flask.
7. Passage culture by transferring a small aliquot to another flask containing 10 ml
Brucella broth/FBS medium.
Passage before viability begins to decline (OD of 0.2 to 0.3 or lower).
The volume of the aliquot used for passage is highly variable and must be based on the
density of the culture and how soon one would like the new culture to reach saturation.
Generally, 100 to 200 l of a healthy culture will generate a saturated culture within
24 to 48 hr. At this point, larger cultures can be started by using 60 ml medium in a
75-cm2 flask or 100 ml in a 150-cm2 flask.
ALTERNATE
PROTOCOL
Static liquid cultures able to grow under standard tissue culture incubator conditions
have only been described for H. pylori (see Basic Protocol 2). Other gastric Helicobacter
species such as H. felis require continuous shaking; therefore, alternative methods for
growth are required. Unavailability of a CO2 incubator or unwillingness of colleagues to
allow the use of a tissue culture incubator for growth of bacterial cultures (because of concerns about contamination) may also favor alternative strategies. Most gastric pathogens
are readily grown in the same liquid culture medium described in Basic Protocol 2 when
placed in a suitable flask within an anaerobic jar on a rotary shaker.
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Current Protocols in Microbiology
5. Place the flask containing the inoculated medium within an anaerobic jar.
6. Cut the corner off of the GasPak envelope, separate the edges, and place inside the
anaerobic jar with the catalyst facing the outside. Add 10 ml water to each envelope,
then seal the top of the anaerobic jar.
7. Seal the jar and place in a 2-liter flask bracket on an orbital shaker at 37 C.
8. Incubate with shaking at 125 rpm for 24 to 48 hr.
9. At the end of this incubation, or earlier, remove an aliquot of the culture and determine
optical density (see Support Protocol 2; see Support Protocol 3 for H. felis).
An optical density of 0.1 at 550 nm should yield 107 cfu/ml, but this should be verified
by means of ones own growth curve (see Support Protocol 2).
10. Prepare a wet mount (see Support Protocol 4) and examine for motility and absence
of contamination with other bacteria.
SUPPORT
PROTOCOL 2
Materials
Laboratory
Maintenance of
Helicobacter
Species
8B.1.6
Brucella broth or other suitable liquid medium supplemented with 10% (v/v)
heat-inactivated FBS
Actively growing liquid culture of H. pylori (see Basic Protocol 2 or Alternate
Protocol)
Freshly prepared blood agar plates containing appropriate antibiotic (see Support
Protocol 1)
25-cm2 tissue culture flask
37 C, 5% CO2 incubator
Current Protocols in Microbiology
3. Determine the OD550 of the culture using Brucella broth/10% FBS as a blank.
4. Using sterile tubes, prepare serial 10-fold dilutions of the aliquot from 1:102 to 1:105 .
5. Inoculate 10 l of each dilution onto a blood agar plate supplemented with antibiotics
and spread uniformly using a bacterial spreader until no liquid remains on the plate.
Mark each plate or fraction of plate used with the time point and dilution.
To reduce the number of plates required, each plate can be divided into half or even thirds
using a marker on the bottom of the plate and distributing each 10-l inoculum only
within the marked boundaries.
10. Repeat this procedure at least twice and average the results in order to obtain a
reasonably accurate growth curve.
SUPPORT
PROTOCOL 3
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8B.1.7
Current Protocols in Microbiology
dilutions of this culture into Brucella broth. Pipet an aliquot of appropriately diluted
culture into a hemacytometer (APPENDIX 4A) and quickly count the number of motile
organisms at 400 total magnification. This is difficult, as a high-power microscope
objective must be used and H. felis bacteria are highly motile and can move into and out
of the hemacytometer grid squares. The authors have found that these two methods give
numbers which are in reasonable agreement with each other.
BASIC
PROTOCOL 3
Materials
Gastric pinch biopsy material
Transport medium (see recipe) in sterile tubes
Brucella broth or other suitable liquid medium supplemented with 10% (v/v) FBS
Freshly prepared blood agar plates containing appropriate antibiotic (see Support
Protocol 1)
Sterile forceps
Sterile disposable pellet pestles and 1.5-ml microcentrifuge tubes (Kontes, cat no.
749520)
Disposable bacterial spreaders (PGC Scientific) or laboratory-made glass spreaders
Additional reagents and equipment for microaerophilic plate culture of
Helicobacter (Basic Protocol 1)
1. Retrieve gastric biopsy material from clinical laboratory in transport medium.
Biopsies should be obtained for culture as soon as possible after endoscopy, although
the authors have had some success in culturing from biopsies stored overnight at room
temperature in transport medium.
2. Using sterile forceps, remove biopsy pieces from the transport medium tubes and
place in sterile 1.5-ml microcentrifuge tube (provided along with a disposable pellet
pestle) with 0.1 ml Brucella broth/10% FBS.
Laboratory
Maintenance of
Helicobacter
Species
3. Grind the tissue using the sterile disposable pellet pestle and add an additional 0.1
ml Brucella broth/FBS.
4. Inoculate 10 l of the homogenate onto a blood agar plate with the appropriate
selective antibiotics and spread with inoculating loop or spreader.
8B.1.8
Current Protocols in Microbiology
5. Cover plates, place in anaerobic jars with GasPaks, and culture as described in Basic
Protocol 1, steps 3 to 5.
SUPPORT
PROTOCOL 4
All of the gastric Helicobacter species produce large amounts of the enzyme urease,
which can cleave urea into carbon dioxide and ammonia. The ammonia is thought to
buffer the acidity of the stomach and thus aid the bacteria in its colonization. It is possible,
particularly in experimental animals, for other urease-positive micro-organisms (e.g.,
Proteus species) to colonize the stomach. Thus, the combination of colony morphology,
urease test, oxidase test, catalase test, and spiral morphology/motility in a wet mount are
used to confirm the identity of H. pylori. As noted above, nucleic acid fingerprinting and
PCR techniques can be used for further verification.
Wet mount/morphology
Viable Helicobacter species will be highly motile, and if enough bacteria are placed on
the slide they can be easily located by phase-contrast microscopy at 400 to 1000 total
magnification. H. felis is relatively long and possesses a distinct corkscrew morphology
with tight spirals. H. pylori and H. mustelae are shorter with less distinct spirals. In
general, they have a comma- or gull winglike shape. In a healthy culture, the bacteria
will appear to be swimming about quite rapidly. An overgrown culture may yield an
optical density in the desired range, but observation of low or no mobility will indicate
lack of viability. Thus it is important to always check cultures for morphology and
motility.
Urease test
Place a loopful of material into 0.3 ml Stuarts urease test broth (see recipe). The broth
should turn pink within minutes.
Catalase test
Place a loopful of material into 0.5 ml of 3% H2 O2 . Viable Helicobacter will form
bubbles.
Oxidase test
Either place a loopful of material into a drop of distilled water on a Bacto Differentiation
Disk Oxidase (BD Biosciences, cat. no. 1633) or place a disk over a colony or area
of growth on a plate with a drop of distilled water. A pink or maroon color within
10 to 20 min, eventually changing to almost black, indicates a positive test.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock
solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Epsilon
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8B.1.9
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Laboratory
Maintenance of
Helicobacter
Species
8B.1.10
Current Protocols in Microbiology
Epsilon
Proteobacteria
8B.1.11
Current Protocols in Microbiology
Anticipated Results
Establishing viable cultures of H. pylori
and other Helicobacter species may take several attempts, especially when starting from a
frozen stock. However, if multiple plates are
inoculated or repeated attempts are made, viable cultures can be obtained as long as the
anaerobic jars are properly sealed. Performing
subsequent passages from a viable culture is
generally much more reliable and the setup is
easy to maintain. One can expect to observe
fairly large and healthy colonies every 3 to 4
days once these cultures are growing. Likewise, once liquid cultures are established, one
should expect to passage cultures every 1 to 2
days. Even if microscopic examination reveals
dense growth but minimal motile organisms,
these cultures may still be viable and suitable
for inoculation into fresh media.
Recovering viable H. pylori from clinical
gastric biopsies can be challenging. It is common for laboratories that perform this function on a regular basis to fail to isolate H.
pylori even when the rapid urease test is positive. The site of biopsy source, freshness of
the biopsy, antibiotic treatment of the subject,
and bacterial density within the biopsy can all
effect the success of isolating H. pylori from
human tissue.
Time Considerations
Laboratory
Maintenance of
Helicobacter
Species
The time necessary to culture Helicobacter species on solid media will vary, depending on whether or not blood agar plates have
been prepared in advance. It will also vary
with the number of plates to be inoculated.
In general, plates can be prepared and ready
for culturing in several hours. Preparation of
the medium and autoclaving takes less than
an hour. The two most time-consuming steps
are allowing the temperature of the media to
cool to 56 C, and allowing the medium to
gel adequately following pouring of the plates.
The whole procedure can be accomplished in
one morning or afternoon. The actual inoculation of plates and preparation of the jars for
incubation take only a few minutes.
The number of bacteria in a stock culture
can be approximated in a few minutes by measuring the OD550 and referring to a growth
curve. Rigorous determination of cfu takes
longer, but by using the OD and a growth
curve, one or two dilutions can be picked for
inoculation onto plates, which takes only a few
minutes. The plates must then be incubated for
8B.1.12
Current Protocols in Microbiology
care in doing it and to think twice about altering culture conditions in a way that would
require constructing a new growth curve. Note
also that, at some time point, the optical density of the growth curve may continue to rise,
but that the number of viable cfu will plateau
and then decrease.
Literature Cited
Alm, R.A., Ling, L.S., Moir, D.T., King, B.L.,
Brown, E.D., Doig, P.C., Smith, D.R., Noonan, B., Guild, B.C., deJonge, B.L., Carmel,
G., Tummino, P.J., Caruso, A., Uria-Nickelsen,
M., Mills, D.M., Ives, C., Gibson, R., Merberg,
D., Mills, S.D., Jiang, Q., Taylor, D.E., Vovis,
G.F., and Trust, T.J. 1999. Genomic-sequence
comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180.
Bereswill, S., Schonenberger, R., van Vliet, A.H.,
Kusters, J.G., and Kist, M. 2005. Novel plasmids for gene expression analysis and for genetic manipulation in the gastric pathogen Helicobacter pylori. FEMS Immunol. Med. Microbiol. 44:157-162.
Jiang, Q., Hiratsuka, K., and Taylor, D.E. 1996.
Variability of gene order in different Helicobacter pylori strains contributes to genome diversity. Mol. Microbiol. 20:833-842.
Kleanthous, H., Clayton, C.L., and Tabaqchali, S.
1991. Characterization of a plasmid from Helicobacter pylori encoding a replication protein
common to plasmids in gram-positive bacteria.
Mol. Microbiol. 5:2377-2389.
Lee, A., ORourke, J., De Ungria, M.C., Robertson,
B., Daskalopoulos, G., and Dixon, M.F. 1997.
A standardized mouse model of Helicobacter
pylori infection: Introducing the Sydney strain.
Gastroenterology 112:1386-1397.
Marshall, B.J. and Warren, J.R. 1984. Unidentified
curved bacilli in the stomach of patients with
gastritis and peptic ulceration. Lancet 1:13111315.
McColm, A.A. 1997. Nonprimate animal models of
H. pylori infection. In Helicobacter pylori Protocols (C.L. Clayton and H.L.T. Mobley, eds.)
pp. 235-251. Humana Press, Totowa, N.J.
McColm, A.A., Bagshaw, J., Wallis, J., and
McLaren, A. 1995. Screening of antiHelicobacter therapies in mice colonized with
H. pylori. Gut 37:A92.
Nedrud, J.G. 1999. Animal models for gastric Helicobacter immunology and vaccine studies.
FEMS Immunol. Med. Microbiol. 24:243-250.
Suerbaum, S. 2000. Genetic variability within Helicobacter pylori. Int. J. Med. Microbiol. 290:175181.
Suerbaum, S. and Michetti, P. 2002. Helicobacter pylori infection. N. Engl. J. Med. 347:11751186.
Tomb, J.F., White, O., Kerlavage, A.R., Clayton,
R.A., Sutton, G.G., Fleischmann, R.D.,
Ketchum, K.A., Klenk, H.P., Gill, S., Dougherty,
B.A., Nelson, K., Quackenbush, J., Zhou, L.,
Kirkness, E.F., Peterson, S., Loftus, B., Richardson, D., Dodson, R., Khalak, H.G., Glodek,
A., McKenney, K., Fitzegerald, L.M., Lee, N.,
Adams, M.D., Hickey, E.K., Berg, D.E., Gocayne, J.D., Utterback, T.R., Peterson, J.D., Kelley, J.M., Cotton, M.D., Weidman, J.M., Fujii,
C., Bowman, C., Watthey, L., Wallin, E., Hayes,
W.S., Borodovsky, M., Karp, P.D., Smith, H.O.,
Fraser, C.M., and Venter, J.C. 1997. The complete genome sequence of the gastric pathogen
Helicobacter pylori. Nature 388:539-547.
Tsuda, M., Karita, M., and Nakazawa, T. 1993. Genetic transformation in Helicobacter pylori. Microbiol. Immunol. 37:85-89.
Windsor, H.M. and ORourke, J. 2000. Bacteriology and taxonomy of Helicobacter pylori. Gastroenterol. Clin. North. Am. 29:633-648.
Key References
Lee, A. and Megraud, F. (eds.) 1996. Helicobacter pylori: Techniques for Clinical Diagnosis
and Basic Research. W. B. Saunders Company,
London.
This monograph contains a collection of clinical,
diagnostic and basic research techniques for Helicobacter species.
Internet Resources
http://www.helico.com
Web site for detailed information about H. pylori
history, epidemiology, and pathogenesis.
http://bmbl.od.nih.gov
Biosafety in Microbiology and Biomedical Laboratories 4th Ed May 1999. Contains details for handling bacteria of differing safety levels and separate
sections for individual pathogens such as H. pylori.
Epsilon
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2, 3
UNIT 9B.1
ABSTRACT
Listeria monocytogenes is an intracellular foodborne pathogen that causes listeriosis,
an infection characterized by gastroenteritis, meningitis, encephalitis, and maternofetal
infections in humans. L. monocytogenes enters the host via contaminated foods, invades
the small intestine, translocates to mesenteric lymph nodes, and spreads to the liver,
spleen, brain and, in pregnant women, the fetoplacental unit. Many pathogenicity tests
for studying L. monocytogenes have been developed, including tests using laboratory
animals. A number of small animal species can be experimentally infected with Listeria.
Mice and guinea pigs can be infected either intragastrically or intravenously, and virulence
evaluated either by enumerating bacteria within infected target organs or by evaluating
the 50% lethal dose (LD50). Although mice and guinea pigs can be infected with Listeria
by a variety of routes, the intragastric route is the most relevant to the human foodborne
C 2008 by John Wiley & Sons,
listeriosis. Curr. Protoc. Microbiol. 10:9B.1.1-9B.1.17.
Inc.
Keywords: Listeria monocytogenes r listeriosis r foodborne pathogen r infection r
animal model
INTRODUCTION
Listeria monocytogenes causes listeriosis, a foodborne infection characterized by bacterial dissemination from the intestinal lumen to the central nervous system and the
fetoplacental unit. Many pathogenicity tests for studying L. monocytogenes have been
developed, including tissue culture assays, fertilized hen egg tests, and tests using laboratory animals. A number of small animal species, including mice and guinea pigs, can
be experimentally infected with Listeria, the mouse species being the most frequently
used. Although mice and guinea pigs can be infected with Listeria by a variety of routes,
the intragastric route is the most relevant to human foodborne listeriosis. Mice can be
infected either intragastrically (see Basic Protocol 1) or intravenously (see Basic Protocol 2), and virulence evaluated either by numerating bacteria within infected target
organs (see Basic Protocol 3) or by evaluating the 50% lethal dose (LD50; see Basic
Protocol 4). The infectious process following intragastric inoculation of Listeria can be
also assessed in the guinea pig model (see Alternate Protocol).
As a proper cultivation of L. monocytogenes is a critical first step for the success of in vivo
experiments, a method for preparing Listeria inoculum is also described (see Support
Protocol).
All strains of Listeria monocytogenes are not equally virulent (Barbour et al., 2001;
Larsen et al., 2002; Roche et al., 2005). Since the L. monocytogenes reference strain
EGDe genome sequence has been determined (Glaser et al., 2001) and EGDe is used
by a large number of laboratories, protocols presented here use this strain, but other
Listeria strains (wild types and mutants) can also be used. The animal model, its genetic
Current Protocols in Microbiology 9B.1.1-9B.1.17, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc09b01s10
C 2008 John Wiley & Sons, Inc.
Copyright
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.1
Supplement 10
background, and the route of infection are critical for the outcome of the infection (Cheers
et al., 1978; Barbour et al., 1996; Lecuit et al., 2001; Czuprynski et al., 2003; Khelef
et al., 2006; Cossart and Toledo-Arana, 2008. Procedures presented here use iFABPhEcad mice and Hartley guinea pigs for intragastric infection, and BALB/c mice for
intravenous infection.
CAUTION: Listeria monocytogenes is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for use and handling of pathogenic microorganisms.
CAUTION: These experiments require Animal Biosafety Level 2 (ABSL-2) conditions.
Follow all appropriate guidelines and regulations for use and handling of infected animals.
See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved
procedures for care and use of laboratory animals.
NOTE: The proper handling and restraint of animals needs to be mastered before any
inoculations. The experimenter needs to be fully aware of inherent dangers such as
accidental needle stick when performing these techniques.
NOTE: The procedure requires familiarity with basic methods for animal care, handling,
and restraint (see Donovan and Brown, 2006a).
IMPORTANT NOTE: The success of these protocols is dependent on proper use of aseptic
techniques.
BASIC
PROTOCOL 1
Materials
6- to 8-week-old sex-matched iFABP-hEcad mice
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
Brain heart infusion (BHI) agar plates
150 mg/ml CaCO3 in PBS, prepare fresh
Animal Models of
Listeria Infection
9B.1.2
Supplement 10
Additional reagents and equipment for the anesthesia of rodents (Donovan and
Brown, 1998)
Prepare animals
1. Acclimate 6- to 8-week-old sex-matched mice to the animal facility environment for
at least 1 week prior to infection.
Animals should be acclimated under standard lighting and temperature conditions with
food and water.
3. Just before injection, thaw an aliquot of bacteria on ice. Dilute bacteria appropriately
in sterile PBS to achieve 0.2 ml per mouse at 2.5 1010 cfu/ml.
The standard dose recommended for L. monocytogenes EGDe via the intragastric route is
5 109 cfu per mouse, delivered in a volume of 0.2 ml. However, lower inocula (109 , 108 )
also lead to systemic infection, with highly reproducible differences between wild-type
(wt) bacteria and isogenic mutants.
4. To confirm the titer of the starting suspension, plate, in duplicate, 0.1 ml of serial
dilutions (107 , 106 , 105 , 104 ) on sterile BHI agar plates, and incubate the plate
24 to 48 hr at 37 C.
5. Fill the syringe with an inoculum of 0.2 ml containing the bacteria number to be
injected, mixed with 0.3 ml of 150 mg/ml CaCO3 suspension in PBS.
Use a 1-ml syringe with a long, bulbous-ended needle (e.g., animal feeding needle 20-G
1-1/2 with silicon tip). The amount of material that can be injected orally is 0.5 ml.
CaCO3 buffering is used to reduce the gastric acidity and to decrease inter-individual
variability which depends upon gastric repletion and pH.
Perform inoculation
6. Remove the mouse from the cage by its tail.
At this point, mice can be anesthetized (Donovan and Brown, 1998) before gavage. The
standard inhalant anesthetics for laboratory animal use are either isoflurane or halothane,
delivered to effect in concentrations up to 5% for initial induction.
7. Hold the mouse by the skin on either side of the base of the neck and exert a slight
downward and forward pressure under the mandible to tilt the head up slightly, thus
aligning the oral cavity and pharynx with the esophagus.
8. Insert the bulbous-ended needle over the tongue into the esophagus and stomach.
9. Slowly inject the inoculum.
Agents to be administered must be in suspension or solution, and at room to body
temperature.
10. After completing the injection, carefully withdraw the needle, place the mouse back
into its cage, and continue injections with additional animals. When injections are
complete, check the animals for normal behavior and then return them to their
housing location with food.
In case of anesthesia, carefully follow the full recovery of the animal.
11. Discard the needle and syringe and disinfect work instruments and area.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.3
Current Protocols in Microbiology
Supplement 10
Animals are then either observed every day for determination of mortality (see Basic
Protocol 4), or they are euthanized and dissected for determination of bacterial counts
from organs (see Basic Protocol 3).
Perform calculations
12. One or two days after incubation at 37 C, count colonies on plates (step 4) and
calculate the initial titer of the suspension as follows: cfu/ml = MCC/(VP DF).
Where MCC is the mean colony count of replicate countable plates, VP is the volume
plated, and DF is the dilution factor of the sample in the plate.
13. Calculate the Listeria load delivered by the IG inoculation as: cfu = cfu/ml DF
VD.
Where cfu/ml is the initial inoculum titer (step 11), DF is the dilution factor used to
prepare the suspension (step 3), and VD is the volume delivered (step 3).
BASIC
PROTOCOL 2
Materials
6- to 8-week-old female BALB/c mice
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
BHI agar plates
Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
37 C incubator
Housing cages for the mice
Heat lamp
Restraining device for mice (Harvard Apparatus)
Sterile 1-ml syringe with a small diameter needle (e.g., 26-G 3/8 in.)
Sterile gauze
Prepare animals
1. Acclimate 6- to 8-week-old female mice to animal facility environment for 1 week
prior to infection procedure.
Animal Models of
Listeria Infection
Animals should be acclimated under standard lighting and temperature conditions with
food and water.
9B.1.4
Supplement 10
2. Just before injection, thaw an aliquot of bacteria on ice. Dilute bacteria appropriately
in sterile PBS to achieve 0.3 ml per mouse at 3.3 104 cfu/ml.
The standard dose for L. monocytogenes EGDe via the intravenous route is 104 cfu per
mouse, delivered in a volume of 0.3 ml.
3. To confirm the titer of the starting suspension, plate, in duplicate, 100 l of serial
dilutions (103 , 102 , 101 , 100 ) on sterile BHI agar plates, and incubate the plate
24 to 48 hr at 37 C.
4. Place the mouse cage under a heat lamp to allow gentle warming in order to promote
vasodilation.
This will increase circulation to the surface blood vessels (as the mouse regulates its
body temperature) and expand the diameter of the veins in the tail. Time will depend on
the intensity of the lamp. It is essential to observe the mice while they are warming and
ensure that they do not become heat stressed while under the lamp. If mice appear to be
agitated or heat stressed, remove the cage immediately from beneath the lamp.
Perform inoculation
6. Fill the syringe with the desired amount of inoculum to be injected. Remove any air
bubbles from the syringe and needle.
Use a 1-ml syringe with a small diameter needle (e.g., 26-G 3/8 in.). The amount of
material that can be injected IV is 0.3 ml. Absolutely no air can be accidentally injected,
as this may cause a fatal air embolism.
7. When the mice are sufficiently warmed, remove a mouse from the cage by its tail.
Holding on by its tail, place the mouse head first into the cylinder of the restraining
device.
8. Locate one of the two lateral veins of the tail. Hold the tail with the thumb and first
finger, curling the end of the tail slightly over the finger.
For more details on the injection procedure, see Donovan and Brown, 2006b.
9. Line up the needle exactly in line with the vein and insert the needle into the vein.
If the needle is in the vein the tip of the needle will be visible inside of the vein. It is
desirable to make the first injection attempt as close to the tip of the tail as possible, so
that a second attempt, if necessary, can be safely made more proximally or on the opposite
vein.
11. After the injection, carefully withdraw the needle and use gauze to apply digital
pressure at the injection site for hemostasis.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.5
Current Protocols in Microbiology
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12. Place the infected mouse in a spare cage and continue injections with the other
animals. When injections are complete, check the animals for normal behavior and
then return them to their housing location.
13. Discard the needle and syringe and disinfect work instruments and area.
Animals are then either observed every day for determination of mortality (see Basic
Protocol 4), or they are euthanized and dissected for determination of bacterial counts
from organs (see Basic Protocol 3).
Perform calculations
14. One or two days after incubation of plates (step 3) at 37 C, calculate the initial titer
of bacterial stock suspension (se Basic Protocol 1, step 12) and then calculate the
Listeria load using the dilution factor of the IV suspension (see step 2 above).
ALTERNATE
PROTOCOL
Materials
300-g, sex-matched Hartley guinea pigs
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), saline
BHI agar plates, sterile
15 mg/ml ketamine in sterile PBS, prepare fresh
25 mg/ml CaCO3 in PBS, prepare fresh
Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
37 C incubator
1-ml syringe with a small diameter needle (e.g., 26-G 3/8 in.), sterile
20-ml syringe
Sterile suction catheter (1.7-mm 270-mm; Vygon, ref. no. 530.05)
Animal cages
Animal Models of
Listeria Infection
Additional reagents and equipment for the anesthesia of rodents (Donovan and
Brown, 1998)
9B.1.6
Supplement 10
Prepare animals
1. Acclimate 300-g, sex-matched guinea pigs to animal facility environment for
1 week prior to infection procedure.
Animals should be acclimated under standard lighting and temperature conditions with
food and water.
3. Just before injection, thaw an aliquot of bacteria on ice. Dilute bacteria appropriately
in sterile PBS to achieve 1 ml per animal at 1010 cfu/ml.
The standard dose for L. monocytogenes EGDe via the intragastric route is 1010 cfu per
animal, delivered in a volume of 1 ml.
4. To confirm the titer of the starting suspension, plate, in duplicate, 0.1 ml of serial
dilutions (107 , 106 , 105 , 104 ) on sterile BHI agar plates, and incubate the plate
24 to 48 hr at 37 C.
5. Remove the guinea pig from the cage.
6. For light anesthesia, inject guinea pig intramuscularly with 1 ml of 15 mg/ml
ketamine in PBS.
Use a 1-ml syringe with a small diameter needle (e.g., 26-G 3/8 in.). For guinea pigs,
ketamine is usually administrated at 50 mg/kg.
Perform inoculation
8. Fill a 20-ml syringe with 5 ml of 25 mg/ml CaCO3 in PBS.
9. Fill a 1-ml syringe with 1 ml containing the desired amount of inoculum to be
injected.
10. When anesthetized, hold the guinea pig by the skin on either side of the base of the
neck and exert a slight downward and forward pressure under the mandible to tilt
the head up slightly, thus aligning the oral cavity and pharynx with the esophagus.
11. Insert a suction catheter intragastrically until feeling a light resistance (15 cm).
This procedure is facilitated if carried out by two experimenters. In our experience, homemade suction catheters should be avoided, because of the harmful effect of their distal
extremity, which is not smooth enough.
12. Adapt the 20-ml syringe to the catheter and slowly inject intragastrically 5 ml of
25 mg/ml CaCO3 solution.
CaCO3 is used to reduce the gastric acidity and to diminish variability between animals.
Agents to be administered must be in suspension or solution, and at room to body
temperature.
13. Replace the 20-ml syringe with the 1-ml syringe without removing the catheter, and
slowly inject intragastrically 1 ml of inoculum.
14. After completing the injection, carefully withdraw the catheter and place the inoculated guinea pig in a spare cage and continue injections with additional animals.
While the animal is anesthetized and nonreactive, pay attention not to put the animals
eyes or nose in contact with the sawdust or other bedding material.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.7
Current Protocols in Microbiology
Supplement 10
15. When injections are complete, check the animals for full recovery and normal behavior after anesthesia, and then return them to their housing location.
16. Discard the needle, syringes, and the catheter. Disinfect work instruments and work
area.
Animals are then either observed every day for determination of mortality (see Basic
Protocol 4), or they are euthanized and dissected for determination of bacterial counts
from organs (see Basic Protocol 3).
Perform calculations
17. One or two days after incubation of plates (step 4) at 37 C, calculate the initial titer
of bacterial stock suspension (see Basic Protocol 1, step 12) and then calculate the
Listeria load using the dilution factor of the IG suspension (see step 3 above).
BASIC
PROTOCOL 3
Materials
Infected 6- to 8-week-old sex-matched mice (Basic Protocol 1 or Basic Protocol 2)
70% ethanol in a spray bottle
Phosphate-buffered saline (PBS), sterile
Dulbeccos minimum essential medium (DMEM)
100 mg/liter gentamicin in sterile PBS, prepare fresh
Sterile Brain Heart Infusion (BHI) medium
BHI agar petri plates, sterile
Animal Models of
Listeria Infection
Personal protective equipment (e.g., laboratory coat, gloves, and eye protection)
15- or 50-ml conical polypropylene centrifuge tubes, sterile
Sterile dissection instruments including:
Medium dissection scissors
Tissue forceps
Small iris scissors
Iris forceps
Small-toothed forceps
Surgical scissors
Dissecting pins
9B.1.8
Supplement 10
Obtain organs
3. Euthanize the infected mouse by CO2 inhalation (Donovan and Brown, 2006c).
Carefully verify that the mouse is dead.
4. In a Class II biohazard hood, Place the mouse on to its back on Styrofoam or cork
board covered with a paper towel, and wet its fur with 70% ethanol to minimize
contamination with hair.
5. Pin extremities to Styrofoam or cork board to immobilize animal.
6. With forceps, grab hold of the skin above the urethral opening. Using small iris
scissors, cut along the midline from the groin to the chin.
Be careful to only cut the skin and not the muscle wall underneath.
7. While still grasping the lower peritoneum, make an incision from the start of the first
incision to down near the knee on both sides of the animal.
You should end up with an incision that looks very similar to an upside down Y. Take care
not to cut the bladder or the intestines.
8. Peel the skin back from the muscle and peritoneal wall underneath.
9. Open up the abdomen by making an incision in the midline of the peritoneal wall
and peeling it back.
10. Remove the spleen, which is behind the stomach and attached to the blood circulation
by a single set of blood vessels (splenic artery and vein). Put the spleen into a 15-ml
(mouse) or 50-ml (guinea pig) ice-cold empty sterile tube and place on ice.
11. With small-toothed forceps, grasp underneath the right lateral lobe of the liver to a
point across the inferior vena cava and the hepatic hilum.
12. Use iris scissors to gently flip these lobes back over the forceps to grasp these lobes
as well as the common bile duct.
13. With a firm hold on these tissues, make an incision with the scissors between the
liver and the diaphragm and cut the inferior vena cava, freeing the liver.
14. Extricate the liver as one organ with a quick but gentle upward motion. Place the
liver in a 15-ml (for mouse) or 50-ml (for guinea pig) ice-cold empty sterile tube and
place on ice.
15. Using forceps, lift the intestines and move them to your left side.
16. Identify the bottom end of the stomach (pylorus), cut through the intestine and
remove the entire small and large intestines.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.9
Current Protocols in Microbiology
Supplement 10
17. Remove the whole mesenteric lymph nodes. Put the mesenteric lymph nodes in a
15-ml (for mouse) or 50-ml (for guinea pig) ice-cold empty sterile tube and place on
ice.
The mesenteric lymph nodes can be seen lying along the blood vessels in the fatty tissue
that connects the intestine to the back wall of the abdomen.
18. Remove the whole small intestine of the mouse, or the central 20-cm-long portion
of the guinea pig small intestine.
19. Open the small intestine longitudinally, remove the intestinal content and put the
small intestine in a 15-ml (mouse) or 50-ml (guinea pig) ice-cold tube containing
5 ml (for the 15-ml tubes) or 10 ml (for the 50-ml tubes) of DMEM and place on ice.
20. Eliminate dead animals following appropriate guidelines and regulations for the
elimination of infected animals.
21. Rinse the small intestine in sterile DMEM four times to remove all the luminal
content.
DMEM is preferable to PBS because it better preserves the integrity of the tissue, which
is critical until the gentamicin treatment ends.
22. Incubate 2 hr at 20 C in DMEM containing 100 mg/liter gentamicin to kill extracellular bacteria from the intestinal lumen.
23. Rinse three times, each time in sterile DMEM. Put the small intestine in a 15-ml
(mouse) or 50-ml (guinea pig) ice-cold tube and place on ice.
24. Add sterile PBS to each tube so that the final volume is equal for each organ series
(usual final volume is 5 ml for mice organs, and 10 ml for guinea pig organs).
25. In a biohazard hood, grind the tissue and homogenize lysate using a rotor-stator
homogenizer (IKA UltraTurrax T25, or similar) until sample is uniformly homogeneous, which usually takes 45 to 60 sec.
Between samples disinfect the homogenizer tip with 70% ethanol and wash with sterile
PBS. Disinfect the homogenizer and the work surfaces of the hood at the end of the
homogenization.
26. Determine the number of viable bacteria released from the organs by performing
serial dilutions of the organ homogenates in sterile BHI.
The total number of dilutions depends on the expected bacterial loads within the organs
and the time points post-infection.
27. Apply 0.1 ml of each dilution in duplicate to BHI agar petri plates, and incubate 24
to 48 hr at 37 C.
Perform calculations
28. After incubation, determine the bacterial load within the organ using the following
formula: cfu/organs = (MCC VH)/(VP DFP).
Where MCC is the mean colony count on duplicate plates, VH is the total volume of the
homogenate, VP is the volume plated, and DFP is the dilution factor of sample in the
plates.
BASIC
PROTOCOL 4
Animal Models of
Listeria Infection
DETERMINATION OF MORTALITY
The classical LD50 test is used to determine the lethal dose of an inoculum that will kill
50% of test animals (LD50). The inoculum is administered at increasing doses, usually
a range of 5, to groups of 5 animals. Mortalities are recorded along the time, and the
9B.1.10
Supplement 10
Materials
Bacterial inoculum (Support Protocol)
Phosphate-buffered saline (PBS), sterile
BHI agar plates, sterile
37 C incubator
Additional reagents and equipment for the euthanasia of rodents (Donovan and
Brown, 2006c)
1. Just before injection, thaw an aliquot of bacteria on ice. Dilute bacteria appropriately
in sterile PBS to achieve increasing doses to be administered.
For BALB/c mice infected intravenously by L. monocytogenes EGDe, typically doses are
103 , 104 , 105 , 106 , 107 cfu per mouse.
2. To confirm the titer of the starting suspension, plate, in duplicate, 100 l of serial
dilutions on sterile BHI agar plates, and incubate the plate 24 to 48 hr at 37 C.
3. Infect animals using one of the inoculation protocols presented in this unit (see Basic
Protocols 1 and 2).
4. Each day following infection, record mortality and eliminate dead animals following
appropriate guidelines and regulations for the elimination of infected animals.
The IACUC of some institutions would ask for euthanasia of moribund animals in an
LD50 assay. Refer to your institutional guidelines for more information.
SUPPORT
PROTOCOL
Materials
Single colony of Listeria cells
Sterile Brain Heart Infusion (BHI) medium
Phosphate-buffered saline (PBS), sterile
BHI agar plates
250-ml Erlenmeyer flask
37 C shaking incubator
2-liter flask
50-ml polypropylene conical tubes, prechilled
Microtubes, prechilled
Current Protocols in Microbiology
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.11
Supplement 10
1. Inoculate a single colony of Listeria cells into 50 ml sterile BHI medium in a 250-ml
Erlenmeyer flask. Grow overnight at 37 C with shaking (200 rpm).
Alternatively, grow a 5 ml culture overnight in a test tube on a roller drum at 37 C.
2. Inoculate 4 ml of the culture into 400 ml sterile BHI medium in a sterile 2-liter flask.
Grow at 37 C, shaking 200 rpm, to an OD600 nm of 0.7 to 0.8.
3. Aliquot culture into eight 50-ml prechilled, sterile polypropylene tubes and leave the
tubes on ice 5 to 10 min.
Cells should be kept cold for all subsequent steps.
Larger tubes or bottles can be used to centrifuge cells if volumes of all subsequent
solutions are increased in direct proportion.
8. To determine the actual number of bacteria per milliliter of the frozen inoculum, thaw
an aliquot of the bacteria on ice, and plate 100 l of serial dilutions, in duplicate, on
BHI agar plates and incubate overnight at 37 C.
9. One or two days after incubation at 37 C, count colonies on plates and calculate the
titer of the suspension as follows: cfu/ml = MCC/(VP DF).
Where MCC is the mean colony count of replicate countable plates, VP is the volume
plated, and DF is the dilution factor of the sample in the plate.
COMMENTARY
Background Information
Animal Models of
Listeria Infection
Listeria monocytogenes is an intracellular foodborne pathogen that causes listeriosis, an infection characterized by gastroenteritis, meningitis, encephalitis, and maternofetal
infections in humans. L. monocytogenes enters the host via contaminated foods, invades
the small intestine, translocates to mesenteric
lymph nodes and spreads to the liver, spleen,
brain and, in pregnant women, the fetoplacental unit. This bacterium has the ability to cross
three tight barriers: the intestinal, the bloodbrain, and the placental barriers. During infection, it enters, survives, and multiplies inside
phagocytic and nonphagocytic cells (Khelef
et al., 2004; Hamon et al., 2006; Cossart and
Toledo-Arana, 2008).
9B.1.12
Supplement 10
is widely used in the study of cellular immunity against Listeria (Pamer, 2004; Zenewicz
and Shen, 2007).
The consequences of foodborne listeriosis being particularly devastating during pregnancy (Mylonakis et al., 2002), and airway
infections with Listeria monocytogenes being implicated in early onset neonatal listeriosis, the fetoplacental transmission of Listeria has been studied in the pregnant mouse
or guinea pig (Abram et al., 2003; Bakardjiev
et al., 2004). In mice, the reproducible infection of the placenta occurred when mice were
intravenously infected at day 14 of pregnancy,
and killed on day 1, 2, 3, or 6 post infection
which corresponded to days 15, 16, 17, and
20 of pregnancy (Abram et al., 2003; Le Monnier et al., 2006). Alternatively, the pregnant
guinea pig animal model can be used to study
the fetoplacental transmission of L. monocytogenes. Pregnant female Hartley guinea pig
of 42 to 52 days gestation are injected intravenously with 108 bacteria after subcutaneous
injection of 0.05 mg/kg atropine and anesthesia with isoflurane (Bakardjiev et al., 2005).
Pregnant guinea pigs can be also used to study
the dose response to L. monocytogenes after
oral exposure (Williams et al., 2007). However, mice and guinea pigs do not appear to
be as susceptible as ovines, caprines, bovines,
and humans to fetoplacental listeriosis (Lecuit,
2007).
While human listeriosis in most cases starts
as an oral infection caused by contaminated
food, focal infections are also observed. Especially in early onset neonatal listeriosis, high
numbers of Listeria are detected in the lung of
the newborn, most likely due to uptake of bacteria from contaminated amniotic fluid. Thus,
although not observed in adults, the lung represents a potential port of entry for L. monocytogenes, at least in newborns contaminated
in utero or during parturition. Listeriosis can
be studied following intratracheal infection of
mice. 10- to 12-week-old female mice were
inoculated with 30 l of bacterial inoculum
via view-controlled intratracheal instillation
through a catheter (0.7 to 19 mm) towards the
bifurcation of the trachea to ensure exclusive
delivery to the lungs. After intratracheal application, bacteria were predominantly localized in the peribronchiolar space and invaded
alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into
the deep organs started almost immediately
after application, although a pulmonary
bacterial reservoir remained during the first
4 days (Munder et al., 2005).
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.13
Current Protocols in Microbiology
Supplement 10
Critical Parameters
Animal Models of
Listeria Infection
Anticipated Results
Bacterial stock suspensions grown on BHI
as described reach titers in the range of 1011
cfu/ml. Repeated freezing and thawing reduces titers and should be avoided.
After infection of animals, the magnitude
of organ titers depends on the bacterial strain
used and on the susceptibility of the animal
strain. Due to the intrinsic inter-experiment
variability of in vivo experiments, it is absolutely critical that adequate controls are
included when performing infection experiments (e.g., comparison to wild-type bacteria
when analyzing bacterial mutants, comparison
to normal or wild-type animals when infecting
immunosuppressed or mutant animals).
Mouse intragastric infection
Female iFABP-hEcad challenged intragastrically with the laboratory strains L. monocytogenes EGDe at a dose of 109 cfu develop, as
of 24 hr after infection, an actively growing infection in the intestinal tissue (106 cfu) that
reaches a load of 107 cfu 72 hr after infection (Fig. 9B.1.1). After invasion of the small
intestine from the intestinal lumen, bacteria
are subsequently transported via the blood to
regional lymph nodes. When they reach the
liver and the spleen, most Listeria are rapidly
killed. Surviving Listeria then multiply and
grow exponentially in the spleen and liver for
the next 48 hr. Maximum counts are observed
72 to 96 hr post-infection (106 cfu in the liver
and spleen). The protective immune response
begins to contain the infection by about hour
96. Bacteria then start to be cleared, leading to
their eradication from all organs if the kinetics
is followed for additional days.
9B.1.14
Supplement 10
Intestine
Lymph nodes
Liver
Spleen
8
7
6
5
4
3
0
24
48 72 96 120 0
24
48 72 96 120 0 24
Time (hr)
48 72 96 120 0
24
48 72 96 120
Figure 9B.1.1 Course of infection in iFABP-hEcad mice following intragastric inoculation with 109 cfu of
L. monocytogenes EGDe.
Liver
Spleen
10
9
8
7
6
5
4
0
24
48 72 96 120 0 24
Time (hr)
48 72 96 120
Figure 9B.1.2 Course of infection in BALB/c mice following intravenous inoculation with 104 cfu
of L. monocytogenes EGDe.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.15
Current Protocols in Microbiology
Supplement 10
Intestine
Lymph nodes
Liver
Spleen
7
6
5
4
3
2
0
24 48 72 96 120 144 0
24 48 72 96 120 144
Figure 9B.1.3 Course of infection in Hartley guinea pigs following intragastric inoculation with 1010 cfu of
L. monocytogenes EGDe.
Time Considerations
Literature Cited
Contact Information
Animal Models of
Listeria Infection
9B.1.16
Supplement 10
Hamon, M., Bierne, H., and Cossart, P. 2006. Listeria monocytogenes: A multifaceted model.
Nat. Rev. Microbiol. 4:423-434.
Firmicutes (Low
G+
+C Gram
Positive)
9B.1.17
Current Protocols in Microbiology
Supplement 10
UNIT 9B.4
Listeria monocytogenes is a facultative intracellular bacterial pathogen that has the ability to invade and multiply in a wide variety of cell types, including professional and
nonprofessional phagocytic cells. Many tissue culture cell assays have been developed
over the years to evaluate the virulence of L. monocytogenes isolates and mutant strains.
These assays enable the dissection of steps leading to a successful infection: invasion,
escape from primary vacuoles, intracellular growth, and cell-to-cell spread.
Invasion of host cells is a primary and essential step of the infection cycle of intracellular bacterial pathogens. L. monocytogenes has the ability to invade nonprofessional
phagocytic cells such as epithelial cells, hepatocytes, fibroblasts, and trophoblasts, as
well as professional phagocytic cells such as macrophages (Portnoy et al., 1988; Gaillard
et al., 1996; Bakardjiev et al., 2004). Invasion of cells by L. monocytogenes occurs by
the zipper mechanism, requiring interaction of a bacterial ligand with a host cell receptor
to cause a cytoskeletal response leading to engulfment of the bacterial cell (Swanson
and Baer, 1995). Once inside a professional or nonprofessional phagocytic host cell, L.
monocytogenes resides in a membrane-bound vacuole for a short period of time. Escape
from this vacuole is a prerequisite for the infection cycle as L. monocytogenes is incapable of multiplying in a vacuole (Gaillard et al., 1987). Following escape from primary
vacuoles, L. monocytogenes efficiently multiplies in the cytosol of its host with an initial
doubling time of <1 hr (Portnoy et al., 1988). Another feature of the L. monocytogenes
intracellular life cycle is its ability to use host cell actin for motility. Actin polymerization
at the bacterial surface begins shortly after bacteria escape from primary vacuoles. Actin
filaments initially form a cloud around each bacterium but eventually form a tail at one
end of the bacterial cell (Tilney and Portnoy, 1989; Mounier et al., 1990). Polarized actin
assembly enables bacteria to move and spread directly from cell to cell by inducing the
formation of membrane filopodia, which are taken up by neighboring cells. Spreading
bacteria are temporarily located in double membrane vacuoles, also called secondary
vacuoles, from which they must escape to access the cytosol of the new host cell and
perpetuate the infection cycle.
This unit is comprised of four tissue culture cell assays evaluating in a quantitative
and qualitative manner every step of the infectious cycle of L. monocytogenes: host
cell invasion, escape from vacuoles, intracellular growth, and cell-to-cell spread. Basic
Protocol 1 describes an assay measuring invasion by L. monocytogenes into Caco-2
cells, nonprofessional phagocytic cells derived from human intestinal epithelia. Basic
Protocol 2 describes an assay to quantify the efficacy of L. monocytogenes to escape
from vacuoles of mouse macrophages; access to a fluorescence microscope is required
for this assay. Basic Protocol 3 is used to determine the kinetics of L. monocytogenes
growth in mouse macrophages. Lastly, Basic Protocol 4 describes a plaquing assay to
evaluate bacteria efficacy to spread directly from cell to cell. These assays can be adapted
for use with other cell lines or with primary cells.
CAUTION: Listeria monocytogenes is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
Firmicutes (Low
G+C Gram
Positive)
Contributed by Hel`ene Marquis
Current Protocols in Microbiology (2006) 9B.4.1-9B.4.25
C 2006 by John Wiley & Sons, Inc.
Copyright
9B.4.1
Supplement 1
CAUTION: No prophylactic measures, except hand washing, are required for personnel
working with L. monocytogenes. However, individuals should be informed of the risk
of acquiring listeriosis, especially for immunocompromised individuals and pregnant
women. If an individual contaminates him- or herself, 70% alcohol or iodine should be
used to decontaminate the infected area. Depending on the circumstances, the individual
may be advised to see a physician. The following CDC and USDA Websites provide information on listeriosis: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis g.htm and
http://www.fsis.usda.gov/Fact Sheets/Listeriosis and Pregnancy What is Your Risk/
index.asp.
BASIC
PROTOCOL 1
Materials
95% ethanol
100 g/ml type I rat tail collagen (see recipe)
CaCl2 and MgCl2 -free Dulbeccos PBS (CMF-DPBS; GIBCO, also see
APPENDIX 2A), sterile
Caco-2 cells (ATCC #HTB-37), trypsinized
Caco-2 medium (see recipe), 4 and 37 C
BHI medium (see recipe)
H2 O, sterile
Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working
with L. monocytogenes, or clinical isolates)
LB plates (APPENDIX 4A)
10 mg/ml gentamicin (see recipe)
Diff-Quick stain set (Dade Behring)
Permount (Fisher)
12-mm round, precleaned glass coverslips in 70% ethanol
35-mm tissue culturetreated dishes (BD Falcon)
Pasteur pipets, sterile
15- and 50-ml polypropylene conical tubes, sterile
37 C, 5% CO2 humidified incubator
37 C water bath
30 and 37 C incubators
1.5-ml microcentrifuge tubes, sterile
15-ml polystyrene conical tubes, sterile
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.2
Supplement 1
Prepare coverslips
1. Dip the tip of a pair of forceps in 95% ethanol and flame to sterilize.
Use caution not to heat the forceps.
2. Pick a single 12-mm round glass coverslip by the edge and dip in 95% ethanol. Blot
excess ethanol by touching the edge of the coverslip to a Kimwipe.
Blot as much ethanol off as possible before flaming.
4. Repeat steps 2 and 3 until there are four coverslips per dish.
5. Cover coverslips with 1.5 to 2 ml of 100 g/ml type I rat tail collagen. Push coverslips
down, as they will tend to float. Incubate for a minimum of 1 hr at room temperature.
6. Aspirate collagen solution with a sterile Pasteur pipet and rinse coverslips once with
2 ml sterile CMF-DPBS. Keep dry and use within 1 hr.
8. Determine volume of cell suspension needed for the experiment. Calculate 3 105
cells per bacterial strain to test.
The number of Caco-2 cells needed will vary according to cell age and growth rate. Young
cells have a slower growth rate. In general, the objective is to obtain a monolayer of cells
that is 70% confluent at time of infection. Fully confluent monolayers do not become
well-infected and tend to lift from the coverslips during washes.
9. Transfer volume of cell suspension needed into a 50-ml conical tube and adjust the
cell concentration to 1.5 105 cells/ml by adding cold Caco-2 medium. Mix well
by pipetting up and down.
10. Distribute 2 ml cell suspension (3 105 cells) per dish containing four coverslips
treated with rat-tail collagen (step 6). Gently swirl the dish to distribute the cells over
the coverslips. Incubate overnight in a 37 C, 5% CO2 humidified incubator.
Coverslips may need to be pushed down with sterile forceps if they move around, float, or
overlap.
11. The next morning, aspirate medium and replace with fresh Caco-2 medium equilibrated to 37 C (i.e., prewarmed in a water bath). Monitor the status of the cells using
an inverted microscope.
Cells should be spread out and 70% confluent.
A compacted cell monolayer tends to lift off from glass coverslips during washes.
9B.4.3
Current Protocols in Microbiology
Supplement 1
13. With the cap on loose, place the tube on its side with a slight slant (5 ) in a 30 C
incubator. Incubate overnight (14 to 16 hr).
L. monocytogenes invades better when grown at 30 C as opposed to 37 C.
The tubes are slanted to avoid spillage of the culture in the incubator. Fold a paper towel
to make a 2- to 3-cm-wide holder. Rest the top of the tube on the folded paper towel.
14. Following incubation, briefly vortex bacterial culture and transfer 1 ml into a sterile
1.5-ml microcentrifuge tube.
15. Pellet bacteria by microcentrifuging 1 min at maximum speed, room temperature.
16. Aspirate supernatant and suspend bacteria in 1 ml CMF-DPBS. Vigorously vortex
to break up bacterial clumps.
17. Repeat steps 15 and 16.
18. Determine the initial inoculum in the following manner.
a. Prepare a 106 dilution of the bacterial suspension, making two series of 1/1000
dilutions into sterile water. Be sure to vortex samples very well and change pipet
tips between dilutions for accuracy.
b. Spread 50 l of this 106 dilution onto an LB plate until all liquid is absorbed.
Prepare in triplicate for accuracy.
c. Repeat for each bacterial strain tested.
L. monocytogenes is a Gram-positive bacterial species with a 25- to 50-nm thick and
highly cross-linked cell wall, which confers a strong resistance to lysis in water.
The surface of the LB agar plates should be dry for better absorption. It helps to let the
plates dry for 24 to 48 hr at room temperature before use. Alternatively, plates can be
cracked open, agar facing down, and dried for 5 to 30 min at 37 C.
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.4
Supplement 1
29. Record numbers of colony forming units (cfu) after incubating the plates for 24 hr
at 37 C or 2 days at room temperature.
Ideally, there should be between 50 and 200 cfu per plate.
Lysates of infected cells can be generated in 2.5 ml of water rather than 5 ml as for
bacterial strains defective in invasion. Alternatively, cells may be infected with more
bacteria (see step 19).
The surface area of one coverslip represents 12% of the surface area of a 35-mm tissue
culture dish. This number needs to be taken into consideration when calculating absolute
invasion levels.
33. Calculate the bacterial invasion level of a test strain (or mutant) relative to a reference
strain (or wild-type) as follows:
Firmicutes (Low
G+C Gram
Positive)
9B.4.5
Current Protocols in Microbiology
Supplement 1
Use relative percentages or ratios to analyze data from different experiments because of
variability in absolute cfu numbers between experiments.
BASIC
PROTOCOL 2
Materials
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
95% ethanol
J774 cells (ATCC #TIB-67)
J774 medium with antibiotics (see recipe for J774 and L2 medium), 37 C
CaCl2 - and MgCl2 -free Dulbeccos PBS (DMF-DPBS, GIBCO; also see APPENDIX
2A), sterile, 4 C and room temperature
J774 medium without antibiotics, 4 and 37 C
BHI medium (see recipe)
Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working
with L. monocytogenes, or clinical isolates)
10 mg/ml gentamicin (see recipe)
Formalin (see recipe)
TBS-TX (see recipe)
9B.4.6
Supplement 1
2. Pick a single 18-mm square glass coverslip by the edge and dip in 95% ethanol. Blot
excess ethanol by touching the edge of the coverslip to a Kimwipe.
Blot as much ethanol off as possible before flaming.
Firmicutes (Low
G+C Gram
Positive)
9B.4.7
Current Protocols in Microbiology
Supplement 1
8. Determine volume of cell suspension needed for the experiment. Calculate 0.8
106 cells per dish to be prepared.
9. Transfer volume of cell suspension needed for the experiment into a 50-ml conical
tube and, using a precooled tabletop centrifuge, centrifuge cells 10 min at 200 g,
4 C.
10. Carefully aspirate CMF-DPBS with a Pasteur pipet by tilting the tube on its side and
keeping the tip of the pipet away from the pellet. Work quickly to avoid aspirating
cells as the pellet is relatively soft.
11. Suspend cells in cold antibiotic-free J774 medium to a final concentration of 0.4
106 cells/ml. Pipet cell suspension up and down twelve to fifteen times to break up
clumps. Avoid generating foam as much as possible.
From this point, always use antibiotic-free J774 medium.
12. Immediately transfer 2 ml cell suspension (0.8 106 cells) into a 35-mm dish
containing one square coverslip (step 3). Gently swirl the dish to distribute the
cells over the coverslip and incubate dishes in a 37 C, 5%CO2 humidified incubator
overnight.
Push coverslip down if necessary.
13. Next morning, push coverslips down but avoid scratching the monolayer. Monitor
the cell status using an inverted microscope.
Ideally, cells should be spread out, not clumpy, and 70% to 80% confluent.
16. Following incubation, briefly vortex the bacterial culture and transfer 1 ml into a
1.5-ml microcentrifuge tube.
17. Pellet bacteria by microcentrifuging 1 min at maximum speed, room temperature.
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.8
Supplement 1
25. Fix the cells by replacing the last CMF-DPBS wash with 2 ml formalin for a minimum
of 10 min at 4 C. If the staining is to be done the next day, seal the dish with Parafilm
and keep the cells in formalin in the dark at 4 C (up to 24 hr).
30. Transfer coverslips into a humid chamber cell-side up and immediately block by
adding 200 to 400 l of detergent-free antibody buffer to each coverslip. Incubate
10 to 30 min at room temperature.
It is important that the coverslips never dry during the staining process.
Firmicutes (Low
G+C Gram
Positive)
9B.4.9
Current Protocols in Microbiology
Supplement 1
31. Aspirate antibody buffer and replace with 50 l rabbit anti-Listeria antibody diluted
1:500 in detergent-free antibody buffer. Incubate 30 to 60 min at room temperature.
There are different antisera for different serotypes of L. monocytogenes.
32a. To wash using the drop/suck method: Drop 200 to 400 l of TBS onto each coverslip
and gently aspirate the liquid from the side of the coverslip using a pipet tip
connected to a vacuum line. Wash with a total of 3 to 6 ml of buffer per coverslip.
32b. To wash by dipping: Dip coverslips several times into a 50-ml conical tube containing TBS.
33. Add 50 l secondary antibody (goat anti-rabbit IgG) conjugated to Alexa Fluor
350 diluted in detergent-free antibody buffer according to the manufacturers recommendations. Incubate 30 to 60 min at room temperature.
There are many different conjugated secondary antibodies commercially available. Likewise, secondary antibodies from animal species other than goat can be used.
42. Dispose of the wet Kimwipes in the humid chamber and dry the coverslips for 5 to
10 min in the dark at room temperature.
43. Mount each coverslip cell-side down on a drop of Prolong antifade in the middle
of a glass slide. Dry overnight in the dark at room temperature. Once dry, seal the
edges of coverslips with nail polish.
Coverslips mounted with Prolong antifade can be stored in a dry slide box for several
months at 20 C.
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
44. Using a fluorescence microscope, count at least 200 intracellular bacteria (stained
with red fluorophore only) per sample and, for each counted intracellular bacterium,
determine if it is decorated with actin filaments (stained with green fluorophore)
by switching to the appropriate filter on the microscope. Do not count extracellular
bacteria, which are stained with blue and red fluorophores.
9B.4.10
Supplement 1
Alexa Fluor 350 is a blue fluorophore that absorbs light at 346 nm and emits at 442
nm. Alexa Fluor 488 is a green fluorophore that absorbs light at 495 nm and emits at
519 nm. Alexa Fluor 568 is a red fluorophore that absorbs light at 568 nm and emits at
603 nm.
BASIC
PROTOCOL 3
Materials
95% ethanol
J774 cells (ATCC #TIB-67)
J774 medium with antibiotics (see recipe for J774 and L2 medium), 37 C
CaCl2 - and MgCl2 -free Dulbeccos PBS (CMF-DPBS, GIBCO; also see
APPENDIX 2A), sterile, 4 C and room temperature
J774 medium without antibiotics, 4 and 37 C
BHI medium (see recipe)
Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researcher working
with L. monocytogenes, or clinical isolates)
10 mg/ml gentamicin (see recipe)
LB plates (APPENDIX 4A)
Diff-Quick stain set (Dade Behring)
Permount (Fisher)
12-mm round pre-cleaned glass coverslips in 70% ethanol
60-mm petri dishes (Nunc)
75-cm2 nontissue culturetreated flasks
Pasteur pipets, sterile
15- and 50-ml polypropylene conical tubes, sterile
Refrigerated tabletop swinging-bucket centrifuge, 4 C
37 C, 5% CO2 incubator
30 and 37 C incubators
1.5-ml microcentrifuge tubes, sterile
37 C water bath
15-ml sterile polystyrene conical tubes
Plotting software (e.g., Prism, Cricket Graph, or Excel)
Additional reagents and equipment for counting cells in a hemacytometer
(APPENDIX 4A)
Prepare coverslips
1. Dip the tip of a pair of forceps in 95% ethanol and flame to sterilize.
Use caution not to heat the forceps.
2. Pick a single 12-mm round glass coverslip by the edge and dip in 95% ethanol. Blot
excess ethanol by touching the edge of the coverslip to a Kimwipe.
Blot as much ethanol off as possible before flaming.
Firmicutes (Low
G+C Gram
Positive)
9B.4.11
Current Protocols in Microbiology
Supplement 1
3. Flame leftover ethanol on coverslip and transfer to a 60-mm petri dish. Prepare one
dish per bacterial strain to be tested.
If the coverslip becomes too hot, it will crack
Up to fifteen 12-mm coverslips can fit in a dish.
8. Determine volume of cell suspension needed for the experiment. Calculate 1.5 106
cells per dish to prepare.
9. Transfer volume of cell suspension needed for the experiment into a 50-ml conical
tube and, using a precooled tabletop centrifuge, centrifuge cells 10 min at 200 g,
4 C.
10. Carefully aspirate CMF-DPBS with a Pasteur pipet by tilting the tube on its side and
keeping the tip of the pipet away from the pellet. Work quickly to avoid aspirating
cells as the pellet is relatively soft.
11. Suspend cells in cold antibiotic-free J774 medium to a final concentration of 0.25
106 cells/ml. Pipet cell suspension up and down twelve to fifteen times to break up
clumps. Avoid generating foam as much as possible.
From this point forward, always use antibiotic-free J774 medium.
If doing a large experiment, it is easier to initially suspend cells and break up the clumps
in 10 to 12 ml medium, then adjust the volume of the cell suspension.
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.12
Supplement 1
12. Immediately transfer 6 ml cell suspension (1.5 106 cells) into a 60-mm dish
containing coverslips (step 3). Gently swirl the dish to distribute the cells over the
coverslips (step 3).
Coverslips will need to be pushed down with sterile forceps. Make sure coverslips do not
overlap.
17. Following incubation, briefly vortex bacterial culture and transfer 1 ml into a 1.5-ml
microcentrifuge tube.
18. Pellet bacteria by microcentrifuging 1 min at maximum speed, room temperature.
19. Aspirate supernatant and suspend bacteria in 1 ml CMF-DPBS. Vigorously vortex
to break up bacterial clumps.
20. Repeat steps 18 and 19.
21. Dilute the suspension of washed bacteria 1:20 in CMF-DPBS.
Firmicutes (Low
G+C Gram
Positive)
9B.4.13
Current Protocols in Microbiology
Supplement 1
t = 2 hr post-infection: 25 to 50 l
t = 5 hr post-infection: 2.5 to 5 l
t = 8 hr post-infection: 25 to 50 l of 1:100 dilution
t = 11 hr post-infection: 10 to 25 l of 1:100 dilution.
It is important to vortex each cell lysate for 10 to 20 sec just prior to taking an aliquot
for plating.
The volume to be plated may vary according to each bacterial strain tested.
28. Using a Diff-Quick stain set, stain coverslip at 4.5, 7.5, and 10.5 hr post-infection
for visual evaluation of the intracellular infection. After staining, allow coverslip to
air dry and mount on glass slide cell-side down on a drop of Permount.
29. Examine stained infected cells by bright-field microscopy using an upright
microscope.
Do not use an oil objective until Permount is solidified (24 hr).
Average numbers for the three coverslips for a specific time point for each strain tested.
31. Plot the growth curve using programs such as Prism, Cricket Graph, or Excel. Use
a log scale to report the number of bacteria per coverslip on the y axis, and a linear
scale to mark time points on the x axis.
32. Calculate the doubling time ( ) between time points using the following equations:
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.14
Supplement 1
where, t is the time elapsed between two time points, y0 is the number of cfu per
coverslip at the first time point, and y(t) is the number of cfu per coverslip at the
second time point.
CELL-TO-CELL SPREAD
This protocol describes a tissue culturecell plaquing assay to evaluate L. monocytogenes
efficacy at spreading from cell to cell (Sun et al., 1990). The most appropriate cell line
to use for this plaquing assay is the mouse fibroblast L2 cell line, which is a variant
of the L929 line (Dales, 1962). Mouse L2 cells are relatively round when grown in
tissue culturetreated dishes and form nicely demarcated plaques when infected with L.
monocytogenes. This cell line can generally be obtained from scientists working with
Corona viruses or with L. monocytogenes. For the assay, mouse L2 cells in six-well tissue
culturetreated plates are infected with L. monocytogenes. Infected cells are washed
with Ca2+ - and Mg2+ -free DPBS (CMF-DPBS) at 60-min post-infection to eliminate
free bacterial cells, then overlaid with tissue culture medium mixed with agarose and
gentamicin. After a 4-day incubation period, a second overlay containing neutral red is
added to each well. Neutral red diffuses down staining live host cells at the bottom of
the wells and revealing clear plaques formed by foci of infection. Plates are scanned and
plaques measured using commercially available software.
BASIC
PROTOCOL 4
Materials
L2 mouse fibroblast cell line (individual researchers working with Corona viruses
or with L. monocytogenes), trypsinized
L2 medium (see recipe for J774 and L2 medium) without antibiotics, 4 C
BHI medium (see recipe)
Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working
with L. monocytogenes or clinical isolates, sterile)
Dulbeccos PBS CaCl2 - and MgCl2 -free (CMF-DPBS, GIBCO; see APPENDIX 2A)
2 L. monocytogenes plaquing medium (see recipe), 37 C
10 mg/ml gentamicin (see recipe)
1.4% (w/v) agarose: autoclave and allow to harden
1 N HCl
0.5% neutral red solution, pH 5.2 (Sigma)
15- and 50-ml conical polypropylene tubes, sterile
6-well tissue culturetreated plates or 35-mm tissue culturetreated dishes
37 C, 5% CO2 humidified incubator
30 C incubator
1.5-ml microcentrifuge tubes, sterile
37 C water bath
50 to 55 C water bath
Pasteur pipets, sterile
Scanner
Plaque measuring software: Adobe Photoshop or equivalent
Additional reagents and solutions for counting cells in a hemacytometer
(APPENDIX 4A)
Prepare L2 cells
1. Suspend trypsinized mouse L2 cells in antibiotic-free L2 medium and count using a
hemacytometer (APPENDIX 4A).
A confluent 25-cm2 flask contains 89 106 cells.
Firmicutes (Low
G+C Gram
Positive)
9B.4.15
Current Protocols in Microbiology
Supplement 1
2. Determine volume of cell suspension needed for the experiment. Calculate 0.7
106 cells per bacterial strain to test.
The number of cells to put down varies according to cell age and growth rate. Young cells
have a slower growth rate in general. The objective is to obtain a monolayer of cells that
is 80% to 90% confluent at the time of infection.
3. Transfer volume of cell suspension needed into a 50-ml conical tube and adjust the
cell concentration to 0.35 106 cells/ml with cold, antibiotic-free L2 medium.
4. Distribute 2 ml cell suspension (0.7 106 cells) per well of a 6-well tissue culture
treated plate or 35-mm tissue culturetreated dish.
5. Incubate 40 to 48 hr in a 37 C, 5% CO2 humidified incubator.
Cell monolayer should be 70% to 90% confluent.
8. On the next morning, briefly vortex bacterial cultures and make a 1:100 dilution in
Ca2+ - and Mg2+ -free DPBS (CMF-DPBS) in 1.5-ml sterile microcentrifuge tubes.
9. Infect cells (step 5) with 10 to 20 l of a 1:100 bacterial dilution (1:10,000 to 1:20,000
final dilution), and incubate 1 hr in a 37 C, 5% CO2 humidified incubator.
During incubation, pepare L2 overlay (steps 10 to 16).
16. Mix the 2 plaquing medium containing gentamicin (step 11) with the 1.4% melted
agarose in a 1:1 ratio. Transfer 3 ml to each well and return plate to the 37 C, 5%
CO2 humidified incubator for 4 days.
9B.4.16
Supplement 1
19. Add 600 l of 0.5% neutral red solution per 10 ml overlay (final neutral red concentration of 0.03% w/v).
Neutral red tends to precipitate if solution is too alkaline or if added to 2 plaquing
solution.
20. Transfer 2 ml to each well on top of the first overlay and return to the 37 C, 5% CO2
humidified incubator for 6 to 8 hr.
Neutral red diffuses in the overlay and stain cells at the bottom of the wells revealing
clear plaques.
Use relative percentages or ratios to analyze data from different experiments because
of variability in absolute plaque size between experiments.
Antibody buffer
10 ml TBS or TBS-TX (see recipe)
100 mg BSA (1% w/v final)
Dissolve on rocker at 4 C
Prepare fresh each day and keep on ice
Brain heart infusion (BHI) medium
100 ml H2 O
3.7 g BHI (BD Biosciences)
Stir to dissolve
Autoclave for 15 min
Store up to 2 months at 4 C
Firmicutes (Low
G+C Gram
Positive)
9B.4.17
Current Protocols in Microbiology
Supplement 1
Caco-2 medium
500 ml EMEM (GIBCO or CellGrow)
130 ml FBS (20% v/v final)
6.5 ml 200 mM L-glutamine (2 mM final)
6.5 ml 10 mM nonessential amino acids (0.1 mM final)
6.5 ml 100 mM sodium pyruvate (1 mM final)
Store in the dark up to 2 months at 4 C
Verify expiration date on EMEM.
Use only sterile solutions to make this medium.
Formalin
40 ml PBS (APPENDIX 2A)
10 ml 16% formaldehyde stock solution, EM-grade (3.2% w/v final)
Wrap tube in aluminum foil
Store up to 1 month at 4 C
Formaldehyde is a known human carcinogen. It is toxic by inhalation, causes eye burn or
blindness, is a strong sensitizer, and irritates the skin, eyes, and respiratory tract.
Gentamicin, 10 mg/ml
10 ml H2 O
100 mg gentamicin (10 mg/ml final)
Pass through a 0.22-m filter to sterilize
Dispense into 1-ml aliquots
Store up to 2 years at 20 C or up to 2 months at 4 C
Always vortex before use
Follow the conversion factor indicated on powder stock container: 100 mg of powder does
not equal 100 mg of gentamicin.
9B.4.18
Supplement 1
TBS-TX
500 ml TBS (see recipe)
0.5 ml Triton X-100 (0.1% v/v final)
In order to dissolve Triton X-100, solution may need to be warmed to 50 to 55 C
Make fresh daily
NP-40 or saponin can substitute for Triton X-100.
COMMENTARY
Background Information
L. monocytogenes is a saprophytic, Grampositive bacterial rod found ubiquitously in nature (Fenlon, 1999). It is the etiologic agent of
listeriosis, a food-borne disease affecting humans and a variety of vertebrates (VazquezBoland et al., 2001). Food-borne infections
by L. monocytogenes initially cause a febrile
gastroenteritis that is self-limiting in healthy
individuals. However, in the immunocompromised or elderly, the infection becomes
systemic, causing a meningoencephalitis and
possibly a septicemia. In pregnant women, L.
monocytogenes infects the fetus, causing abortion. As a food-borne pathogen, the bacterium
has become a significant public health problem and has caused several epidemics in the
United States and Europe (CDC, 1999).
The tissue culture cell assays that are described in this unit represent an in vitro approach to evaluate the virulence of L. monocytogenes isolates and mutant strains. These
assays enable the dissection of steps leading to a successful infection: invasion, escape
from primary vacuoles, intracellular growth,
and cell-to-cell spread. These assays have
been used to screen libraries of transposoninsertion mutants and to examine the phenotype of specific mutants leading to the discovery and characterization of important virulence factors (Portnoy et al., 1992; Dussurget et al., 2004). These assays have also been
used to phenotype the intracellular behavior of
isolates from clinical and food sources (Gray
et al., 2004).
Critical Parameters
For every assay described in this unit, it
is always important to repeat them more than
once to verify reproducibility and to acquire
enough data points for statistical analysis. In
addition, as the physiological and biochemical status of tissue culture cells may change
with the number of passages, one should
try to perform replicate experiments within a
short period of time or to use cells that have
not been passaged excessively. Another potential source of variability is the FBS. Before buying a new lot of FBS, each assay
should be performed with the old and new
lots of FBS in parallel to verify reproducibility. It is preferable to buy a large quantity of
FBS from a single lot to avoid reproducibility
problems.
Stocks of L. monocytogenes should be
stored at 80 C in 50% glycerol. Cultures of
L. monocytogenes on BHI agar plates can be
stored for up to 4 weeks at 4 C. However,
it is preferable to streak a fresh plate every
week. Spontaneous rough variants will appear
on older cultures (Lenz and Portnoy, 2002).
These variants have a defect in cell septation
and will show deficiency in many of the assays
described in this unit.
Troubleshooting
Table 9B.4.1 presents some commonly encountered problems with the assays discussed
in this unit, as well as their possible causes and
potential solutions.
Firmicutes (Low
G+C Gram
Positive)
9B.4.19
Current Protocols in Microbiology
Supplement 1
Table 9B.4.1 Troubleshooting Guide for Tissue Culture Cell Assays Used to Analyze Listeria monocytogenes
Problem
Possible cause
Solution
Coverslips break
Dirty coverslips
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.20
Supplement 1
Table 9B.4.1 Troubleshooting Guide for Tissue Culture Cell Assays Used to Analyze Listeria monocytogenes,
continued
Problem
Possible cause
Solution
No bacteria recovered
Diff-Quickstained samples look Coverslip mounted cell side up Put a drop of permount on the coverslip
fuzzy under the microscope
and add a clean coverslip on top.
Diffuse staining
Detergent present
Mounting medium
incompatible with fluor
Oxidation of fluor
Bacterial growth
Unknown
Firmicutes (Low
G+C Gram
Positive)
9B.4.21
Current Protocols in Microbiology
Supplement 1
Figure 9B.4.1 Fluorescence micrograph of infected J774 cells showing bacteria decorated with
actin laments. (A) Bacteria were labeled with anti-L. monocytogenesspecic antibodies and with
rhodamine-conjugated goat anti-rabbit IgG. (B) Actin was labeled with Alexa Fluor 488 phalloidin.
(C) Overlay: most of the bacteria are yellow because of co-localizion with a cloud of actin. A few
bacteria are red because they have not yet escaped the vacuole. For the color version of this gure
go to http://www.currentprotocols.com.
Figure 9B.4.2
Anticipated Results
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.22
Supplement 1
Figure 9B.4.3 Micrograph of J774 cells infected with L. monocytogenes and stained with DiffQuick. Bacteria show as dark small rods. Notice the primary infected cell in the middle with
neighboring cells in the process of becoming infected. For the color version of this gure go to
http://www.currentprotocols.com.
Figure 9B.4.4 L2 cells stained with neutral red 4 days after infection with L. monocytogenes.
Clear zones in the monolayer represent plaques formed by foci of infection.
Time Considerations
The time required for preparing the coverslips depends on the experience of the person. Initially, it may take as long as 30
min for a dish with fifteen coverslips, but
eventually it will take 10 min or less. For
Basic Protocols 1 to 3, the cells are seeded the
day before the infection is to be performed. For
Firmicutes (Low
G+C Gram
Positive)
9B.4.23
Current Protocols in Microbiology
Supplement 1
Literature Cited
Tissue Culture
Cell Assays Used
to Analyze
L. monocytogenes
9B.4.24
Supplement 1
Firmicutes (Low
G+C Gram
Positive)
9B.4.25
Current Protocols in Microbiology
Supplement 1
UNIT 9C.4
S. aureus is an important human pathogen that has the ability to invade many types of
host tissues including skin, lung, cardiac endothelium, bone, and soft tissues, particularly
in patients with immunosuppression and those harboring intravascular devices. Despite
its maligned character, this bacterium normally does not cause disease and is frequently
found as a commensal in the nares of 25% of healthy individuals. However, in diabetics,
i.v. drug abusers, and patients with cystic fibrosis, renal failure, or immunosuppression,
the incidence of colonization and disease by this pathogen increases. Although it has
long been held that S. aureus is an extracellular pathogen, recent experimental evidence
suggests that S. aureus is capable of invading and surviving in a broad range of host cells
including keratinocytes, pulmonary epithelial cells, osteoblasts, and cultured endothelial
cells (Cheung et al., 1991; Bayles et al., 1998; Lowy, 1998). These intracellular sites can
serve as reservoirs that enable the microorganism to avoid innate immunity and extraneous antimicrobial agents. Additionally, quorum sensing (Section 1C) and virulence gene
expression inside professional phagocytes such as neutrophils and macrophages would
confer additional survival advantages upon the bacteria, leading to apoptosis or lysis of
host cells.
The culture of mammalian cells and tissues is now a widely accepted technique for
studying the interactions of bacterial pathogens with host cells. This unit describes
some of the cell lines commonly used to study S. aureus adherence, internalization, and
intracellular trafficking, and the induction of apoptosis in host cells.
CAUTION: Staphylococcus aureus is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: The protocols described in the unit involve the use of human and/or murine
tissues that may be infected with S. aureus. Follow all appropriate guidelines and regulations for the use and handling of human-derived materials and blood-borne pathogens,
as well as for storage and disposal of sharps. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
BASIC
PROTOCOL 1
Firmicutes (Low
G+C Gram
Positive)
Contributed by Ambrose L. Cheung and Kenneth W. Bayles
Current Protocols in Microbiology (2007) 9C.4.1-9C.4.7
C 2007 by John Wiley & Sons, Inc.
Copyright
9C.4.1
Supplement 4
Materials
MAC-T cells (contact Kenneth W. Bayles, kbayles@unmc.edu)
MAC-T growth medium (see recipe)
0.25% trypsin in HBSS (Sigma)
Invasion medium: MAC-T growth medium (see recipe) without antibiotics or FBS
S. aureus (strain RN6390; Network on Antimicrobial Resistance in Staphylococcus
aureus; http://www.narsa.net)
Tryptic soy broth (TSB; see recipe) as bacterial growth medium
Hanks balanced salt solution (HBSS; APPENDIX 2A), filter sterilized
Gentamicin sulfate (Invitrogen)
0.025% (v/v) Triton X-100 (U.S. Biochemical) in sterile distilled water
Tryptic soy agar plates (see recipe)
24-well tissue culture plates (Costar)
Shaking bacteriological incubator
Additional reagents and equipment for counting cells using a hemacytometer
(APPENDIX 4A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
aseptic technique should be used accordingly.
NOTE: All culture incubations should be performed in a humidified 37 C, 5% CO2
incubator unless otherwise specified.
1. Seed MAC-T cells at 6 104 cells/well (containing 1 ml of MAC-T growth medium)
in 24-well tissue culture plates and grow until the density reaches 5 105 cells/well
(3 days). Periodically determine cell density by incubating the cells from a control
well with 0.25% trypsin for 5 min at 37 C to release the cells from the surface of the
plate, then counting using a hemacytometer (APPENDIX 4A).
2. At a time point 16 hr prior to the invasion assay (and prior to reaching 5 105
cells/well), replace the growth medium in each well with 1 ml of invasion medium.
3. Inoculate the S. aureus bacteria into 10 ml TSB and incubate overnight at 37 C with
vigorous shaking (250 rpm).
This should give a final bacterial cell density of 1010 cfu/ml.
7. Discard the supernatants of the cocultures and wash the monolayers in each well
three times, each time with 1 ml HBSS.
9C.4.2
Supplement 4
8. Add 100 l of 0.25% trypsin to each well and incubate 5 min at 37 C. Lyse the
MAC-T cells by adding 900 l of 0.025% (v/v) Triton X-100 and vortexing briefly.
9. Prepare 10-fold serial dilutions of the cell lysates in TSB and plate in triplicate on
tryptic soy agar plates to quantify the bacteria.
BASIC
PROTOCOL 2
S. aureus bacteremia and dissemination into organ tissues are essential steps in the pathogenesis of staphylococcal disease (Lowy, 1998). One unique aspect of the pathogenesis of
S. aureus bacteremic infections in humans is the ability of the organism to adhere to and
colonize endothelial cells during transient bacteremia. To define this interaction, several
investigators have used cultured human umbilical vein endothelial cells (HUVEC) as a
model. A consequence of this interaction is the internalization of S. aureus by HUVEC,
eventuating in apoptosis of the endothelial cells (Kahl et al., 2000). The protocol below
describes the use of human umbilical cord endothelial cells in adherence and apoptosis
assays (Jaffe et al., 1973; Jaffe, 1980).
Materials
S. aureus (strain RN6390; Network on Antimicrobial Resistance in Staphylococcus
aureus; http://www.narsa.net)
Bacterial growth medium: e.g., tryptic soy broth (TSB; see recipe) or brain-heart
infusion medium (e.g., Difco)
Phosphate-buffered saline (PBS; APPENDIX 2A)
M199 medium (Bio-Whittaker)
Tryptic soy agar for growing S. aureus (see recipe)
Human umbilical vein epithelial cells (HUVEC) grown to confluence in
gelatin-coated 96-well plates (APPENDIX 4B)
0.05% (w/v) collagenase/0.01% (w/v) EDTA
0.25% trypsin/0.1% EDTA (Cellgro) in M199 medium (Bio-Whittaker)
2% (v/v) Triton X-100 in H2 O
10 g/ml lysostaphin (Ambi Biologicals)
18-mm glass culture tube
Humidified 37 C, 5% CO2 incubator
Microtip sonicator (e.g., Branson)
Additional reagents and equipment for culture of HUVEC (APPENDIX 4B) and trypan
blue exclusion test of cell viability (Strober, 1997)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
aseptic technique should be used accordingly.
NOTE: Unless otherwise stated, all reagents and incubations are at 37 C.
Firmicutes (Low
G+C Gram
Positive)
9C.4.3
Current Protocols in Microbiology
Supplement 4
4. Confirm the bacterial density by plating serial dilutions on tryptic soy agar plates
and counting colonies to determine the number of cfu.
Enumerate HUVEC
5. Enumerate the number of HUVEC in each of three wells of a confluent 96-well
plate by adding 200 l of 0.05% collagenase/0.01% EDTA, incubating 5 min, then
pipetting up and down to mix and counting viable cells by trypan blue exclusion
(Strober, 1997).
A viability of 95% is anticipated as confirmed by the exclusion of the trypan blue dye.
In these studies, the authors grow HUVEC to confluence on gelatin-coated 96-well microtiter plates (Corning) in culture medium in the absence of antibiotics (APPENDIX 4B).
Similar studies can also be performed in 24-well or 6-well plates (Tables A.4B.1 to
A.4B.3).
Separate sets of triplicate wells of confluent HUVEC will be required for enumeration
of total bacteria (adherent plus internalized; steps 6 to 10) and for enumeration of
internalized bacteria (steps 11 to 12).
7. After incubation, wash the monolayers gently with 200 l/well of M199 medium,
tilting the microtiter plate slightly and aspirating the wash medium under gentle
suction with a Pasteur pipet. Repeat this wash three times.
Take special care to avoid direct contact of the pipet tip with the cells.
8. Detach the HUVEC and adherent bacteria by adding 200 l of 0.25% trypsin/0.1%
EDTA in M199 medium to each well.
9. Mix well by pipetting up and down with a 1000-l pipet tip several times. Transfer
to a microcentrifuge tube containing 200 l of 2% Triton X-100 in water.
10. Sonicate for 20 sec with a microtip sonicator set at maximum power to disrupt
bacterial clusters. Plate serial dilutions on tryptic soy agar plates and count colonies
to determine the number of cfu, reported as the mean from triplicate wells.
This assay will yield both adherent and internalized S. aureus bacteria.
Tissue Culture
Assays Used to
Analyze Invasion
by S. aureus
9C.4.4
Supplement 4
COMMENTARY
Background Information
S. aureus is an opportunistic pathogen that
can infect humans and other mammals. In humans, S. aureus can cause a variety of superficial infections (e.g., cellulitis or skin abscesses) and deep-seated infections ranging
from bacteremia to abscesses in almost any
organ, or multiple organs, of the body. A commonly occurring scenario is that the microorganism gains access to the bloodstream via
an infected catheter or other intravascular device. Dissemination to distal organs from the
bloodstream would require bacterial attachment to endothelial cells followed by invasion
Firmicutes (Low
G+C Gram
Positive)
9C.4.5
Current Protocols in Microbiology
Supplement 4
Tissue Culture
Assays Used to
Analyze Invasion
by S. aureus
Critical Parameters
In adherence assays with HUVEC, take
extreme care in washing between incubation steps to avoid dislodging HUVEC from
the gelatin-coated wells. An alternative is to
lightly fix the HUVEC with 0.05% glutaraldehyde for 5 min followed by washes; however, a
pilot study should be performed to ensure that
bacterial adherence to HUVEC is not affected
by the fixing procedure.
A recent study by Qazi et al. (2004) indicates that gentamicin can inhibit the growth
of the intracellular bacteria and, thus, may impact the normal metabolic processes that occur within the intracellular niche. Although the
authors of this unit have not observed these effects on bacterial growth, incubation at 37 C
with 50 g/ml lysostaphin for 10 min can be
used in place of gentamicin treatment.
Troubleshooting
If enumeration of cfu leads to significant
variations in results of the adherence assays
with HUVEC, one can deploy bacteria labeled with methyl-[3 H]thymidine. These labeled bacteria, once prepared and stored at
4 C, can be used for several assays and, if
heat-killed (by incubating at 55 C for 1 hr),
can last for as long as 3 to 4 weeks. After
adherence, the HUVEC and adherent bacteria
can be lysed with 200 l of 2.5% SDS and 0.2
M NaOH (if in microtiter wells) and 100 l of
this lysate is neutralized with 400 l of 0.05
M acetic acid and counted for radioactivity in
the presence of scintillation fluid (Readysafe,
Beckman Instruments).
Anticipated Results
In the authors experience, the adherence
varies significantly among clinical isolates.
With most studies, 1% of the bacteria
showed nonspecific adherence to tissue culture
wells. Adherence to HUVEC can be increased
by precoating the wells with plasma, probably simulating what happens in vivo (10%
to 20% of the inoculum can adhere in this
manner).
The percent survival of MAC-T cells is
quite variable, ranging from 5% to 40%, depending on the specific bacterial strain used
and the calculated MOI (Hyunh et al., 1991;
Bayles et al., 1998; Fox et al., 2000). Presumably due to the induction of apoptosis and
subsequent permeabilization of the host cell
9C.4.6
Supplement 4
Time Considerations
Literature Cited
Firmicutes (Low
G+C Gram
Positive)
9C.4.7
Current Protocols in Microbiology
Supplement 4
UNIT 9D.5
Because S. pyogenes is a strictly human pathogen (with the exception of one strain,
see below), there is no really good animal model for studying its virulence. In the
absence of such a complete model, methods have been devised to model different aspects
of the human disease. The three infection models presented are valuable in assessing
different aspects of S. pyogenes virulence. The long-term throat colonization model
(Basic Protocol 1) uses the single S. pyogenes strain isolated as a mouse pathogen by
Lancefields group (Hook et al., 1960). It also causes disease in humans. This strain was
chosen in the hope that it would initiate a more natural infection in the mouse. This
model is valuable for studies of the early colonization steps of infection and for studies
of long-term persistence (at least one month). Basic Protocol 2 (pneumonia model) also
uses the mouse-pathogenic strain. Although it bypasses the initial steps of infection by
using intratracheal inoculation, it gives rapid, reproducible results about virulence of the
strain due to systemic dissemination. It may serve as a more natural alternative than
the model employing intraperitoneal inoculation. The third model (Basic Protocol 3)
uses a human isolate of S. pyogenes and introduction of the bacteria by subcutaneous
inoculation into out-bred mice. Many virulent strains of S. pyogenes can be used in Basic
Protocol 3. This model mimics one major type of human disease: infection through the
skin followed by systemic dissemination of the bacteria and possible death. Additional
protocols (Basic Protocols 4 to 11) present methods for determining the amount of S.
pyogenes in the different mouse tissues following infection.
The procedures presented in this unit use healthy mice, and no additives (e.g., antibiotics,
culture media, or beads) are introduced during the inoculation. These models are generally
useful in testing the virulence of mutants constructed in the laboratory. For example, to
determine whether the capsule is important for virulence and which step(s) of infection
it affects, a capsule-deficient mutant can be used in these models. In addition, the models
provide assays to assess the effectiveness of potential vaccines or therapeutics. It should
be remembered that different strains of S. pyogenes behave very differently in many
assays, including animal models. Because S. pyogenes secretes many known and potential
virulence factors, experiments performed with washed bacteria (as presented below) will
give different results from those performed with unwashed inocula.
CAUTION: Streptococcus pyogenes is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: This experiment requires Animal Biosafety Level 2 (ABSL-2) conditions.
Follow all appropriate guidelines for the use and handling of infected animals. See UNIT
1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: Protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aseptic technique should be used accordingly.
NOTE: When performing counts on a hemacytometer (APPENDIX 4A) for S. pyogenes, each
chain should be considered a single colony forming unit (cfu). Be certain that only cocci
Contributed by Samantha Roberts, June R. Scott, Linda K. Husmann, and Christine A. Zurawski
Current Protocols in Microbiology (2006) 9D.5.1-9D.5.15
C 2006 by John Wiley & Sons, Inc.
Copyright
Firmicutes (Low
G+C Gram
Positive)
9D.5.1
Supplement 2
in chains are present in the culture. Cultures should be abandoned if other species are
present.
BASIC
PROTOCOL 1
Materials
Streptococcus pyogenes strain B514-Sm (Hook et al., 1960)
2 brain heart infusion (BHI)/2% (v/v) supplement B (see recipe)
0.9% saline (APPENDIX 2A)
7-week-old female (preferred) C57BL10/SnJ mice (Jackson Laboratories): place
order to allow for acclimatization one week prior to inoculation at the age
indicated (see Critical Parameters and Troubleshooting)
Solution containing 1.5 mg/ml xylazine and 100 mg/ml ketamine
THY plates containing 1000 g/ml streptomycin (see recipe)
85 g/ml hyaluronidase (optional)
15-ml screw-cap conical tubes
Klett flasks, screw-cap (Bellco)
Klett colorimeter with red filter
Refrigerated centrifuge (e.g., Beckman with JA-14 rotor) and centrifuge bottles
Rodent ear punch
Balance accurate to 0.01 g
20-l micropipettor and round-tipped sequencing pipet tips
Additional reagents and equipment for counting cells (APPENDIX 4A) and for
anesthesia (Donovan and Brown, 1998)
1. Grow a 6-ml overnight culture of S. pyogenes strain B514-Sm in 2 BHI/2% supplement B. Incubate at 37 C, static, in a 15-ml screw-cap conical tube.
IMPORTANT NOTE: DO NOT use hyaluronidase. Capsule production by the bacteria
is essential for induction of throat colonization.
Murine Infection
Models of
S. pyogenes
5. Place the culture pellet on ice and keep on ice throughout the rest of the inoculation
procedure.
6. Resuspend the bacteria in the small amount of liquid remaining in the centrifuge
bottle.
9D.5.2
Supplement 2
8. Adjust the density of the bacterial suspension with 0.9% saline so that the number
of cfu to be delivered to each mouse is contained in a 10-l volume.
For example, if each mouse is to be given a dose of 1 107 cfu, the bacterial suspension
should be at a density of 1 109 cfu/ml.
9. Anesthetize each 7-week-old female (preferred) C57BL10/SnJ mouse by intraperitoneal injection with 30 l of a solution containing 1.5 mg/ml xylazine and 100 mg/ml
ketamine (see Donovan and Brown, 1998).
Female mice are preferred because they are less aggressive.
For alternate anesthesia, see Basic Protocol 3, steps 11 and 12.
10. Once the animals become unresponsive to having their hind feet pinched, punch
their ears for identification using a standard rodent ear punch. If necessary, weigh
the animals at this time using a balance accurate to 0.01 g.
Use the system of no punches, one in the right ear, one in the left ear, two in the right ear,
etc.
13. Allow the animals to remain on their backs, undisturbed, in their cages until they
recover from the anesthesia.
This will take 30 min.
14. Plate in quadruplicate 10-fold serial dilutions of the inoculum on THY plates containing 1000 g/ml streptomycin to perform viable plate counts and confirm the cfu
present in the suspensions used for inoculation.
Countable plates should have only 25 to 50 colonies per plate because the colonies are
very mucoid. Alternatively, plate on THY plates containing 85 g/ml hyaluronidase and
1000 mg/ml streptomycin to allow counting of higher numbers on more plates with lower
dilutions.
THY plates containing 1000 g/ml streptomycin are used for growing group A Streptococcus (GAS) streptomycin-resistant strains to prevent growth of contaminating Staphylococcus. If using wild-type Streptococcus, isolate a streptomycin-resistant strain by plating
>109 bacteria on THY medium containing 1000 g/ml streptomycin.
BASIC
PROTOCOL 2
Firmicutes (Low
G+C Gram
Positive)
9D.5.3
Current Protocols in Microbiology
Supplement 2
Materials
S. pyogenes strain B514-Sm (Hook et al., 1960)
2 BHI/2% supplement B (see recipe)
0.9% saline (APPENDIX 2A)
5-week-old female C3HeB/FeJ mice (Jackson Laboratories; http://www.jax.org):
place order to allow for acclimatization one week prior to inoculation at the age
indicated (see Critical Parameters and Troubleshooting)
Solution containing 1.5 mg/ml xylazine and 100 mg/ml ketamine
THY plates containing 1000 g/ml streptomycin (see recipe)
85 g/ml hyaluronidase (optional)
15-ml screw-cap conical tubes
Klett flask, screw-cap (Bellco)
Klett colorimeter with red filter
Refrigerated centrifuge (e.g., Beckman with JA-14 rotor) and centrifuge bottles
Rodent ear punch
Balance accurate to 0.01 g
22-G straight feeding needle (Popper and Sons) and 1-cc tuberculin syringe
Additional reagents and equipment for counting cells (APPENDIX 4A) and for
anesthesia (Donovan and Brown, 1998)
1. Grow a 6-ml overnight culture of S. pyogenes strain B514-Sm in 2 BHI/2% supplement B. Incubate at 37 C, static, in a 15-ml screw-cap conical tube.
IMPORTANT NOTE: DO NOT use hyaluronidase (capsule production by the bacteria is
essential for induction of pneumonia).
5. Place the culture pellet on ice and keep on ice throughout the rest of the inoculation
procedure.
6. Resuspend the bacteria in the small amount of liquid remaining in the centrifuge
bottle.
7. Determine cfu per milliliter by counting in a hemacytometer (APPENDIX 4A).
Be certain that only cocci in chains are present in the culture.
Note that each bacterial chain is considered a single cfu (see Critical Parameters and
Troubleshooting).
Murine Infection
Models of
S. pyogenes
9D.5.4
Supplement 2
8. Adjust the density of the bacterial suspension with 0.9% saline so that the number
of cfu to be delivered to each mouse is contained in a 20-l volume.
For example, if each mouse is to be given a dose of 1.0 107 cfu, the bacterial suspension
should be at a density of 5.0 108 cfu/ml.
Current Protocols in Microbiology
10. Once the animals become unresponsive to having their hind feet pinched, punch
their ears for identification using a standard rodent ear punch. If necessary, weigh
the animals at this time using a balance accurate to 0.01 g.
Use the system of no punches, then one in the right ear, one in the left ear, two in the right
ear, etc.
11. Deposit 20 l bacterial suspension into the lower trachea by holding the mouse
vertically with its head up, inserting a 22-G straight feeding needle attached to a 1-cc
tuberculin syringe into the mouth and then into the lower trachea.
Keep in mind that the trachea has a rough vacuum cleaner hose texture while the
esophagus is very smooth. Upon placing the needle into the trachea, the rounded tip of
the needle should be felt from the outside. When the tip of the needle reaches the bronchi
of the mouse, resistance will be felt. Once this happens, do not insert the needle any
further; pull the needle up just slightly.
12. Place the mouse on its back and allow it to remain in that position until it recovers
from anesthesia.
This will take 30 min.
13. Plate in quadruplicate 10-fold serial dilutions of the inoculum on THY plates containing 1000 g/ml streptomycin to perform viable plate counts and confirm the cfu
present in the suspensions used for inoculation.
Countable plates should have only 25 to 50 colonies per plate because the colonies are
very mucoid. Alternatively, plate on THY plates containing 85 g/ml hyaluronidase and
1000 g/ml streptomycin to allow counting of higher numbers on more plates with lower
dilutions.
THY plates containing 1000 g/ml streptomycin are used for growing group A Streptococcus (GAS) streptomycin-resistant strains to prevent growth of contaminating Staphylococcus. If using wild-type Streptococcus, isolate a streptomycin-resistant strain by plating
>109 bacteria on THY medium containing 1000 g/ml streptomycin.
BASIC
PROTOCOL 3
Firmicutes (Low
G+C Gram
Positive)
9D.5.5
Current Protocols in Microbiology
Supplement 2
Materials
6-week-old female CD1 (outbred) mice, 21 to 23 g (Charles River Laboratories;
http://www.criver.com): place order to allow for acclimatization one week prior
to inoculation at the age indicated (see Critical Parameters and Troubleshooting)
THY medium (see recipe)
Group A Streptococcus (GAS) strain, preferably streptomycin-resistant (e.g., AM3;
Stamp and Hendry, 1937)
0.9% saline (APPENDIX 2A), 4 C, sterile
2.5% Avertin (see recipe)
Depilatory agent (e.g., Nair)
THY plates containing 1000 g/ml streptomycin (see recipe)
85 g/ml hyaluronidase (optional)
Isoflurane
15-ml conical tube
Klett flask (Bellco)
Klett colorimeter and with red filter
Refrigerated centrifuge (e.g., Beckman with JA-20 rotor)
Balance accurate to 0.01 g
Rodent ear punch
Cotton-tipped applicators
Gauze pads
Alcohol wipes
27-G needle and 1-ml insulin/tuberculin syringe
Calipers (optional)
Urogenital swab, single-use (Puritan)
Dissecting tools, sterile
Dounce tissue homogenizer
Additional reagents and equipment for counting cells (APPENDIX 4A), removal of
spleen (Basic Protocol 6, steps 1 to 3), and anesthesia (Donovan and Brown,
1998)
Prepare bacterial cultures
1. Inoculate 5 ml THY medium with a GAS strain in a 15-ml conical tube. Incubate
overnight at 37 C, static.
The authors use AM3 because a streptomycin-resistant strain is preferable.
2. Inoculate 2.5 ml overnight culture into a Klett flask containing 50 ml THY medium
(a 1:20 dilution). Incubate in a 37 C static water bath until the Klett reading is 50 to
55 Klett (mid-log phase).
This usually takes 4 to 5 hr.
5. Resuspend pellet in 10 ml 0.9% saline and centrifuge 15 min at 6000 g (7,000 rpm
in a JA-20 rotor), 4 C.
6. Carefully pour off supernatant.
Murine Infection
Models of
S. pyogenes
7. Place the culture on ice and keep on ice throughout the remainder of protocol.
8. Resuspend the cell pellet in 4 ml of 0.9% saline.
9D.5.6
Supplement 2
10. Adjust the density of the culture with 0.9% saline so that the desired cfu are delivered
in a 100-l volume.
For example, if delivering 108 cfu, the culture should be at 109 cfu/ml.
The authors use 2 107 cfu/mouse for inoculation. This is above the LD50 for GAS strain
AM3.
Inoculate mice
11. Weigh each mouse using a balance accurate to 0.01 g and multiply the weight in
grams by fifteen to determine the number of microliters of 2.5% Avertin to administer.
12. Anesthetize each mouse by intraperitoneal injection of the calculated volume of
2.5% Avertin (see Donovan and Brown, 1998).
13. Once the animals have become unresponsive to having their hind feet pinched, punch
their ears for identification using a standard rodent ear punch.
Use system of zero punch, 1 left, 1 right, 2 left, 2 right.
14. Lay each mouse on its stomach and apply a quarter-sized amount of depilatory agent
to the back of the back haunch of the mouse with a cotton-tipped applicator. Wait
5 min, then remove hair with a gauze pad. Swab depilated spot with alcohol wipe.
15. Inoculate 100 l bacterial suspension just under the skin surface in the center of the
bald spot using a 27-G needle and 1-ml insulin/tuberculin syringe.
Keep the syringe at an 45 angle to the skin, bevel side up. Insert the needle just under
the skin. If done correctly, this should result in a small bleb under the skin with no
bleeding.
16. Allow animals to remain on their stomachs in cages until they recover from
anesthesia.
This should take 30 min.
17. Plate in quadruplicate 10-fold serial dilutions of the inoculum on THY plates containing 1000 g/ml streptomycin to perform viable plate counts and confirm the cfu
present in the suspensions used for inoculation.
Countable plates should have only 25 to 50 colonies per plate because the colonies are
very mucoid. Alternatively, plate on THY plates containing 85 g/ml hyaluronidase and
1000 g/ml streptomycin to allow counting of higher numbers on more plates with lower
dilutions.
THY plates containing 1000 g/ml streptomycin are used for growing group A Streptococcus (GAS) streptomycin-resistant strains to prevent growth of contaminating Staphylococcus. If using wild-type Streptococcus, isolate a streptomycin-resistant strain by plating
>109 bacteria on THY medium containing 1000 g/ml streptomycin.
Firmicutes (Low
G+C Gram
Positive)
9D.5.7
Current Protocols in Microbiology
Supplement 2
20. Remove spleens (see Basic Protocol 6, steps 1 to 3) using sterile dissecting tools and
transfer aseptically to a Dounce tissue homogenizer.
21. Homogenize in 1 ml of 0.9% saline and plate (undiluted) in duplicate for viable
bacteria on THY plates containing 1000 g/ml streptomycin.
22. Wipe the wound site area with an alcohol wipe. Lift the scab with sterile forceps
and swab with a single-use urogenital swab placed in 1 ml THY medium containing
1000 g/ml streptomycin.
23. Incubate 2 days at 37 C and evaluate by microscopic examination to determine if
streptococci are present.
Materials
Mouse infected with S. pyogenes strain B514-SM (Basic Protocol 1, 2, or 3),
sacrificed
THY medium (see recipe)
Surgical scissors, sterile
Sterile swab, calcium alginate urogenital
1. Cut off the head of the mouse with surgical scissors. Insert the point of the scissors
into the skull portion of the spinal column, which is very white in color.
2. Using the spinal column as a guide, cut up into the skull between the two main brain
hemispheres.
3. Using fingers, peel back the skull on each side, exposing the brain.
4. Swab the surface of the brain (i.e., the meninges) and the area between the two
hemispheres with a sterile swab.
Murine Infection
Models of
S. pyogenes
9D.5.8
Supplement 2
BASIC
PROTOCOL 5
Materials
Mouse infected with S. pyogenes strain B514-SM (Basic Protocol 1, 2, or 3),
sacrificed
THY medium and plates (see recipe)
0.9% saline (APPENDIX 2A)
Surgical scissors and forceps, sterile
1. Cut into the throat area of the mouse, using sterile surgical scissors, to expose the
trachea.
2. Remove the three tracheal rings immediately below the larynx. Place in 500 l THY
medium.
3. Vortex 10 sec on high speed.
4. Perform serial 10-fold dilutions in 0.9% saline and plate (in duplicate) the undiluted
and 1/10 dilutions.
5. Incubate 1 to 2 days at 37 C and count colonies to determine cfu present in the
trachea.
BASIC
PROTOCOL 6
Materials
Mouse infected with S. pyogenes strain B514-SM or AM3 (Basic Protocol 1, 2, or
3), sacrificed
THY medium and plates (see recipe)
Surgical scissors and forceps, sterile
Dounce tissue homogenizer, sterile
1. Pull the skin up using forceps and cut through the skin and into the peritoneal cavity
using surgical scissors. Take care not to cut into the intestines.
2. Remove a 3 3mm section of the spleen and place in 500 l THY medium.
The spleen is located behind the stomach (and is actually attached to the stomach) and is
brick red in color.
3. Carefully transfer the entire spleen and medium to a sterile Dounce tissue homogenizer.
The homogenizer should be thoroughly cleaned (with soap and water), rinsed, and autoclaved before use.
4. Thoroughly homogenize the tissue and plate (undiluted) in duplicate on THY plates.
5. Incubate 1 to 2 days at 37 C and count colonies to determine cfu present in the
spleen.
BASIC
PROTOCOL 7
Firmicutes (Low
G+C Gram
Positive)
9D.5.9
Current Protocols in Microbiology
Supplement 2
Materials
Mouse infected with S. pyogenes strain B514-SM (Basic Protocols 1 or 2),
sacrificed
THY medium (see recipe)
0.9% saline (APPENDIX 2A)
Surgical scissors and forceps, sterile
Dounce tissue homogenizer
1. Open the chest cavity of the mouse and peel back the ribs, exposing the heart and
lungs.
2. Remove the entire left lung lobe and place in 500 l THY medium.
3. Carefully transfer the entire lung and medium to a Dounce tissue homogenizer.
4. Thoroughly homogenize the lung in the medium, remove a 0.1 ml portion of the
homogenate, and perform 10-fold serial dilutions in 0.9% saline.
5. Plate the undiluted and 1/10 dilutions (in duplicate) on THY plates.
6. Incubate 1 to 2 days at 37 C and count colonies to determine if streptococci were
present in the lungs.
BASIC
PROTOCOL 8
Materials
Mouse infected with S. pyogenes strain B514-SM (Basic Protocols 1, 2, or 3),
sacrificed
THY medium (see recipe)
Surgical scissors, sterile
200-l micropipettor and appropriate tips
15-ml screw-cap conical tube
1. Cut into the large axillary artery of the mouse using surgical scissors.
This artery is located in the arm pit area under either front leg.
3. Add the entire blood sample to 10 ml THY medium in a screw-cap conical tube.
4. Incubate 2 days at 37 C and evaluate by microscopic examination to determine if
streptococci had entered the bloodstream of the mouse.
BASIC
PROTOCOL 9
Materials
Murine Infection
Models of
S. pyogenes
9D.5.10
Supplement 2
BASIC
PROTOCOL 10
Materials
Carbamyl chloride (carbachol; Sigma-Aldrich)
Soybean trypsin inhibitor (Sigma-Aldrich)
Phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich)
BSA (Sigma-Aldrich)
Sodium azide (NaN3 )
Mouse infected with S. pyogenes strain B514-SM (Basic Protocols 1 or 2)
22-G straight feeding needle (Popper and Sons) and 1-cc tuberculin syringe
200-l micropipettor
80 C freezer
1. Prepare a fresh solution of 20 g/ml carbamyl chloride (carbachol) in water
2. Prepare a protease inhibitor solution containing 2 mg/ml of each of the following in
water:
3. Inject 0.1 ml carbachol solution into the mouse intraperitoneally (see Donovan and
Brown, 1998).
About 5 min after the injection, the mouse will start to salivate (drool).
4. Collect the saliva with a 200-l micropipettor. Keep the saliva sample on ice.
Usually, the mouse will continue to salivate for 15 min and it is possible to collect
200 l of saliva.
4 C.
Firmicutes (Low
G+C Gram
Positive)
9D.5.11
Current Protocols in Microbiology
Supplement 2
Materials
Mouse infected with S. pyogenes strain B514-SM (Basic Protocols 1 or 2)
Calcium alginate swab (urethro-genital applicator; Baxter Healthcare)
THY plates and medium containing 1000 g/ml streptomycin (see recipe)
1. Hold the conscious mouse firmly by its dorsal neck surface and place its tail between
the ring finger and the little finger to aid in immobilizing the mouse.
2. Insert a sterile calcium alginate swab into the mouth far enough so that the back of
the throat is reached.
Resistance can be felt when the swab reaches the back of the throat (that area of the oral
cavity is fairly hard). Another good indicator that the swab has been inserted far enough
is when all of the white portion of the swab is inside of the mouth and none is visible from
the outside.
3. Rotate the swab from one side of the throat to the other at least three times.
4. Rotate the swab over the surface of a THY plate containing 1000 g/ml streptomycin.
Break off the metal end of the swab and place it in a snap-cap tube containing 1 ml
THY medium containing 1000 g/ml streptomycin.
5. Incubate 2 days at 37 C.
6. Examine the plates for the presence of mucoid, streptomycin-resistant colonies.
7. If the plate culture is negative, lightly vortex the medium culture, and place a few
drops of the culture on a glass slide and examine using a microscope.
A positive medium culture is indicated by the presence of many cocci in chains within a
single field.
Avertin, 2.5%
Dilute 1 ml Avertin stock solution (see recipe) in 39 ml PBS (APPENDIX 2A). Mix
thoroughly at room temperature for at least 10 min. Pass through a 0.22-m filter
to sterilize. Store in the dark up to 3 months at room temperature.
Do not place on ice because it will precipitate.
9D.5.12
Supplement 2
COMMENTARY
Background Information
Streptococcus pyogenes is an important
Gram-positive human pathogen that is responsible for a spectrum of mild to severe infections
of throat, skin, and deep tissues. S. pyogenes
causes pharyngitis (strep throat), which is
characterized by a reddened sore throat, difficulty swallowing, fever, and swollen lymph
nodes. A butterfly facial rash accompanying these symptoms is an indication of scarlet fever, which is caused by certain strains
of S. pyogenes. Infection of the epidermis
by S. pyogenes (impetigo) is highly contagious and characterized by inflamed and purulent lesions on the skin. Both pharyngitis
and impetigo are mild infections that can be
diagnosed in the clinic by culturing the infected site and assaying for the presence of S.
pyogenes. More severe local infections caused
by S. pyogenes include erysipelas, which involves dermal layers of the skin, and cellulitis, which can involve subcutaneous tissue. Analysis of serum for anti-streptococcal
antibodies is often necessary to diagnose S.
pyogenes as the causative agent of erysipelas
or cellulitis. Localized streptococcal infections can and should be treated with antibiotics (usually penicillin or clindamycin) to
limit the potential for development of invasive
streptococcal disease or post-streptococcal
sequelae (such as rheumatic fever or
glomerulonephritis).
In the last twenty years, S. pyogenes has
gained notoriety due to an increase in the
incidence of necrotizing fasciitis and the
emergence of streptococcal toxic shock syndrome (reviewed by Bisno and Stevens, 1996).
Necrotizing fasciitis is a rapidly spreading in-
Firmicutes (Low
G+C Gram
Positive)
9D.5.13
Current Protocols in Microbiology
Supplement 2
and throat-colonization models offer the additional advantage that the strain used is a natural pathogen of mice as well as of humans.
The murine models can be used to assess the
virulence of different strains of S. pyogenes
by comparing the ability to colonize and persist in the mouse, the ability to disseminate
and cause a systemic infection, and the time to
death. These models can also be used in competition experiments where two streptococcal
strains are co-inoculated and bacteria are isolated after the infection to determine which
strain had a selective advantage in vivo.
Murine Infection
Models of
S. pyogenes
Anticipated Results
Anticipated results and signs of illness are
discussed to some extent in the protocol section. In the throat colonization model (Basic
Protocol 1), it is expected that the wild-type
strain (B514-Sm) should be able to establish
long-term colonization of the throat in up to
60% of infected mice. Colonization is assayed
by throat culture as described in Basic Protocol 11. While this model does not have a
linear dose-response relationship, the ability
of isogenic strains to colonize the mice can
be compared. In the pneumonia model (Basic Protocol 2), it is expected that the wildtype B514-Sm strain will produce pneumonia in 50% of inoculated mice within 72 hr
when administered at a dose of 1 107 cfu. In
the subcutaneous infection model (Basic Protocol 3), it is expected that all mice will develop skin lesions within 1 to 2 days, and 50%
will develop a lethal systemic infection within
4 days when wild-type strain AM3 is administered at a dose of 2 107 cfu. The time
to death induced in mice can be compared
between isogenic strains of AM3. Isolation
and culturing of the spleen as described in
Basic Protocol 6 can be used to determine if
the mouse suffered from systemic infection.
Time Considerations
There are several important considerations
when planning the timing of experiments. One
week must be allowed for the mice to acclimate to their environment before inoculation.
On the day of inoculation, it will take several
9D.5.14
Supplement 2
Literature Cited
Ashbaugh, C.D., Moser, T.J., Shearer, M.H., White,
G.L., Kennedy, R.C., and Wessels, M.R. 2000.
Bacterial determinants of persistent throat colonization and the associated immune response
in a primate model of human group A streptococcal pharyngeal infection. Cell Microbiol.
2:283-292.
Bisno, A.L. and Stevens, D.L. 1996. Streptococcal
infections of skin and soft tissues. New Engl. J.
Med. 334: 240-245.
Bunce, C., Wheeler, L., Reed, G., Musser, J., and
Barg, N. 1992. Murine model of cutaneous infection with gram-positive cocci. Infect. Immun.
60: 2636-2640.
Dalton, T.L., Hobb, R.I., and Scott, J.R. 2006.
Analysis of the role of CovR and CovS in the dissemination of Steptococcus pyogenes in invasive
skin disease. Microb. Pathogenesis. In press.
Donavan, J. and Brown, P. 1995. Blood Collection. In Current Protocols in Immunology (J.E.
Coligan, A.M. Kruisbeek, D.H. Margulies, and
W. Strober, eds.) pp. 1.7.1-1.7.8. John Wiley &
Sons, Hoboken, N.J.
Donovan, J. and Brown, P. 1998. Anesthesia.
In Current Protocols in Immunology (J.E.
Coligan, A.M. Kruisbeek, D.H. Margulies, and
W. Strober, eds.) pp. 1.4.1-1.4.5. John Wiley &
Sons, Hoboken, N.J.
Hollingshead, S.K., Simecka, J.W., and Michalek,
S.M. 1993. Role of M protein in pharyngeal
colonization by group A streptococci in rats.
Infect. Immun. 61:2277-2283.
Firmicutes (Low
G+C Gram
Positive)
9D.5.15
Current Protocols in Microbiology
Supplement 2
Laboratory Maintenance of
Mycobacterium tuberculosis
UNIT 10A.1
ABSTRACT
This unit includes protocols for the laboratory maintenance of Mycobacterium tuberculosis, including growth on liquid and solid media as well as recommendations for
long-term strain storage. Considerations for working with M. tuberculosis at Biosafety
C
Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.1.1-10A.1.8.
2007 by John Wiley & Sons, Inc.
Keywords: tuberculosis r medium r Middlebrook r 7H9 r 7H10 r 7H11 r Sauton r
strain storage
INTRODUCTION
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects almost one
third of the worlds population. Yearly, there are an estimated 10 million new cases of TB,
resulting in 2 to 3 million deaths. Over the last fifteen years, genetic tools to manipulate
M. tuberculosis have proliferated (UNIT 10A.2). Furthermore, availability of the complete
M. tuberculosis DNA genome sequence (as well as genome sequences of other related
mycobacteria) has yielded tremendous information allowing a better understanding of
the bacterium and the disease it causes.
The protocols in this unit will detail common medium preparation considerations and
strain storage.
CAUTION: Mycobacterium tuberculosis is a Biosafety Level 3 (BSL-3) pathogen. Follow
all appropriate guidelines for the use and handling of pathogenic microorganisms. See
Strategic Planning, Safety Considerations, for further discussion. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for additional information.
CAUTION: Use disposable plastic inoculation loops and plate spreaders in the BSL-3
setting, since the alternative of sterilizing glass or metal inoculation loops or plate
spreaders with ethanol and an open flame generates aerosols.
STRATEGIC PLANNING
Safety Considerations
Standards of BSL-3 containment should be followed for work with M. tuberculosis.
Investigators should consult with the local safety officer and/or institutional biosafety
committee for additional guidance. See http://www.cdc.gov/OD/ohs/symp5/jyrtext.htm
for a brief description of general BSL-3 laboratory features. Complete inactivation of M.
tuberculosis cells is necessary to perform any studies outside the BSL-3 laboratory. All
methods for inactivation should be validated in individual laboratories, taking into consideration the concentration of cells used. An article by Schwebach et al. (2001) provides
some guidance on determining if particular conditions are adequate for inactivation of
mycobacteria. For many procedures, the authors of this unit have found the following
conditions sufficient for inactivation of M. tuberculosis: incubation at 65 C for 12 hr,
Actinobacteria
(High G+C
Gram Positive)
10A.1.1
Supplement 6
Antibiotic
Suggested supplier
50 mg/liter hygromycin B
80 C for 2 hr, or 95 C for 15 min; alternatively, incubation for 1 hr in a final concentration of 2.5% glutaraldehyde, 2% paraformaldehyde, or 5% formalin, or in Vesphene IIse.
During heat inactivation, care should be taken to ensure that the entire tube is immersed
in a water bath preheated to the appropriate temperature. Incubation of M. tuberculosis
cultures with glutaraldehyde, paraformaldehyde, formalin, or Vesphene IIse should be
done in a matter that ensures that the entire interior surface of the tube has been coated
by gently rolling and inverting the tube.
Materials
Middlebrook 7H9 liquid medium (see recipe)
M. tuberculosis
Laboratory
Maintenance of
M. tuberculosis
10A.1.2
Supplement 6
3a. Close lids on bottles enough to keep them from coming off, but leave loose enough
to allow for exchange of gases.
4a. Incubate the square medium bottle cultures in a plastic box on a platform shaker
with mild agitation (100 rpm) in an incubator at 37 C.
ALTERNATE
PROTOCOL 1
Materials
Sauton medium (see recipe)
M. tuberculosis
30-ml square medium bottles (Nalgene, cat. no. 2019-0030), sterile
Disposable plastic inoculation loops, sterile
Incubator containing platform shaker or roller-bottle apparatus
490-cm2 roller bottles (Corning, cat. no. 430195)
Additional reagents and equipment for bacterial culture techniques including
inoculation of liquid medium (APPENDIX 4A)
For small-volume cultures
1a. Transfer 10 ml Sauton medium to a sterile, square medium bottle.
2a. Transfer a colony of M. tuberculosis to the bottle with a disposable inoculating
loop, or add 0.1 to 0.2 ml of a thawed strain stock.
APPENDIX 4A
3a. Close lids on bottles enough to keep them from coming off, but leave loose enough
to allow for exchange of gases.
Actinobacteria
(High G+C
Gram Positive)
10A.1.3
Current Protocols in Microbiology
Supplement 6
4a. Incubate the square medium bottle cultures in a plastic box on a platform shaker
with mild agitation (100 rpm) in an incubator at 37 C.
Materials
M. tuberculosis
Middlebrook 7H10 or 7H11 agar plates (see recipes and Strategic Planning)
Vesphene IIse disinfectant (Fisher)
Sterile plastic inoculating loops
Sterile plastic plate spreaders
Heavy-duty aluminum foil or sterile metal-plate canisters
Additional reagents and equipment for bacterial culture techniques, including
spreading and streaking plates (APPENDIX 4A)
1a. Usual procedure: Using sterile, plastic inoculating loops, streak M. tuberculosis
across Middlebrook 7H10 or 7H11 agar plates (see Strategic Planning) as described
in APPENDIX 4A.
1b. For plating larger volumes of bacterial culture (e.g., serial dilutions, transductions,
transformations): Put 100 to 200 l of the culture in the center of an agar plate.
Using a sterile, plastic plate spreader, distribute the liquid using a circular motion,
focusing the liquid towards the center of the plate (also see APPENDIX 4A). When
most of the liquid has been absorbed, expand the circular motion to distribute all
the way to the edges of the plate.
If liquid is pushed too early to the edges, a large fraction of cells will grow along the
rim, making counting and picking colonies for later applications difficult.
2. Invert a stack consisting of <10 inoculated agar plates and wrap tightly in heavyduty aluminum foil or place in a sterile metal canister to prevent plates from drying
out during prolonged incubation. Moisten a disposable paper towel or shopcloth
and lightly coat the inside layer of aluminum foil with a dilute solution of Vesphene
IIse, to prevent contamination during incubation.
3. Incubate 3 to 6 weeks at 37 C.
Laboratory
Maintenance of
M. tuberculosis
10A.1.4
Supplement 6
STORAGE OF M. TUBERCULOSIS
Typically, mycobacteria can be stored as lyophilized stocks, agar slants, or frozen stocks.
Since lyophilization is not a cost-effective product-storage alternative for virulent M.
tuberculosis, and agar slants can take up considerable BSL-3 storage space, the best
alternative for strain storage is frozen stocks. It is recommended that, for frequently used
strains, a working stock of a few dozen vials be made. For all strains, save two vials of
each strain and, where possible, distribute the strain duplicates between two different
70 C freezers.
BASIC
PROTOCOL 3
Materials
M. tuberculosis culture (Basic Protocol 1 or Alternate Protocol 1)
M. tuberculosis strain storage medium (see recipe)
Sterile 2-ml cryotubes
1. Transfer 1 ml of mycobacteria culture to a sterile microcentrifuge tube and microcentrifuge 10 min at 11,000 rpm, 4 C, to pellet cells.
2. Discard supernatant and resuspend cell pellet in 1 ml M. tuberculosis strain storage
medium.
3. Transfer to a 2-ml cryotube. Store up to 2 years at 70 C in a freezer in a BSL-3
laboratory.
Actinobacteria
(High G+C
Gram Positive)
10A.1.5
Supplement 6
10A.1.6
Supplement 6
Tyloxapol, 20%
20 ml Tyloxapol (Sigma, cat. no. T-8761)
80 ml H2 O
Sterilize by filtration through 0.22-m membrane
Store up to 1 year at 4 C
The final concentration of Tyloxapol used in the media in this unit is 0.05% (v/v).
COMMENTARY
Background Information
Tuberculosis (TB) is a disease caused by M.
tuberculosis. M. tuberculosis is spread via the
aerosol route and predominately causes disease in lung tissue, although it can spread to
other organs. It is estimated that nearly onethird of the worlds population is infected with
Actinobacteria
(High G+C
Gram Positive)
10A.1.7
Current Protocols in Microbiology
Supplement 6
Time Considerations
One of the most important aspects of working with M. tuberculosis is to minimize any
possible aerosol exposure. Second in importance to safety considerations, scrupulous attention to detail in preparing medium and
reagents is crucial. Given the long doubling
time of the organism (3 weeks for colonies
to be visible on solid medium), care should
be taken to minimize any possible sources of
contamination of the medium.
Strain storage
Care should be taken to save strains when
they are fresh. Also, some strains are more
stable than other strains (i.e., titers for frozen
stocks may remain stable for some strains,
while others will show a 10- to 100-fold drop
in titer over 6 months to 1 year).
Anticipated Results
Figure 10A.1.1 provides an example of the
colonies that arise from growth on Middle-
Acknowledgments
Special thanks to Kieran Bhatt, Bing Chen,
Zhiyan (Annie) Dai, Dee Dao, Robert Glover,
Morad Hassani, Tsugunda (Ko) Hsu, Jordan Kriakov, Sunhee Lee, Catherine Vilcheze,
Brian Weinrick, and Torin Weisbrod for contributions and helpful discussions.
Literature Cited
Cohn, M.L., Oda, U., Kovitz, C., and Middlebrook, G. 1954. Studies on isoniazid and tubercle bacilli. I. The isolation of isoniazid-resistant
mutants in vitro. Am. Rev. Tuberc. 70:465475.
Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall,
A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a biosafety level 3 laboratory. J. Clin.
Microbiol. 39:769771.
Laboratory
Maintenance of
M. tuberculosis
10A.1.8
Supplement 6
UNIT 10A.2
ABSTRACT
This unit includes protocols for the genetic manipulation of Mycobacterium tuberculosis,
including nucleic acid extraction (plasmid DNA, genomic DNA, and mRNA), and
methods for electroporation (transformation), transduction (including allelic exchange),
and transposon mutagenesis. Considerations for working with M. tuberculosis at
Biosafety Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.2.1C 2007 by John Wiley & Sons, Inc.
10A.2.21.
Keywords: tuberculosis r plasmid r genomic DNA r mRNA r transformation r
transduction r bacteriophage r mutagenesis
INTRODUCTION
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects almost one
third of the worlds population. Yearly, there are an estimated 10 million new cases of TB
from which 2 to 3 million people die. Over the last 15 years, the development of genetic
tools to manipulate M. tuberculosis has proliferated. Furthermore, completion of the
M. tuberculosis DNA sequence (Cole et al., 1998), as well as other related mycobacteria,
has yielded tremendous information for better understanding the bacterium and the
disease it causes.
The three biggest challenges in working with M. tuberculosis are first, the risk of aerosol
infection, second, the long doubling time of the bacterium, and third, the consequences
of a cell wall rich in mycolic acids, which leads to cells that clump. Since M. tuberculosis
is a human pathogen and can infect via the aerosol route, it is important to minimize the
production of aerosols (see Strategic Planning, Safety Considerations). The long doubling
time (18 to 22 hr for wild-type strains, longer for some mutant strains) presents specific
challenges in aseptic technique (refer to UNIT 10A.1). Finally, mycolic acids, a hallmark
of mycobacteria, are hydrophobic and cause bacteria to clump together, interfering with
the establishment of clonal isolates. (See UNIT 10A.3 for more information about mycolic
acids and their isolation.)
CAUTION: Mycobacterium tuberculosis is a Biosafety Level 3 (BSL-3) pathogen. Follow
all appropriate guidelines for the use and handling of pathogenic microorganisms. See
Strategic Planning for further discussion. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
STRATEGIC PLANNING
Growth
discusses laboratory maintenance of M. tuberculosis including media preparation, inoculation of solid and liquid media, and strain storage.
UNIT 10A.1
Actinobacteria
(High G+C
Gram Positive)
Current Protocols in Microbiology 10A.2.1-10A.2.21, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc10a02s6
C 2007 John Wiley & Sons, Inc.
Copyright
10A.2.1
Supplement 6
Safety Considerations
Standards of BSL-3 containment should be followed for work with M. tuberculosis.
Consult with the Safety Officer and/or Institutional Biosafety Committee for additional
guidance. See http://www.cdc.gov/OD/ohs/symp5/jyrtext.htm for a brief description of
general BSL-3 laboratory features. Inactivation of M. tuberculosis cells is necessary to
continue some studies outside the BSL-3 laboratory. All inactivation methods should be
validated in individual laboratories, taking into consideration the concentration of cells
used. To test if conditions are adequate for inactivation of M. tuberculosis, an article
by Schwebach et al. (2001) provides some guidance. For many of the procedures, the
authors have found the following conditions sufficient for inactivation of M. tuberculosis:
incubation for either 12 hr at 65 C, 2 hr at 80 C, or 15 min at 95 C; incubation in a
final concentration of either 2.5% gluteraldehyde, 2% paraformaldehyde, 5% formalin,
or Vesphene IIse for 1 hr. Care should be taken to ensure that the entire tube is immersed
in the water bath. Likewise, incubation of M. tuberculosis cultures with gluteraldehyde,
paraformaldehyde, formalin, or Vesphene IIse should be done in a matter that ensures
that the entire interior surface of the tube has been coated by gently rolling and inverting
the tube.
BASIC
PROTOCOL 1
Materials
10% (w/v) glycine (see recipe)
Cell culture
GTE solution (see recipe)
Lysozyme solution (see recipe)
10% SDS (see recipe)
10 mg/ml proteinase K (see recipe)
5 M NaCl
CTAB solution (see recipe), 65 C
24:1 (v/v) chloroform/isoamyl alcohol
Isopropanol
70% ethanol
TE buffer (APPENDIX 2A)
37 C incubator
15-ml polypropylene conical tubes
Refrigerated tabletop centrifuge
2-ml microcentrifuge tubes
Microcentrifuge
Genetic
Manipulation of
M. tuberculosis
10A.2.2
Supplement 6
7. Preheat CTAB solution to 65 C, add 160 l, and mix gently. Incubate 10 min at
65 C.
8. Add an equal volume (1 ml) 24:1 (v/v) chloroform/isoamyl alcohol, shake vigorously to mix, and microcentrifuge for 5 min.
9. Transfer 900 l aqueous layer to a fresh 2-ml microcentrifuge tube.
10. Repeat extraction with 24:1 (v/v) chloroform/isoamyl alcohol, shake vigorously to
mix, and spin in microcentrifuge for 5 min.
11. Transfer 800 l to fresh 2-ml microcentrifuge tube.
CAUTION: For BSL3 organisms, dip tube in a disinfectant such as Vesphene IIse to
disinfect outer surface. From this point on, the supernatant can be processed in a BSL-2
laboratory (see Strategic Planning).
12. To 800 l aqueous layer, add 560 l (0.7 vol) isopropanol, mix gently by inversion
until the DNA has precipitated out of solution.
13. Incubate 5 min at room temperature. Microcentrifuge for 10 min, room temperature.
14. Aspirate supernatant and add 1 ml of 70% ethanol to wash DNA pellet. Mix gently
by inversion and microcentrifuge 10 min at room temperature.
15. Carefully aspirate supernatant, avoiding the pellet, and air-dry DNA pellet for 15 min.
Do not overdry.
16. Add 50 l TE buffer to dried DNA pellet and store overnight at room temperature
or 4 C to allow pellet to dissolve. Store up to 1 year at 20 C.
RNase A can be added to TE (1 g/ml) to reduce RNA contamination.
ALTERNATE
PROTOCOL 1
Actinobacteria
(High G+C
Gram Positive)
10A.2.3
Current Protocols in Microbiology
Supplement 6
Materials
M. tuberculosis cell culture
3:1 (v/v) chloroform/methanol (using water-saturated chloroform)
Phenol, Tris-buffered (APPENDIX 2A)
GTC solution (see recipe)
2-Mercaptoethanol
Isopropanol
70% ethanol
TE buffer (APPENDIX 2A)
Spectrophotometer
50-ml polypropylene conical tubes
Tabletop centrifuge
Vortex
1. Grow cells to late log phase (OD600 of 0.8 to 1.0).
2. Transfer 50 ml cell culture to a 50-ml conical tube and centrifuge 10 min at 2000
g, room temperature.
Use polypropylene tips and tubesno polystyrene plastic.
Genetic
Manipulation of
M. tuberculosis
10A.2.4
Supplement 6
Materials
Plated colonies or liquid cultures
24:1 (v/v) chloroform/isoamyl alcohol
3 M ammonium acetate
95% ethanol
Plastic loop
200 l microcentrifuge tubes with screw-caps and O-rings
Vortex
95 C heating blocks
Microcentrifuge
1. For colonies, scrape a clump of cells off a plate using a plastic loop. Transfer the
cells to a 200-l sterile water in screw-cap (O-ring) microcentrifuge tube. For liquid
culture, transfer 200 l of culture to a 200-l microcentrifuge tube.
2. Briefly vortex to resuspend cells. Incubate 30 min in a 95 C heating block with
another heating block placed on top.
For BSL3 organisms, dip closed tube in a disinfectant such as Vesphene IIse to disinfect
the outer surface of the tube. From this point on, the filtrate can be processed in a BSL2
laboratory.
BASIC
PROTOCOL 2
NOTE: The organic solvents will not harm the column or matrix, but ensure that the
plastic pipets and receptacles are made of polypropylene, not polystyrene.
NOTE: The yield of an 8-kb plasmid from a 50-ml culture of M. tuberculosis is 1/10
what is expected from a similar culture in E. coli, most likely because of profound
Actinobacteria
(High G+C
Gram Positive)
10A.2.5
Current Protocols in Microbiology
Supplement 6
differences in cell wall composition and subsequent cell lysis and DNA recovery, and
also because of lower plasmid copy number in M. tuberculosis.
Materials
10% (w/v) glycine (see recipe)
Cell culture
Qiagen midi-prep kit containing:
P1 buffer
10 mg/ml lysozyme
1:1 (v/v) chloroform/methanol
1. Grow a culture of mycobacteria to log phase (OD600 = 1). Prior to harvest (3 to 24
hr), add glycine to the culture at a final concentration of 1% (w/v) from a 10% stock
solution in water. Incubate culture at 37 C.
The addition of glycine serves to weaken the cell wall, making it easier to lyse the cells
with lysozyme.
Materials
M. tuberculosis culture
3:1 (v/v) chloroform/methanol, prepare fresh
Trizol
Chloroform
Isopropanol
70% ethanol (made with DEPC-treated water), cold
Diethylpyrocarbonate (DEPC)-treated water
Spectrophotometer
15-ml polypropylene tubes
Tabletop centrifuge
Vortex
13-ml Sarstedt tube
1.5- and 2-ml RNase-free tubes (Ambion)
1. Grow 50 ml culture of M. tuberculosis to an OD600 of 0.5 to 1.0.
Genetic
Manipulation of
M. tuberculosis
10A.2.6
Supplement 6
3. Discard supernatant and centrifuge tubes 1 min at 2000 g to bring down residual
supernatant to be discarded.
4. Add 1 ml freshly prepared 3:1 (v/v) chloroform/methanol mix to each pellet and
immediately mix well using a 1-ml disposable pipet tip.
5. Vortex 1 min, then add 5 vol (5 ml) Trizol to each tube.
6. Vortex each sample for 15 sec to mix well
7. Incubate 10 min at room temperature (layers should separate).
8. Centrifuge 15 min at 2000 g, 4 C.
9. Rinse 13-ml Sartstedt tubes with chloroform. Transfer aqueous upper level into the
rinsed Sartstedt tube and add an equal volume of isopropanol.
For BSL3 organisms, dip closed tube in a disinfectant such as Vesphene IIse to disinfect
the outer surface of the tube. From this point on, the filtrate can be processed in a BSL2
laboratory.
ALTERNATE
PROTOCOL 3
This protocol for RNA preparation uses the Fast RNA prep machine (FastPrep; FP120,
Bio101, Thermo Electron Corporation). In brief, using the FastRNA Pro Blue kit in
conjunction with the FastPrep machine allows for homogenization of mycobacterial cells
that allows for release of RNA into the FastPrep RNApro solution. For mycobacteria, the
key point is not to overload the matrix provided in the kituse 5 ml of a mycobacterial
culture with an OD600 of 0.5 to 1.0 or 10 ml culture with a lower OD600 .
Materials
Mycobacterial culture
Qiagen RNA protect reagent (Qiagen cat. no. 76506)
Qiagen RNeasy kit (Qiagen cat. no. 74104)
Absolute ethanol
Spectrophotometer
2-ml microcentrifuge tubes
Microcentrifuge
Lysing Matrix B tube (MP Bio cat. no. 6911-100)
Fast RNA prep machine (FastPrep; FP120, Bio101, Thermo Electron Corporation)
1. Culture 10 ml mycobacterial culture grown to an OD600 of 0.2 and collect by
centrifuging for 10 min at 2000 g, room temperature.
2. Decant the supernatant and resuspend the cell pellet in 1 ml of Qiagen RNA protect
reagent. Incubate the cell suspension 1 hr at room temperature.
CAUTION: Although this treatment has been found to sterilize M. tuberculosis and M.
bovis cultures such that subsequent steps can be performed in a BSL-2 laboratory, this
should be confirmed in each laboratory.
At this point, the cells can be stored for 6 months at 80 C or RNA extraction can begin.
Current Protocols in Microbiology
Actinobacteria
(High G+C
Gram Positive)
10A.2.7
Supplement 6
Materials
M. tuberculosis culture
GTC solution (for RNA prep; see recipe)
15- or 50-ml conical tubes
Tabletop centrifuge
1. Grow M. tuberculosis to desired growth phase.
2. In a 15- or 50-ml conical tube, prepare a volume of GTC solution equal to the cell
culture volume ready.
3. Add culture straight into the tube and mix by vortexing or inverting the capped tube.
4. Centrifuge 10 min at 2000 g, room temperature.
5. If processing by Trizol for RNA extraction (see Basic Protocol 3), resuspend well in
Trizol (1 ml/25 ml culture), then proceed with RNA preparation in Basic Protocol 3,
step 6 or store at 80 C.
Alternatively pellet can also be processed using the Fast RNA prep (see Alternative
Protocol 2, beginning at step 4), taking care not to overload the matrix.
BASIC
PROTOCOL 4
Genetic
Manipulation of
M. tuberculosis
10A.2.8
Supplement 6
Materials
M. tuberculosis culture
Middlebrook 7H9 broth
10% glycerol with Tween-80 (see recipe), sterile
DNA
Selective plates
Spectrophotometer
490-cm2 roller bottle (Corning cat. no. 430195)
37 C incubator
50-ml conical tubes
Tabletop centrifuge
1.5-ml microcentrifuge tubes
0.2-cm electroporation cuvettes
Electroporator (e.g., BioRad Gene Pulser)
15-ml snap-cap plastic tube (Falcon, cat. no. 2059 or equivalent)
1. Grow M. tuberculosis to mid-log phase (OD600 should be between 0.5 and 1.0) in
Middlebrook 7H9 broth in a 490-cm2 roller bottle at 37 C.
A culture volume of 100 ml is sufficient for five electroporations if the culture is resuspended in 1/100 volume or 25 electroporations; if resuspended in 1/10 the volume, see
step 5. Check the OD600 the day before preparing the cells to make sure that the culture
is still in log phase (i.e., the OD600 has approximately doubled in 1 day).
2. Transfer cells to 50-ml conical tubes and centrifuge 15 min at 2000 g, room
temperature.
3. Discard supernatant and wash cells with an equal volume of sterile room temperature
10% glycerol with Tween-80. Pellet cells by centrifugation for 15 min at 2000 g,
room temperature.
4. Repeat the wash and centrifugation two additional times.
5. Resuspend the cell pellet in sterile, room temperature, 10% glycerol with Tween-80,
using 1/100 the original volume of the culture. Use 200 l of cells per transformation.
Alternatively, resuspend the cell pellet in 1/10 of the original volume of the culture and
use 400 l of cells per transformation.
6. Mix DNA and bacteria in a 1.5-ml microcentrifuge tube and incubate for 10 min or
longer at room temperature.
7. Transfer bacteria and DNA to 0.2-cm cuvette and electroporate (BioRad Gene Pulser,
settings: 2500 mV, 1000 , 25 F). To avoid overheating the electroporator, allow
the electroporator to cool for 1 to 2 min between transformations.
8. Add 1 ml of Middlebrook 7H9 medium to each cuvette and transfer cells to a 15-ml
snap-cap plastic tube.
Alternatively, add 1 ml Middlebrok 7H9 medium to each cuvette and do not transfer to
another tube (replace the cuvette cap).
10A.2.9
Current Protocols in Microbiology
Supplement 6
BASIC
PROTOCOL 5
Figure 10A.2.1 Specialized transduction. Regions anking the upstream and downstream regions of your
favorite gene (yfg) are cloned into a hygromycin-containing cassette on a plasmid containing both lambda cos
sites and mycobacteriophage cos sites. M. smegmatis is transfected at 30 C to allow for production of a hightiter phage lysate containing the yfgX::HygR allele. The high-titer phage lysate is then used to transduce M.
tuberculosis at 37 C (non-permissive temperature for phage lysis) to deliver yfgX::HygR substrate to the cell for
subsequent allelic exchange.
Genetic
Manipulation of
M. tuberculosis
10A.2.10
Supplement 6
substrate (AES) cosmid, Bardarov et al. (2002) reviews the necessary features of an
AES cosmid. Briefly, the AES plasmid has multiple cloning sites (MCS) upstream and
downstream of an antibiotic-resistance marker (hygromycin), and a counter-selectable
marker (sacB). Also on the AES plasmid is oriE, an E. coli plasmid origin of replication,
a -cos site for lambda packaging, and a unique PacI restriction site for eventual ligation
to a modified, temperature-sensitive mycobacteriophage such as TM4 (Bardarov et al.,
2002). The lengthy protocol for generating knockout strains of M. tuberculosis includes:
(1) construction of allelic exchange subsrates (AES) phasmids, (2) phasmid screening,
(3) phage preparation and verification, (4) preparation of high-titer phage lysates, and
(5) phage transduction. Only the final step of phage transduction that utilizes an M.
tuberculosis strain is performed at BSL-3 (M. smegmatis is a BSL-1 organism; see UNIT
1A.1 for more information on biosafety levels).
Materials
Upstream and downstream regions (1-kb each) of gene of interest
pYUB854 plasmid or equivalent (jacobsw@hhmi.org; Bardarov et al., 2002)
PacI restriction endonuclease and buffer 1
phAE159 or equivalent (jacobsw@hhmi.org)
Shrimp alkaline phosphotase (SAP)
1% agarose gel
T4 DNA ligase and 10 T4 ligase buffer
Lambda in vitro packaging mix (e.g., MaxPlax, Epicentre cat. no. MP5110)
Mycobacteriophage buffer (MP buffer; see recipe)
E. coli HB101 cells (see Support Protocol 2)
LB broth with and without hygromycin (150 mg/liter)
LB plates with hygromycin (150 mg/liter)
Qiagen miniprep kit (Qiagen cat. no. 27106)
10 BSA
M. smegmatis mc2 155
Middlebrook 7H9 broth without Tween-80
Middlebrook top agar (see recipe)
Middlebrook 7H10 plates without Tween-80
M. tuberculosis
Mycobacteriophage wash medium (see recipe)
37 and 65 C water baths
Agarose gel electrophoresis apparatus
Microcentrifuge
15-ml snap-cap tubes (Falcon cat. no. 2059 or equivalent)
Oak Ridge centrifuge tubes (Nalgene cat. no. 3115 or equivalent)
0.45- and 5-m syringe filters
Spectrophotometer
37 to 39 C heating block
2-ml screw-cap tubes
Additional reagents and equipment for electroporation (see Support Protocol 3)
Construct allelic exchange substrate (AES) phasmid
1. For the target DNA sequence to be deleted, clone 0.5 to 1 kb of sequence upstream and downstream of the region into a plasmid such as pYUB854 (described in
Bardarov et al., 2002).
Upstream and downstream regions are typically each amplified by PCR, cloned into a
vector such as pTOPO (Invitrogen, cat. no. K4500-40), sequenced to confirm the insert,
then sequentially subcloned into pYUB854.
Actinobacteria
(High G+C
Gram Positive)
10A.2.11
Current Protocols in Microbiology
Supplement 6
2 to 4 l phAE159
4 to 6 l plasmid
1 l 10 T4 ligase buffer
0.5 l T4 DNA ligase (400 U/l)
Bring up to 10 l with water
7. Add 5 l ligation mixture to a MaxPlax in vitro packaging extract on dry ice. Remove
and mix gently by tapping lightly with finger as the sample thaws.
8. Incubate for up to, but no more than, 2 hr at room temperature.
9. Stop reaction by adding 400 l MP buffer.
10. Add 1 ml E. coli HB101 host cells directly to the packaging tube, mix, and incubate
1 hr at 37 C without shaking (see Support Protocol 2 for preparation of HB101 host
cells).
Alternatively, remove half of packaging mixture and store for future use (in the case where
HB101 cells are not competent).
16. Digest the phasmid minipreps with PacI to screen phasmids that have the correct
insert. For example, for a total volume of 10 l, add:
5 l DNA
1 l 10 NEB buffer 1
1 l 10 BSA
1 l PacI
2 l water.
Genetic
Manipulation of
M. tuberculosis
10A.2.12
Supplement 6
Prepare phage
18. Eighteen to 24 hr prior to the electroporation, start a fresh culture of M. smegmatis
mc2 155 for use in step 21.
19. Electroporate 5 to 10 l phasmid DNA into 400 l competent M. smegmatis mc2 155
cells (see Support Protocol 3, steps 1 through 7).
20. Add 1 ml Middlebrook 7H9 broth (without Tween-80) and incubate 1 hr at 37 C.
21. Set up two dilutions:
(a) Add 400 l transformed mc2 155 to 400 l actively growing mc2 155.
(b) Add 110 l transformed mc2 155 to 1 ml actively growing mc2 155.
22. Add 300 to 600 l of dilutions a and b, separately, to 3 ml Middlebrook top agar in
15-ml snap-cap tubes, vortex to mix, and plate onto prewarmed 7H10 plates (without
Tween-80).
23. Incubate the plates 3 days at 30 C.
24. With a sterile pipet tip, core out one to two plaques each into 200 l MP buffer to
recover phage; incubate at 1.5 hr at room temperature, then store at 4 C.
30. Harvest the phage lysate by pipetting up the MP buffer from each plate and pooling.
Centrifuge this agar-phage mix in an Oak Ridge centrifuge tube 15 min at 15,800
g, 4 C.
31. Filter the phage lysate through a 5-m filter and then a 0.45-m filter.
32. Make serial dilutions of the phage lysate in MP buffer. In duplicate, mix 300 l
phage lysate dilutions with 300 l mc2 155 cells and incubate 30 min at 30 C.
33. To each phage dilution, add 3 ml Middlebrook top agar and plate on Middlebrook
7H10 plates.
34. For each dilution, incubate one plate at 30 C and one plate at 37 C to confirm
temperature-sensitive phenotype and to determine phage titeri.e., plaque forming
units (pfu)/ml.
Actinobacteria
(High G+C
Gram Positive)
10A.2.13
Current Protocols in Microbiology
Supplement 6
36. Transfer 10 ml culture to a 15-ml conical tube and centrifuge 15 min at 2000 g,
4 C.
37. Decant supernatant and wash pellet once with an equal volume of mycobacteriophage
wash medium.
38. Centrifuge 15 min at 2000 g, decant supernatant and resuspend cell pellet in 1 ml
MP buffer.
39. Prewarm phage at 37 C to 39 C in a heating block containing water in the wells.
40. In a 2-ml screw-cap tube, mix 1 ml cells in MP buffer and 1 ml high-titer phage.
Gently mix the cells and phage using a 1000-l tip. Incubate overnight in a 37 to
39 C heating block. Include cells only and phage only controls.
41. Microcentrifuge 10 min at 15,800 g, 4 C, to pellet cells. Remove supernatant and
resuspend pellet in 0.2 ml Middlebrook 7H9 broth.
42. Plate on one selective plate. Incubate 3 to 4 weeks at 37 C.
SUPPORT
PROTOCOL 2
Materials
E. coli HB101 (ATCC #33694)
LB broth supplemented with 10 MgSO4 and 0.2% maltose
10 mM MgSO4
37 C incubator with shaker
Spectrophotometer
50-ml conical tubes
1. To prepare E. coli HB101 for transduction, grow the HB101 strain overnight in LB
broth supplemented with 10 mM MgSO4 and 0.2% maltose at 37 C with shaking.
2. Inoculate 25 ml fresh medium with 0.5 ml overnight culture.
3. Shake culture at 200 rpm at 37 C until OD600 reaches 0.8 to 1.0.
4. Transfer cells to a 50-ml conical tube and centrifuge 10 min at 2000 g (3000 rpm),
4 C.
5. Decant supernatant and resuspend cell pellet in 12.5 ml of 10 mM MgSO4 .
6. Store cells up to 2 days at 4 C until ready for use.
Genetic
Manipulation of
M. tuberculosis
10A.2.14
Supplement 6
Preparation of M. smegmatis
cells for electroporation is similar to the electroporation protocol for M. tuberculosis (see Basic Protocol 4) with some modifications.
SUPPORT
PROTOCOL 3
Materials
M. smegmatis mc2 155 cells
Middlebrook 7H9 broth or LB broth with 0.05% Tween
10% glycerol with 0.05% Tween, 4 C
Transforming DNA
Medium plates with antibiotic (e.g., 10-20 g/ml Kanamycin at; at 50 g/ml
Hygromycin B)
Spectrophotometer
50-ml conical tubes, prechilled
0.2-cm electroporation cuvettes (GenePulser, BioRad cat. no. 165-2086)
GenePulser electroporator (BioRad cat. no. 1652076)
15-ml snap-cap plastic tube (Falcon cat. no. 2059 or equivalent)
Tabletop centrifuge
1. Grow fresh 50 ml culture of M. smegmatis mc2 155 in either Middlebrook 7H9 broth
or LB with Tween (0.05%) to mid-log phase (OD600 0.5 to 1.0).
2. Incubate cells 10 min (no longer than 2 hr) on ice.
For the following steps, keep cells as close to 0 C (ice bath) as possible. The electroporation cuvettes, 50-ml conical tubes, and 10% glycerol should be pre-chilled to 4 C.
3. Transfer chilled cells to a 50-ml prechilled conical tube. Centrifuge cells 10 min at
2000 g, 4 C. Decant supernatant from cell pellet.
4. Wash cells two times with 40 ml ice-cold 10% glycerol.
For optimal results, 10% glycerol should be prepared fresh weekly with distilled water.
Sterilize by filtration through 0.2-m membrane pore, do not autoclave.
5. Resuspend the washed cell pellet in 5 ml ice-cold 10% glycerol (1/10 culture volume)
and store on ice (0.4 ml per sample).
6. Dispense transforming DNA (1 to 10 l) into a prechilled electroporation cuvette
with 0.2-cm electrode gap and incubate on ice. To each aliquot of DNA, add 400 l
of prepared cell suspension and mix by gently pipetting up and down (use prechilled
pipet tips for cell transfer).
7. Pulse one time with a GenePulser electroporator set at 2.5 V, resistance 1000 ,
capacitance 25 FD.
The time constant reading should be 19 to 21 for control cells with no DNA added.
8. Immediately dilute transformed cell mixture with 2 ml medium. Transfer cell suspension to a 15-ml snap-cap plastic tube.
9. Incubate 2 hr at 37 C (with or without shaking) to allow cell recovery and expression
of antibiotic resistance.
10. Centrifuge 10 min at 2000 g, 4 C. Aspirate supernatant from cell pellet; resuspend
cell pellet in 0.5 ml medium.
11. Plate desired volume of cell resuspension on plates containing the antibiotic of choice
(e.g., 10 to 20 g/ml kanamycin or 50 g/ml hygromycin B).
Actinobacteria
(High G+C
Gram Positive)
10A.2.15
Current Protocols in Microbiology
Supplement 6
Materials
M. tuberculosis culture
Mycobacteriophage (MP) buffer (see recipe)
High-titer phage lysate containing transposon
Middlebrook 7H9 broth
Middlebrook 7H10 plates with 50 mg/liter hygromycin
Spectrophotometer
15-ml conical tube
Centrifuge (Sorvall RT 600 tabletop centrifuge or equivalent)
37 C incubator
1. Grow 10 ml M. tuberculosis culture to an OD600 of 0.8.
2. Transfer to a 15-ml conical tube and centrifuge 10 min at 3450 g (4000 rpm),
room temperature.
3. Decant supernatant and resuspend pellet in 10 ml MP buffer.
4. Centrifuge cells 10 min at 3450 g (4000 rpm), room temperature.
5. Decant supernatant and resuspend cell pellet in 200 l MP buffer.
6. In a sterile 15-ml conical tube, mix 200 l of cells with 1 ml of high-titer phage
lysate (1010 to 1011 pfu/ml) containing transposon.
7. Incubate 4 hr at 37 C.
8. Add 1 ml of Middlebrook 7H9 broth to the transduction mixture and incubate 24 hr
at 37 C.
9. Centrifuge cells 10 min at 3450 g (4000 rpm), room temperature.
10. Decant supernatant and resuspend cell pellet in 1 ml Middlebrook 7H9 broth.
11. Plate the cell suspension on eleven plates of Middlebrook 7H10 with 50 mg/liter
hygromycin B: one plate with 10 l, one plate with 50 l, and nine plates with 100 l
each of cell suspension.
12. Incubate plates 3 weeks at 37 C
Genetic
Manipulation of
M. tuberculosis
13. After the 3-week incubation, count the colonies to determine the number of transposition events and screen as needed.
10A.2.16
Supplement 6
CTAB solution
Dissolve 4.1 g sodium chloride in 90 ml water. While stirring on a magnetic stirrer,
add 10 g cetrimide (hexadecyltrimethylammonium bromide, Sigma cat. no. H5882).
Incubate in 65 C water bath to dissolve cetrimide into solution. Store 1 year at room
temperature.
Dextrose, 20%
Dissolve 20 g dextrose in water and bring up to 100 ml with water. Sterilize by
autoclaving or by filtration through a 0.22-m pore membrane. Store 1 year at room
temperature.
GTE solution
25 mM TrisCl, pH 8.0
10 mM EDTA
50 mM glucose
Autoclave and store 1 year at room temperature
Lysozyme solution
For optimal lysozyme activity, make a fresh 10 mg/ml solution of lysozyme (Sigma
cat. no. L6876) in 25 mM TrisCl, pH 8.5 buffer.
Actinobacteria
(High G+C
Gram Positive)
10A.2.17
Current Protocols in Microbiology
Supplement 6
Genetic
Manipulation of
M. tuberculosis
One of the most important aspects of working with M. tuberculosis is to minimize any
possible aerosol exposure to this pathogen (see
Strategic Planning, Safety Considerations).
Second to that, scrupulous attention to detail in
preparing media and reagents is crucial. Given
the long doubling time (3 weeks for colonies
10A.2.18
Supplement 6
Anticipated Results
Extraction of genomic DNA
Yield of DNA from this method is lower
than the CTAB method but may be adequate
for PCR and some Southern analysis. In contrast, the quality of the DNA from the CTAB
method is higher than the GTC prep, but the
time investment is much greater, which is a
consideration when there are many cultures to
screen (see Time Considerations).
Figure 10A.2.2 Southern analysis. Chromosomal loci for M. tuberculosis (A) wild type, (B) marked lysA mutant, and
(C) unmarked lysA mutant. See Basic Protocol 5 for construction of the knock-out phages and phage transduction. The
probe is the upstream anking sequence (UFS) of lysA that was used for the knock-out construct and represented as a
black box. The arrows identify the fragments that will be detected by Southern analysis. (D) Southern blot of NcoI-digested
genomic DNA (see Basic Protocol 1 for DNA isolation) probed with the UFS of lysA that was used for the knock-out
construct. Lanes 1 and 2, molecular weight = 3 (unmarked lysA mutant); lanes 4 and 5, fragment size indicated (marked
lysA mutant).
Actinobacteria
(High G+C
Gram Positive)
10A.2.19
Current Protocols in Microbiology
Supplement 6
Time Considerations
Extraction of genomic DNA
The GTC method takes 1 hr in the BSL-3
laboratory and then another 1 hr in the BSL-2
laboratory. In contrast, the CTAB method has
at least one to two overnight incubations. If
glycine pretreatment is used, then it requires
<1 hr on day 1, 30 min on day 2, and 2 hr on
day 3. For some strains, better DNA yield is
observed if the pellet is allowed to dissolve
overnight so further analysis could begin
on day 4 (or day 3 without the overnight
incubation).
Extraction of plasmid DNA
This protocol is spread over 3 days depending on the length of the incubation with
glycine. Day 1 takes <10 min, day 2 requires
30 min, and day 3 takes 2 hr.
PCR template preparation
This protocol takes 1 hr for less than ten
samples.
Genetic
Manipulation of
M. tuberculosis
Extraction of RNA
The main protocol takes 1 hr followed
by an overnight incubation. If there are more
than four to six samples to process, the time
required will be longer.
Electrotransformation
Preparation of the cells for electroporation
by repeated washes with glycerol is key for
removing salts that might interfere with electroporation. Day 1 of this protocol takes 2 hr
followed by an overnight incubation. The plating on day 2 takes <30 min. Transformants
will not be evident for another 3 to 4 weeks.
Generation of knockouts with specialized
transduction
To knockout a gene with specialized transduction can take weeks to months. Beginning with target DNA sequence already cloned
in pYUB854, construction of the allelic exchange phasmid is spread out over 3 days, with
2 hr of work each day. Phasmid screening
takes another 2 days. Day 1 of phage preparation takes 10 min, day 2 (electroporation
of M. smegmatis) requires 2 to 3 hr, and day
5 requires 2 hr. For preparation of high titer
phage lysate, day 1 requires 1 hr and day
4 requires 10 hr (not all hands-on time, as
there are a number of incubation steps). For
phage transduction, day 1 requires 1 hr, and
day 2 requires 30 min. Transductants will not
be evident for another 3 to 4 weeks following
plating.
Transposon mutagenesis
Preparation of the transposon transduction
takes 2 hr on day 1 with a 4-hr incubation
followed by an overnight incubation, day 2
only requires 30 min. Colonies will not be
evident for 3 to 4 weeks.
Acknowledgments
Special thanks to Kieran Bhatt, Bing Chen,
Zhiyan (Annie) Dai, Dee Dao, Robert Glover,
Morad Hassani, Jordan Kriakov, Sunhee Lee,
Catherine Vilcheze (see UNIT 10A.3), Brian
Weinrick, and Torin Weisbrod for contributions and helpful discussions.
Literature Cited
Bardarov, S., Bardarov, S. Jr., Pavelka, M.S. Jr.,
Sambandamurthy, V., Larsen, M., Tufariello, J.,
Chan, J., Hatfull, G., and Jacobs, W.R. Jr. 2002.
Specialized transduction: An efficient method
for generating marked and unmarked targeted
gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007-3017.
Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:15131523.
Butcher, P.D. 2004. Microarrays for Mycobacterium tuberculosis. Tuberculosis (Edinb)
84:131-137.
10A.2.20
Supplement 6
Kriakov, J., Lee, S., and Jacobs, W.R. Jr. 2003. Identification of a regulated alkaline phosphatase,
a cell surface-associated lipoprotein, in
Mycobacterium smegmatis. J. Bacteriol. 185:
4983-4991.
Wards, B.J. and Collins, D.M. 1996. Electroporation at elevated temperatures substantially
improves transformation efficiency of slowgrowing mycobacteria. FEMS Microbiol. Lett.
145:101-105.
Actinobacteria
(High G+C
Gram Positive)
10A.2.21
Current Protocols in Microbiology
Supplement 6
UNIT 10A.3
This unit describes the isolation of Mycobacterium tuberculosis mycolic acids and their
analysis by either thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). The term mycolic acid was given by Stodola et al. (1938) to
designate the principal ether-soluble fraction of the wax isolated from M. tuberculosis (Stodola et al., 1938). Mycolic acids are long-chain -alkyl -hydroxy fatty acids
(70 to 90 carbons) that are present within the mycobacterial cell wall. M. tuberculosis
comprises three major mycolic acid classes (Fig. 10A.3.1): the -mycolic acids (containing cyclopropane rings and/or double bonds with either a cis or trans configuration),
methoxy-mycolic acids (containing methoxy groups and cyclopropane rings), and ketomycolic acids (containing keto groups and cyclopropane rings).
Mycolic acids are either covalently bound to arabinogalactan or found as free lipids
esterified to trehalose or glucose in the mycobacterial cell wall. They have been shown to
modulate cell wall fluidity, protect mycobacteria against chemicals such as antibacterials
or stress, play a role in pathogenecity and persistence, and have diverse immunological
functions (Kremer and Besra, 2005).
Analysis of mycolic acids by TLC has been widely used to study mycolic acid biosynthesis, the formation of unusual mycolic acids in mycobacterial mutant strains, and the
effect of antimycobacterial drugs targeting the cell wall. It is mainly a qualitative method,
which allows for an easy identification of the three major mycolic acid classes. HPLC
analysis is a quantitative method that efficiently separates different structures based on
functional groups and chain length. It has been extensively used to classify and identify
mycobacterial species. When linked to a mass spectrometer, HPLC is a powerful tool to
isolate and characterize mycolic acids.
Both protocols utilize the same initial steps: the mycolic acids are hydrolyzed from the
cell wall by saponification and then derivatized. In the first method, the saponified mycolic
acids are methylated and then extracted prior to TLC analysis. In the second method, the
mycolic acids are extracted as free fatty acids and then derivatized to UV-absorbing esters
for HPLC analysis. Both methods will work for the extraction and analysis of mycolic
acids from other mycobacteria, such as M. smegmatis, M. avium, or M. leprae, or other
bacterium that contain mycolic acids, such as Corynebacterium, Gordonia, Nocardia,
and Rhodococcus. However, mycolic acids from other bacteria have different structures
OH
-mycolates
CO2H
OMe
OH
methoxy-mycolates
CO2H
OH
keto-mycolates
CO2H
Figure 10A.3.1 Structure of -, methoxy-, and keto-mycolates from M. tuberculosis. Only one
type of mycolic acid for each class is represented. Reprinted from Minnikin et al. (2002), with
permission from Elsevier.
Actinobacteria
(High G+C
Gram Positive)
Contributed by Catherine Vilch`eze and William R. Jacobs
Current Protocols in Microbiology (2007) 10A.3.1-10A.3.11
C 2007 by John Wiley & Sons, Inc.
Copyright
10A.3.1
Supplement 5
Isolation and
Analysis of
M. tuberculosis
Mycolic Acids
Figure 10A.3.2 Autoradiography TLC of [14 C]-labeled fatty acid methyl esters (FAMEs) and
mycolic acid methyl esters (MAMEs) isolated from M. tuberculosis. FAMEs are a mixture of fatty
acids having from 14 to 26 carbons in length, and are usually not separated. Using three elutions
in a nonpolar solvent system allows for the separation of the three major classes of mycolic acids:
-, methoxy-, and keto-mycolic acids.
10A.3.2
Supplement 5
Materials
M. tuberculosis culture (UNIT 10A.1)
[1-14 C]-acetic acid, sodium salt in ethanol (1.0 mCi/ml)
Vesphene
40% aqueous tetrabutylammonium hydroxide solution
Methylene chloride (optima-grade)
Methyl iodide
3 N aqueous hydrochloric acid solution
Anhydrous sodium sulfate
Nitrogen gas
Liquid scintillation counting solution
Hexanes (HPLC-grade)
Ethyl acetate (HPLC-grade)
30-ml PETG media bottles (Fisher)
37 C incubator with shaker
15-ml conical tubes
Pyrex glass tube (13-mm o.d. 100-mm length) with Teflon-lined screw caps
50 and 100 C heating block
Tube rotator (e.g., Glas Col)
Cotton-plugged Pasteur pipets (optional)
4-ml vial with screw-thread cap
5-l capillary pipets
Silica gel 60 F254 250-m glass plates (5 10cm or 10 10cm)
TLC tank with lid
Autoradiography cassette
X-ray film
Culture and label M. tuberculosis cells
1. Under BSL-3 conditions (UNIT 1A.1), grow a 10-ml culture of M. tuberculosis
(UNIT 10A.1) to log-phase (OD600 = 0.6) in a 30-ml sterile PETG media bottle.
2. Label the culture by adding 10 Ci of [1-14 C]-acetic acid, sodium salt (1 Ci/ml
culture).
3. Incubate 20 hr (one generation time) at 37 C with shaking (100 rpm).
4. Transfer the culture to a 15-ml conical tube and centrifuge 10 min in a tabletop
centrifuge at 2000 g, 20 C. Discard the supernatant in a bottle containing Vesphene
and labeled for radioactive waste.
M. tuberculosis is extremely resistant to chemical germicides due mostly to its cell wall
composition. Vesphene has been shown to effectively sterilize M. tuberculosis cultures
(Schwebach et al., 2001).
5. Resuspend the cell pellet in 10 ml distilled water and centrifuge 10 min at 2000
g, 20 C. Discard the supernatant in a bottle containing Vesphene and labeled for
radioactive waste.
10A.3.3
Current Protocols in Microbiology
Supplement 5
8. Remove tube from heat and allow it to cool to room temperature before opening.
Add 2 ml methylene chloride and 100 l methyl iodide. Close the cap tightly.
At this point, the outside of the tube can be washed with Vesphene and the tube removed
from the BSL-3 facility according to standard procedures. Continue the protocol in the
fume hood of a BSL-2 laboratory.
Extract
10. Centrifuge the tube 1 min at 1400 g, room temperature, to allow for phase
separation. Using a Pasteur pipet, discard the upper aqueous phase.
11. Add 1 ml of 3 N aqueous hydrochloric acid solution. Mix the phases well by vortexing
the tube for 10 sec.
12. Let the phases separate or centrifuge the tube 1 min at 1400 g, room temperature,
to achieve phase separation. Discard the upper aqueous phase.
13. Add 1 ml of distilled water to the organic phase. Mix the phases well by vortexing
the tube for 10 sec.
14. Let the phases separate (5 min) or centrifuge the tube 1 min at 1400 g, room
temperature, to achieve phase separation. Discard the upper aqueous phase.
15. Remove any residual water in the organic phase by adding 2 to 4 g anhydrous sodium
sulfate. Vortex and let stand in the hood for 5 min.
16. Filter the suspension through fluted filter paper and transfer the organic phase into a
4-ml vial.
Alternatively, filter the sample using a cotton-plugged Pasteur pipet. Push the cotton
to the tapered part of the pipet with another pipet and use it as a disposable filtering
funnel.
17. Evaporate the organic phase to dryness under nitrogen by placing the vial in a 50 C
heating block and directing a gentle stream of nitrogen gas into the vial.
The residue, which contains FAMEs and MAMEs, can be stored at 20 C for several
months.
Analyze by TLC
18. Resuspend the residue in 200 l methylene chloride.
19. Measure cpm for the sample (ratio sample/scintillation liquid: 1/100).
This step gives the cpm/ml of each sample and allows, if required, the loading of equal
amounts of counts (cpm) for each sample on the TLC plate. In that case, the volume
spotted on the TLC plate might differ for each sample.
20. Using a 5-l capillary pipet, load a 5-l aliquot onto a Silica Gel 60 F254 plate. Allow
the aliquot to dry before placing the TLC plate into the TLC chamber.
The aliquot should be spotted 1 cm from the bottom of the plate. If more than one sample
needs to be spotted, leave at least 1 cm between samples.
21. Add the elution solvent: 75 ml of 95:5 (v/v) hexane/ethyl acetate to the TLC chamber.
Ensure that the solvent level in the TLC chamber is below the loading lane.
Isolation and
Analysis of
M. tuberculosis
Mycolic Acids
22. Place the TLC plate into the chamber. Let the elution solvent migrate until it is 0.5 cm
from the top of the TLC plate. Remove the plate from the chamber.
10A.3.4
Supplement 5
23. Let the plate dry (leave it in the fume hood for 5 min), then repeat the elution two
additional times (three elutions total).
The three elutions are necessary for the separation of the major classes of mycolic acids
by TLC.
24. In a dark room, place the dry TLC plate into an autoradiography cassette. Place a
sheet of X-ray film on top of the TLC plate. Close the lid and place the cassette in a
80 C freezer.
25. Expose for 24 to 36 hr at 80 C to obtain the autoradiogram.
ALTERNATE
PROTOCOL 1
Materials
0.6 M aqueous silver nitrate solution
Petroleum ether (b.p. 35 to 60 C, ACS-certified)
Anhydrous diethyl ether (ACS-certified)
125 C oven
Additional reagents and equipment for culturing, labeling, saponifying,
methylating, and extracting mycolic acids (see Basic Protocol 1)
Culture, label, saponify, methylate, and extract mycolic acids
1. Culture, label, saponify, methylate, and extract mycolic acids as described in Basic
Protocol 1 steps 1 to 18.
Prepare silver-impregnated TLC
2. Use a 10 10cm silica gel 60 TLC plate. Place the plate in a TLC tank containing
75 ml of 10% aqueous silver nitrate solution. Allow the solution to migrate until it
is 2 cm from the top. Remove the TLC plate, dry in a fume hood for 5 min, and then
activate 15 min in a 125 C oven.
The aqueous silver nitrate solution can be stored up to 1 year at room temperature in the
dark and reused until it runs out.
3. Load a 5-l aliquot of the mycolic acid extract onto the non-silver nitrate
impregnated section of the TLC plate. Allow the aliquot to dry before placing the
TLC plate into the TLC chamber.
Make sure the sample is loaded 1 cm from the bottom of the plate and 1 cm from the
left side of the plate, in the non-silver nitrateimpregnated part of the TLC (see Fig.
10A.3.3A).
Actinobacteria
(High G+C
Gram Positive)
10A.3.5
Current Protocols in Microbiology
Supplement 5
Figure 10A.3.3 (A-C) Illustration of the protocol for 2-D TLC. (D) Autoradiography 2-D TLC of
[14 C]-labeled mycolic acid methyl esters (MAMEs) isolated from M. tuberculosis. The different
mycolic acid classes, -, methoxy-, and keto-mycolic acids, are indicated.
Isolation and
Analysis of
M. tuberculosis
Mycolic Acids
10A.3.6
Supplement 5
BASIC
PROTOCOL 2
As in Basic Protocol 1, this protocol also extracts and derivatizes shorter-chain fatty acids
(C14 to C26 ), which elute in the first 5 min of the HPLC run. These shorter-chain fatty
acids can be separated by HPLC using the same reversed-phase C18 column with the
following settings: column temperature at 45 C, flow rate at 2 ml/min, and wavelength
at 260 nm. The mobile phase is 83:17 (v/v) acetonitrile/water used as an isocratic elution
for 20 min, followed by a linear increase to 100% acetonitrile in 2 min, and held at 100%
acetonitrile for 18 min (Zimhony et al., 2000).
Materials
M. tuberculosis (UNIT 10A.3)
Vesphene
50% (w/v) aqueous potassium hydroxide solution (9 N)
Methanol (HPLC-grade)
6 N aqueous hydrochloric acid solution
Chloroform (HPLC-grade)
Anhydrous sodium sulfate
Nitrogen gas
p-bromophenacyl ester derivatization kit (Alltech Associates)
Potassium bicarbonate
2-Propanol (HPLC-grade)
490-cm2 sterile roller bottle
Pyrex glass tube (16-mm o.d. 150-mm length) with Teflon-lined screw caps
50 , 85 , and 95 C heating blocks
Conical funnel
2-ml screw-thread vial with cap
Reversed-phase C18 column (4.6 150mm, 3-m particle size)
Culture M. tuberculosis
1. Grow a 30-ml culture of M. tuberculosis (UNIT 10A.3) to an optical density of 1 in a
490-cm2 sterile roller bottle.
2. Centrifuge 10 min at 2000 g, 20 C. Discard the supernatant in a waste bottle
containing Vesphene.
3. Resuspend the cell pellet in 10 ml distilled water. Centrifuge 10 min at 2000 g,
room temperature, and discard the supernatant in a waste bottle containing Vesphene.
Perform saponification
4. Resuspend the cell pellet in 2 ml of 50% aqueous potassium hydroxide solution.
Transfer into a Pyrex glass tube with Teflon-lined screw cap. Add 2 ml methanol to
the tube.
5. Close the cap and heat 16 hr in a 95 C heating block.
Make sure that only the part of the Pyrex glass tube containing the solution is in the heating
block and that at least the upper half of the glass tube is outside of the heating block.
This allows for cooling and condensation of any vapor coming from the saponification
mixture and prevents possible leakage.
Actinobacteria
(High G+C
Gram Positive)
10A.3.7
Current Protocols in Microbiology
Supplement 5
Perform extraction
7. Slowly, add 6 N aqueous hydrochloric acid solution until the pH is below 3 (3 to
4 ml), checking the pH with pH indicator paper.
8. Add 2 ml chloroform and carefully vortex.
9. Allow the phases to separate by gravity. If phase separation does not occur in 5 min,
the sample can be centrifuged 5 min at 1000 g, 20 C.
10. Using a Pasteur pipet, transfer the lower organic phase into a new Pyrex glass tube
and set aside. Extract the aqueous phase a second time with chloroform, repeating
steps 8 and 9. Combine the organic phases.
11. Remove any residual water in the organic phase by adding anhydrous sodium sulfate
(3 g). Vortex and allow it to stand for 5 min.
The sodium sulfate will clump while being hydrated.
12. Filter the suspension through a piece of fluted filter paper in a conical funnel to
remove the sodium sulfate clumps and transfer the organic phase into a new Pyrex
Glass tube.
Alternatively, filter the sample using a cotton-plugged Pasteur pipet. Push the cotton
to the tapered part of the pipet with another pipet and use it as a disposable filtering
funnel.
13. Evaporate the organic phase to dryness under nitrogen by placing the tube in a 50 C
heating block and direct a gentle stream of nitrogen gas into the tube (10 to 15 min
is usually necessary to obtain a dry sample).
Analyze by HPLC
16. Using a reversed-phase C18 column, set the flow rate at 1.5 ml/min, and the wavelength at 260 nm.
17. Equilibrate the column with 60:40 (v/v) methanol/2-propanol using isocratic elution.
18. Inject the sample.
Isolation and
Analysis of
M. tuberculosis
Mycolic Acids
10A.3.8
Supplement 5
Figure 10A.3.4 HPLC chromatogram of mycolic acids isolated from M. tuberculosis H37Rv. The
mycolic acid species elute between 15 and 25 min.
19. Elute isocratically in 60:40 (v/v) methanol/2-propanol for 3 min followed by a linear
increase to 6:94 (v/v) methanol/2-propanol over 18 min. Elute isocratically with
30:70 (v/v) methanol/2-propanol for 4 min.
The mycolates will elute between 15 and 22 min. In the first 5 min of the elution, the
byproducts of the derivatization reaction and the shorter-chain fatty acids are present in
the eluate (see Fig. 10A.3.4).
ALTERNATE
PROTOCOL 2
Butler and Guthertz (2001) have published a general method for isolation and analysis
of mycolic acids by HPLC. The advantage of their method is time: their saponification
requires only 1 hr (versus overnight) and the HPLC analysis is quicker with mycolic
acids eluting between 7 and 9 min (versus 15 to 25 min). The disadvantages are: (1) the
HPLC elution system uses a chlorinated solvent (methylene chloride) and, therefore, the
seals of the HPLC pump and the HPLC tubing must be resistant to chlorinated solvents,
(2) autoclaving tubes containing organic solvents (step 5) may not be authorized in some
institutions for safety purposes, and (3) a quicker elution of mycolic acids might result
in a poorer resolution of peaks and compromise the HPLC mycolic acid analysis. Follow
Basic Protocol 2 with alterations in the following steps.
5. Close the cap and autoclave 1 hr at 121 C.
Autoclaving glass tubes containing organic solvents may not be authorized in some
institutions.
Perform elution
The HPLC elution system uses methanol and methylene chloride instead of methanol
and 2-propanol as mobile phase and proceeds as follows.
17. Set the flow rate at 1.5 ml/min and equilibrate the column with 98:2 (v/v)
methanol/methylene chloride using isocratic elution.
18. Inject the sample.
Actinobacteria
(High G+C
Gram Positive)
10A.3.9
Current Protocols in Microbiology
Supplement 5
19. Elute isocratically by increasing linearly the mobile phase to 80:20 (v/v)
methanol/methylene chloride for 1 min followed by a second linear increase to
35:65 (v/v) methanol/methylene chloride for 9 min. Elute isocratically for 2 min
using 98:2 (v/v) methanol/methylene chloride.
COMMENTARY
Background Information
Isolation and
Analysis of
M. tuberculosis
Mycolic Acids
Mycobacterial mycolic acids are key constituents of the mycobacterial cell wall. They
play an important role in maintaining the structure and permeability of the cell wall and
thus preventing the entry of many antibiotics.
Changes in mycolic acid structures have been
associated with variation in cell wall permeability and attenuation of pathogenic mycobacterial strains. Mycolic acids are synthesized by the combined action of a type I
fatty acid synthase (FASI) and a type II fatty
acid synthase (FASII). FASII elongates the
short-chain fatty acids arising from FASI into
meromycolates (C56 ). Mycolic acids are then
formed by a claisen condensation between
the meromycolates and the major FASI endproduct (C26 ). Takayama et al. (2005) have
recently published a review on the biosynthesis of mycolic acids and their processing in
M. tuberculosis and presented an assessment
of all previously published data.
The protocols in this unit describe the
extraction of M. tuberculosis mycolic acids
from the cell wall and their analysis by TLC
or HPLC. These analyses can be used to
study mycolic acid biosynthesis and detect unusual mycolic acids accumulating due to mutations or environmental conditions. Isolation
and quantification of individual mycolic acid
species is readily done by HPLC.
A 10-ml M. tuberculosis culture does not
form enough mycolic acids to be easily visualized on TLC. Therefore, the first step in the
TLC protocol is labeling with [1-14 C]-acetate,
which allows for the visualization of mycolic
acids by autoradiography. After labeling, the
mycolic acids are saponified and then methylated using methyl iodide. Other groups have
used diazomethane for the methylation reaction but due to the highly explosive character of diazomethane, this is not recommended.
The recommended procedure for TLC analysis of MAMEs is to use 95:5 (v/v) hexane/ethyl acetate as the elution solvent. The
use of low-boiling-point solvents such as acetone or diethyl ether in chromatographic systems should also be avoided, as these solvents
are too volatile to ensure a constant ratio between elution solvents.
Anticipated Results
The autoradiogram shown in Figure
10A.3.2 was obtained from a 10-ml M. tuberculosis H37Rv culture labeled when the culture had reached an OD600 of 0.47. Labeling
lasted 20 hr followed by a 20-hr alkaline treatment. MAMEs were resuspended in 200 l
of methylene chloride and 5 l of the solution (6000 cpm) were spotted onto the TLC
plate. The autoradiogram was obtained after
exposure for 36 hr at 80 C. Figure 10A.3.4
shows the HPLC chromatograph of the mycolic acids from M. tuberculosis H37Rv. Chloroform was added to the derivatization mixture
and 95 l of the final solution was injected onto
the HPLC reversed-phase C18 column (4.6
150mm, 3-m particle size).
10A.3.10
Supplement 5
Time Considerations
Once the culture is ready (3 to 7 days of
culturing, depending on the inoculum size),
the TLC protocol takes 3.5 days: 20 hr for
the labeling, 20 hr for the alkaline treatment,
and 2 to 3 hr for the methylation and extraction.
At this point, the dried sample can be stored
in a 20 C freezer. The three elutions of the
TLC plate take between 30 and 45 min, and
then the film is exposed for 24 to 36 hr. The
HPLC protocol takes 1 day, the longest step
being the saponification. The protocol can be
stopped after the drying step (step 13) and the
dried sample can be stored in a 20 C freezer.
The p-bromophenacyl mycolic acid esters in
solution in acetonitrile (step 15) can be stored
at 4 C.
Literature Cited
Butler, W.R. and Guthertz, L.S. 2001. Mycolic
acid analysis by high-performance liquid chromatography for identification of Mycobacterium
species. Clin. Microbiol. Rev. 14:704-726.
Kremer, L. and Besra, G.S. 2005. A waxy tale, by
Mycobacterium tuberculosis. In Tuberculosis
and the Tubercle Bacillus (S.T. Cole, K. Davis
Eisenach, D.N. McMurray, and W.R.J. Jacobs,
eds.) pp. 287-305. ASM Press, Washington,
D.C.
Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall, A. 2001. Sterilization of Mycobacterium
tuberculosis Erdman samples by antimicrobial
fixation in a biosafety level 3 laboratory. J. Clin.
Microbiol. 39:769-771.
Stodola, F.H., Lesuk, A., and Anderson, R.J. 1938.
The chemistry of the lipids of Tubercle bacilli
liv. The isolation and properties of mycolic acid.
J. Biol. Chem. 126:505-513.
Takayama, K., Wang, C., and Besra, G.S. 2005.
Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin.
Microbiol. Rev. 18:81-101.
Zimhony, O., Cox, J.S., Welch, J.T., Vilcheze, C.,
and Jacobs, W.R. Jr. 2000. Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase
I (FASI) of Mycobacterium tuberculosis. Nat.
Med. 6:1043-1047.
Internet Resources
http://www.cdc.gov/nchstp/tb/Laboratory Services/
maps tagged.pdf
Mycolic acid pattern standards for HPLC identification of mycobacteria.
Actinobacteria
(High G+C
Gram Positive)
10A.3.11
Current Protocols in Microbiology
Supplement 5
UNIT 10A.4
ABSTRACT
This unit includes protocols for the isolation of proteins from Mycobacterium
tuberculosis. Considerations for working with M. tuberculosis at Biosafety Level 3
C 2007 by
containment are also discussed. Curr. Protoc. Microbiol. 6:10A.4.1-10A.4.5.
John Wiley & Sons, Inc.
Keywords: tuberculosis r TB r protein r culture filtrate r Sauton
INTRODUCTION
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects almost one
third of the worlds population. Yearly, there are an estimated 10 million new cases of TB
resulting in 2 to 3 million deaths. Over the last 15 years, the development of genetic tools
to manipulate M. tuberculosis has proliferated (UNIT 10A.2). Furthermore, availability of the
complete M. tuberculosis DNA genome sequence (as well as other related mycobacteria)
has yielded tremendous information towards a better understanding of the bacterium and
the disease it causes.
The protocols in this unit will detail bacterial protein extraction and culture filtrate
preparation. These proteins may then be analyzed using standard methods such as SDSPAGE or 2-D electrophoresis. See Braunstein et al., 2003, and Kriakov et al., 2003, for
examples of M. tuberculosis protein analyses.
CAUTION: Mycobacterium tuberculosis is a Biosafety Level 3 (BSL-3) pathogen. Follow
all appropriate guidelines for the use and handling of pathogenic microorganisms. See
Strategic Planning, Safety Considerations for further discussion. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for additional information.
SAFETY CONSIDERATIONS
Standards of BSL-3 containment should be followed for work with M. tuberculosis.
Consult with the Safety Officer and/or Institutional Biosafety Committee for additional
guidance. See http://www.cdc.gov/OD/ohs/symp5/jyrtext.htm and UNIT 1A.1 for a brief description of general BSL-3 laboratory features. Complete inactivation of M. tuberculosis
cells is necessary to perform some studies outside the BSL-3 laboratory. All methods
for inactivation should be validated in individual laboratories, taking into consideration
the concentration of cells used. Schwebach et al. (2001) provides some guidance on
determining if particular conditions are adequate for inactivation of mycobacteria. For
many of the procedures, the authors have found the following conditions sufficient for
inactivation of M. tuberculosis: incubation for 12 hr at 65 C, 2 hr at 80 C, or 15 min at
95 C; or incubation for 1 hr in a final concentration of one of the following solutions:
2.5% glutaraldehyde, 2% paraformaldehyde, 5% formalin, or Vesphene IIse. During heat
inactivation, care should be taken to ensure that the entire tube is immersed in a water
bath preheated to the appropriate temperature. Incubation of M. tuberculosis cultures
with gluteraldehyde, paraformaldehyde, formalin, or Vesphene IIse should be done in a
Actinobacteria
(High G+C
Gram Positive)
10A.4.1
Supplement 6
matter that ensures that the entire interior surface of the tube has been coated by gently
rolling and inverting the tube.
BASIC
PROTOCOL 1
Materials
Mycobacteria (UNIT 10A.1)
Phosphate buffered saline (PBS; APPENDIX 2A)
Protein extraction buffer (see recipe), ice cold
2 SDS sample buffer (APPENDIX 2A)
15-ml screw-cap conical tubes
Refrigerated tabletop centrifuge
Vortex and tube adaptor
2-ml screw-cap tubes (Sarstedt)
Microcentrifuge
Glass beads (106-m, Sigma cat. no. G4649)
95 C water bath
1. Wash cells two times with an equal volume of PBS to remove BSA that might
interfere with resolution of mycobacterial proteins by SDS-PAGE.
2. Centrifuge culture 10 min at 2000 g, 4 C, in 15-ml screw-cap conical tubes.
3. Discard supernatant and add 10 ml PBS to cell pellet. Vortex to resuspend cell pellet
then centrifuge 10 min at 2000 g, 4 C.
4. Discard supernatant and resuspend cell pellet in 1.5 ml PBS. Vortex to resuspend
cell pellet.
5. Transfer cell suspension to a 2-ml Sarstedt screw-cap tube. Microcentrifuge 5 min
at 15,800 g, 4 C.
6. Discard supernatant and resuspend cell pellet in 300 to 400 l of ice-cold protein
extraction buffer.
7. Add 0.4 g of 106-m glass beads.
8. Vortex 5 min at top speed using an adaptor to hold the tubes.
9. Microcentrifuge 5 min at 15,800 g, 4 C, to pellet glass beads and debris at the
bottom of the tube.
10. Transfer supernatant to a fresh 2-ml Sarstedt screw-cap tube.
11. Add an equal volume of 2 SDS sample buffer.
12. Vortex briefly to mix sample, then incubate 5 min in a 95 C water bath.
Analysis of
M. tuberculosis
Proteins
13. Store sample up to 2 weeks at 70 C; thaw at 2 C (10 min) just prior to use.
14. Perform SDS-PAGE (CPMB UNIT 10A.2).
10A.4.2
Supplement 6
BASIC
PROTOCOL 2
Materials
Bacterial culture in Sauton medium (UNIT 10A.1)
50-ml conical tubes
Sterile cell scraper
Refrigerated tabletop centrifuge
Filter units (0.45- and 0.2-m)
Cell concentrator (Amicon Ultrafiltration unit or equivalent)
1. Grow 100 to 200 ml bacterial culture in Sauton medium.
2. Transfer culture to 50-ml conical tubes.
Bacteria will be difficult to manipulate because of a lack of albumin and detergent in the
medium; the culture will grow as a pellicle on the surface of the medium and will stick
to pipets. For most analysis, it is useful to compare the supernatant and pellet; therefore,
it is important to collect as much of both components as possible. Decanting the culture
directly to 50-ml conical tubes and using a sterile cell scraper will help in increasing the
amount of material recovered.
Actinobacteria
(High G+C
Gram Positive)
10A.4.3
Current Protocols in Microbiology
Supplement 6
COMMENTARY
Background Information
Tuberculosis (TB) is a disease caused by M.
tuberculosis. M. tuberculosis is spread via the
aerosol route and predominately causes disease in lung tissue, although it can spread to
other organs. It is estimated that nearly onethird of the worlds population is infected with
TB, many of those are cases of latent infection.
Upon waning of the immune system through
aging or infection with HIV, TB can reactivate from its latent form, even after decades of
quiescence. Antibiotic therapy is available for
the treatment of TB, although due to the slowgrowth of the organism, long courses of treatment (6 months to 1 year) are recommended.
Analysis of
M. tuberculosis
Proteins
Anticipated Results
Extraction of proteins
Yield can be altered by increasing the
amount of starting culture and/or adjusting the
Figure 10A.4.1 SDS-PAGE gel. Typical distribution of M. tuberculosis proteins from culture
ltrates and whole-cell lysates. Cells were grown to an OD600 of 0.4 in Sauton medium without
detergent. Proteins were extracted following Basic Protocols 1 and 2.
10A.4.4
Supplement 6
Time Considerations
Extraction of proteins
The protocol takes 2 hr.
Preparation of culture filtrates
Centrifugation and filtering of the culture
filtrate take <1 hr. Depending on the cutoff
filter used, application of the supernatant to
the cell concentrator can take 2 to 8 hr.
Acknowledgments
Special thanks to Kieran Bhatt, Bing Chen,
Zhiyan (Annie) Dai, Dee Dao, Robert Glover,
Literature Cited
Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall,
A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a Biosafety Level 3 laboratory. J. Clin.
Microbiol. 39:769-771.
Braunstein, M., Espinosa, B.J., Chan, J., Belisle,
J.T., and Jacobs, W.R. Jr. 2003. SecA2 functions
in the secretion of superoxide dismutase A and
in the virulence of Mycobacterium tuberculosis.
Mol. Microbiol. 48:453-464.
Kriakov, J., Lee, S., and Jacobs, W.R. Jr. 2003. Identification of a regulated alkaline phosphatase,
a cell surfaceassociated lipoprotein, in Mycobacterium smegmatis. J. Bacteriol. 185:49834991.
Actinobacteria
(High G+C
Gram Positive)
10A.4.5
Current Protocols in Microbiology
Supplement 6
UNIT 10A.5
ABSTRACT
Animal models of tuberculosis infection continue to provide useful information about the
nature of the disease process, including specific information about the immune response
to the infection and the disease pathology. In addition, standardized animal models are
now used extensively to test the capacity of new vaccines to inhibit the course of the
infection, as well as test the capacity of new drugs to sterilize the infection. This unit
describes some basic protocols illustrating established protocols for infecting animals
with tuberculosis, subsequent processes for analysis, and various aspects of biosafety
that must be observed. Using these protocols the course of infection, the basic immune
response, and the extent of lung pathology can be determined in mouse and guinea
pig models of experimental tuberculosis. Curr. Protoc. Microbiol. 7:10A.5.1-10A.5.29.
C 2007 by John Wiley & Sons, Inc.
Keywords: mouse r guinea pig r tuberculosis r animal models r immune response r
pathology
INTRODUCTION
The past decade has seen an explosion of knowledge regarding the immunological and
pathological basis of the host response to Mycobacterium tuberculosis, much of it coming
from the mouse model and, to a lesser extent, from the guinea pig model. As more and
more laboratories have acquired aerosol generation devices, studies have shifted away
from intravenous and other routes of infection towards the much more realistic low-dose
aerosol infection model.
The mouse remains the chosen model for basic immunological investigations because
of the wealth of available reagents and, over the past two decades, much has been
learned about the T cell response, including its various subsets, soluble mediators
such as cytokines and chemokines, signaling pathways, and basic pathology, as well
as a growing appreciation of the layers of innate immunity that comprise the earliest
responses in the mouse lung. In contrast, far fewer immunological reagents are available
for the guinea pig, although this is now changing. This animal model remains an
important step for new vaccine testing, reflecting similarities in the pathology of the
disease process to that seen in humans.
Whereas the early literature focused on changes in the bacterial load in the two models
after various manipulations, the focus of present day has been pathology of tissues,
information about RNA message, or data obtained by flow cytometry, and so forth. As a
result, this unit includes some basic protocols describing how to prepare lungs and other
tissues for such analyses.
SAFETY CONSIDERATIONS
The basis of safety in a laboratory is facility design, fail-safes, precise training, and an
exposure control plan. Biosafety Level III laboratories have become more commonplace,
Current Protocols in Microbiology 10A.5.1-10A.5.29, November 2007
Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc10a05s7
C 2007 John Wiley & Sons, Inc.
Copyright
Actinobacteria
(High G+C
Gram Positive)
10A.5.1
Supplement 7
driven by the current interest in biodefense. These laboratories vary between laboratories
working with small inocula in which M. tuberculosis can be safely handled in appropriate
Class II biosafety cabinets located within restricted access areas, and laboratories working
with large numbers of animals infected with this organism.
Biosafety Level III laboratories by definition have defined air handling systems that
remove air under negative pressure many times per minute. These can be solid structures
or movable (e.g., biobubbles within a larger room) and operate best as stand-alone
buildings. This is not always possible, and where Biosafety Level III laboratories arise
from the renovation of regular laboratories, great care must be taken in terms of the design,
air flow, access, adjacent traffic, and so forth. At a minimum, tandem HEPA filter systems
are required, and these should be checked on a regular basis. Renovating laboratories to
become Biosafety Level III laboratories where there are regular laboratories on the floors
above is not recommended.
Fail-safes refer to levels of safety. For example, a technician loading a nebulizer of an
aerosol chamber should be wearing an OSHA-approved mask with a respirator (e.g.,
powered air purifying respirator, PAPR) on top. The technician may be in a biobubble
surrounding the generator, but should be in a room that has the greatest negative air
pressure in relation to the Biosafety Level III facility as a whole. The technician may
work with a colleague to check procedures and to make sure that there are no distractions.
After completing the aerosol run and decontaminating equipment, the technician should
shower out of the facility, leaving all protective clothing (e.g., surgical scrubs, etc.)
behind for autoclaving. The technician should undergo extensive training before directly
working with M. tuberculosis, including knowledge of required actions to take should
anything go wrong while in the Biosafety Level III area.
The central figure in any institution is the Biosafety Officer. This individual should review procedures on a regular basis and pre-approve the agents used. Clinical samples of
M. tuberculosis, which may include drug-resistant isolates, should be listed by the
Biosafety Officer, and the laboratory responsible should know their location (usually
in a 70 C freezer) and the number of vials present at any time.
Official guidelines for the United States are readily available from the National Institutes of Health, from the Centers for Disease Control (which now regard drug-resistant
M. tuberculosis as a Class C Select Agent), and from the Occupational Safety and Health
Organization. Other national and international agencies have guidelines that cover other
countries (refer to APPENDIX 1B). These organizations cannot be responsible for individual
laboratory conduct, therefore, it is the responsibility of the faculty and staff that work
each day in the Biosafety Level III laboratories to establish their own guidelines and
strictly adhere to them.
IMPORTANT NOTE: Refer to Section 1A for more information on laboratory safety
issues.
BASIC
PROTOCOL 1
Animal Models of
M. tuberculosis
Infection
10A.5.2
Supplement 7
Figure 10A.5.1 Glas-Col aerosol device. This instrument is based upon earlier models originally
developed by Middlebrook. The central gasket contains a large steel cage in which up to 80 to
100 mice can be exposed to aerosol at any one time. The device attached on the right is a heat
sterilizer to incinerate any bacteria escaping the internal HEPA-lter systems.
pie-shaped compartments for the animals. Each of the compartments can accommodate
as many as 25 mice. The aerosol chamber has a heavy acrylic lid with two locking
handles that lock the lid tightly against a heavy-duty rubber gasket. The lid also has two
ultraviolet lamps on its underside and these lamps come on during the decontamination
cycle of instrument operation. The front of the instrument consists of a control panel
with four timers for the various cycles of operation; two air flow meters, two air control
knobs, as well as an on/off switch for the exhaust air incinerator. On the front of the
instrument, there are three hoses with clamps used to attach the Venturi nebulizer. When
the instrument is in operation, compressed air flows through the nebulizer and produces
a very fine mist of the bacterial suspension, which is then carried by a larger volume of
air flowing into the aerosol chamber. This fine mist is then inhaled by the animals within
the basket. It then exits the chamber to flow through two HEPA filters and then a superheated exhaust stack where any residual particles are incinerated. After the nebulization
procedure, the machine runs a cloud decay process in which the aerosol chamber is
purged with fresh air. This may also help in drying any bacilli attached to the fur of
the animal. A final step is an ultraviolet cycle in which the UV lamps come on and
decontaminate the top surfaces of the basket.
Materials
Suspension of bacteria in 5-ml syringe with 18-G needle
Mice
5% Lysol wash bottle
70% ethanol
Airborne infection apparatus (Glas-Col)
Venturi nebulizer (Glas-Col)
Large and extra-large autoclave bags
Autoclave tape
Prepare apparatus and load animals
1. Ensure that the aerosol basket in the airborne infection apparatus has been cleaned
and is ready for use.
Actinobacteria
(High G+C
Gram Positive)
10A.5.3
Current Protocols in Microbiology
Supplement 7
2. Place an In Use light switch just outside the aerosol room and turn it on.
3. Fill the nebulizer with the bacterial suspension.
4. Secure nebulizer to airborne infection apparatus by clamping to inlet ports. Ensure
that the nebulizer is secure to avoid the possibility of the aerosol being released into
the room.
5. Place aerosol basket stand and basket into airborne infection apparatus.
6. Load animals into basket and secure lid. If loading different groups of mice, mark
the tail of each mouse with an indelible pen designating group name. Place no more
than 25 mice into each chamber of the basket.
Run apparatus
7. Turn Power switch on airborne infection apparatus to On position.
8. When LED screen reads Glas-Col Apparatus Co., turn on UV Light switch and
Program switch.
9. When LED screen reads Enter When Ready, press Enter.
10. When LED screen reads Enter Preheat Time. . . 900, press Enter to accept.
11. When LED screen reads Enter NEB Time. . . 1800, change value to 2400 and press
Enter.
12. When LED screen reads Enter CD Time. . . 1800, change value to 2400 and press
Enter.
13. When LED screen reads Enter DEC Time. . . 900, press Enter to accept, LED screen
will read Preheat Cycle In Process.
The right-hand (compressor) gauge should read 40-60 psi; if it does not, adjust upward
with corresponding control beneath.
10A.5.4
Supplement 7
BASIC
PROTOCOL 2
Materials
5% Lysol wash bottle
70% ethanol wash bottle
M. tuberculosis H37Rv stock
Guinea pigs
Biosafety Class II cabinet
50-ml conical tubes
1- and 20-ml syringes
18- and 26-G needles
Madison infection chamber
Autoclaved glass nebulizer jars
Guinea pig cages
Stainless-steel container(s)
Absorbent bench-top paper
Disposable surgical gown
Infection basket(s)
5-ml tubes (Falcon)
Powered air purifying respirator (PAPR)
Large/extra-large biohazard bags
Autoclave tape
Prepare work area and run test
1. Sterilize the surfaces of the Biosafety Class II cabinet with paper towels wet with
5% Lysol, followed by wiping the surfaces two times with 70% ethanol.
2. Transport all materials from airlock into the barrier, and take the M. tuberculosis
stock out of the dry icecontaining ice chest and place into a 50-ml conical tube, and
begin thawing the stock inside the biosafety cabinet.
3. Load 15 ml of sterile distilled water into a 20-ml syringe affixed to an 18-G needle
(this will be used for the test run).
4. Begin the test run in the Madison infection chamber (15-min cycle) to ensure that
the pressure driving the vacuum is adequate.
a. Dispense the 15 ml of sterile distilled water into the autoclaved glass nebulizer jar
by removing the plug screw on the Collison nebulizer (Fig. 10A.5.2), unsheathing
the needle, and placing the needle tip in through the orifice to rest against the side
of the jar (so as not to create any aerosols when dispensing).
b. After dispensing the distilled water into the jar, refasten the plug screw on the
nebulizer.
c. For the test run only, unlatch the Control Box lid and check that the time delay
relay, TD1, is set to 900 sec/On Delay, and that the second time delay, TD2, is set
to 300 sec/Interval Delay.
d. Close/latch the Control Box lid, and supply power to the chamber system, which
powers the compressor/vacuum pump unit.
Actinobacteria
(High G+C
Gram Positive)
10A.5.5
Current Protocols in Microbiology
Supplement 7
Figure 10A.5.2
Collison nebulizer jar for the Madison guinea pig aerosol chamber.
Figure 10A.5.3
e. Check for the proper amount of airflow into the system (secondary air, 40 to 45
liters/min) indicated on the flow meter on the right side of the flow panel. Also,
be sure to check the Photohelic unit that the proper amount of vacuum is being
maintained (consistently 9 to 10 in. water; Fig. 10A.5.3).
5. Proceed with the test run. Observe the flow pressures on the Control Box
(Fig. 10A.5.4).
Animal Models of
M. tuberculosis
Infection
10A.5.6
Supplement 7
Figure 10A.5.4
c. Unlatch the chamber door (eight latches around the perimeter) and open the door
to visualize the operation of the mixing fanstwo at the rear and two at the front
of the chamber.
d. Test the mixing fan(s) operation by depressing the Fan Test button on the Control
Box.
e. If everything is operational, reseal the airtight door with all eight latches.
f. Initiate the test run by depressing the Cycle Start button on the Control Box.
g. Log both the reading on the primary airflow meter into nebulizer (left side of flow
panel) and the reading on the pressure gauge beneath the regulator into a log book.
h. Also, before leaving the room, be sure that the nebulizer is fully functional by
checking for spray coming out of each of the three jets of the nebulizer probe.
i. Illuminate the In Use light.
Load bacteria
10. Fit a 1-ml syringe with a 26-G needle, insert needle into the M. tuberculosis vial,
and invert vial.
11. Pull bacteria in and out of syringe at least ten times; do not allow frothing to occur.
Actinobacteria
(High G+C
Gram Positive)
10A.5.7
Current Protocols in Microbiology
Supplement 7
12. Pull 1 ml into the syringe, then, turn the bottle upright and pull 100 l of air into
the syringe (so that the vial does not expel any liquid when needle is removed), and
withdraw needle.
13. Slowly dispense the 1 ml of bacteria into a 5-ml tube so as not to create an aerosol.
14. In another 5-ml tube, perform the necessary dilution(s) from concentrated stock to
arrive at the working stock concentration needed to place x ml into 16 ml x ml of
sterile distilled water such that the final concentration is 1 106 cfu/ml.
15. Put aside 1 ml of the stock solution for plating on agar to verify the inoculum
concentration.
16. Transport the diluted inoculum in a 20-ml syringe to the aerosol chamber.
17. Put on the PAPR.
When the End Cycle indicator sounds for the test run, press Reset on the Control Box and
remove a sterile autoclaved nebulizer.
18. Carefully place the inoculum into the nebulizer and screw into place. Place the
syringe into Lysol.
20. Relatch the door, and be sure that all the eight latches are fastened on the door so as
to create the necessary airtight seal.
21. Recheck for the proper amount of airflow being channeled through the chamber (40
to 45 liters/min). Also, be sure to check that the vacuum gauge reads 9 to 10 in. water
(after opening/closing the chamber door this may take 1 to 2 min).
22. Initiate the cycle by depressing the Start button, and set a timer for 15 min.
23. Be sure to check the three jets on the nebulizer probe for proper spray/dispersion
before leaving the room.
29. Gently clean the probe with a paper towel stack wet with 70% ethanol.
10A.5.8
Supplement 7
30. Pour 20 ml water (from a sterile 50-ml conical tube) inside an additional autoclaved
nebulizer jar and screw into the apparatus.
31. Check for the proper amount of airflow being channeled through the chamber (40 to
45 liters/min). Also, be sure to check that the vacuum gauge reads 9 to 10 in. water.
32. Initialize the Cleaning run by depressing the Start button on the Control box.
33. Following the Cleaning run, turn off all switches.
34. Replace the nebulizer jar with the final autoclaved nebulizer jar.
35. Place the used jar into 5% Lysol solution.
36. Close the lid to the container, place into a large biohazard bag, and mark
FRAGILEglass nebulizer jars
37. Remove the infection basket(s) from the cart(s), double bag with extra-large biohazard bags, and seal with autoclave tape.
38. Open the chamber door, and wipe inside of chamber apparatus with 5% Lysol; repeat
process with 70% ethanol (while always wearing the PAPR).
39. Sweep up and dispose of any debris from animal cages, etc.
40. Autoclave the packed nebulizer/aerosol baskets.
Although these chambers are well made, resist the temptation to run an aerosol infection
experiment when you first receive the instrument. Run water alone at least three or
four times before you do anything else. Look carefully at the various joints for bubbling
when run under pressure; some joints may need tightening. Only when you are sure the
instrument is operating well should you then run the instrument containing mycobacteria.
BASIC
PROTOCOL 3
To verify the concentration of the inoculum used in the nebulizer, an aliquot of the
mycobacterial suspension is diluted and plated on 7H11 agar (see UNIT 10A.1). The dilutions
should be ten-fold serial dilutions, and are easily done using 24-well plates (100 l of
the bacterial suspension in 900 l saline or PBS). A volume of 100 l of each dilution
(102 , 103 , 104 , 105 , 106 , 107 , 108 ) is plated onto quadrants of petri dishes containing
7H11 agar. Colonies are counted 3 weeks later to verify the inoculum size.
NOTE: Take great care when pipetting the serial dilutions to prevent aerosol generation.
Any small spills or drops should be disinfected with 5% Lysol. All pipet tips used to
serially dilute the mycobacteria are placed in a pipet boat containing 5% Lysol solution.
Actinobacteria
(High G+C
Gram Positive)
10A.5.9
Current Protocols in Microbiology
Supplement 7
Materials
M. tuberculosis bacterial stock
5% Lysol solution
70% ethanol
50-ml conical tubes
Tube rack
Pipet boat, optional
Small biohazard bag
3- and 10-ml syringes
18- and 26 1/2G needles
1. Retrieve bacterial stock from freezer and place into 50-ml conical tube, close tube,
and write your initials. Transfer to biosafety cabinet, place tube into rack, and allow
stock to thaw.
2. Fill one 50-ml conical tube (or fill pipet boat about half full) with 5% Lysol solution.
Place a small biohazard bag inside of hood.
3. Remove aluminum seal from bacterial stock, and spray top of bacteria vial with 70%
ethanol.
4. Fit a 3-ml syringe with a 26 1/2G needle, insert needle into vial, and invert vial.
5. Pull bacteria in and out of syringe at least three times; do not allow frothing to occur.
6. Pull 1 ml of bacteria into syringe, revert tube, and pull 100 l air into syringe (so that
excess liquid does not come out when needle is removed), then withdraw needle.
7. Remove cap from diluent tube, move hand away from tube.
8. Place needle tip just below meniscus of diluent and slowly add bacteria. Avoid
touching the side of the needle to the sides of the diluent tube.
If required, make serial dilutions in additional conical tubes to reach desired dilution for
inoculum.
Animal Models of
M. tuberculosis
Infection
10A.5.10
Supplement 7
Materials
Bacterial suspension at desired concentration (see Basic Protocol 3)
Mice
70% ethanol
1-ml syringe with 25- to 30-G needle
Restraint device (e.g., Braintree Scientific)
Heat lamp
Gauze sponge or swab
IMPORTANT NOTE: Restraint devices vary and are often homemade; they are also
available commercially from suppliers such as Braintree Scientific. Most are metal or
plastic holders with an opening at one end to expose the tail of the mouse without harming
the animal. The procedure must be done in a BSL-3 facility.
1. Fill a 1-ml syringe with the injectate and be careful to remove all air bubbles to avoid
risk of air embolism.
2. Gently place the mouse in a restraint device so that the tail can be immobilized
without the mouse being able to turn.
3. Warm the tail with a heat lamp by placing the lamp 6 to 9 in. away. Look for tail
vein vasodilation, when adequate vasodilation is observed, move the lamp away. Do
not expose the animal to any more heat than necessary.
4. Swab the tail with 70% ethanol.
5. Find the lateral vein (one on each side of the tail) and insert the needle parallel to the
vein a few millimeters into the vessel. Keep the bevel of the needle facing up.
6. Inject slowly. No bleb should form if needle is properly located in the vein. If a bleb
appears indicating failure to properly enter the vein, try again further up the tail.
7. Withdraw the needle and apply digital pressure if necessary to achieve hemostasis.
If you feel any resistance to the injection, stop because the vein was probably missed.
Never force the injection, if forced, the mycobacteria will spray back at you. Always wear
a plastic face protector in addition to a mask and usual protective clothing.
Actinobacteria
(High G+C
Gram Positive)
10A.5.11
Current Protocols in Microbiology
Supplement 7
BASIC
PROTOCOL 5
Materials
Sodium pentobarbital (120 mg/kg)
1- to 3-ml syringe with 20- to 25-G needle
1. Weigh the animal and calculate dose of sodium pentobarbital at 120 mg/kg.
2. Gently restrain the animal.
3. Inject sodium pentobarbital via the intraperitoneal route using a 1- to 3-ml syringe
and 20- to 25-G neeedle.
4. Verify death by lack of cardiac pulse, respiration, and eye pupil response/dilation.
10A.5.12
Supplement 7
5. Plating
6. Counting bacterial colonies
Current Protocols in Microbiology
BASIC
PROTOCOL 7
Mouse organ-sized pestles are normally packaged in five- or ten-packs. Pestle packs are
placed plastic side down inside a red autoclave bag and packed in a cooler with dry ice
in a bag placed on top of them. The pestle packs must not be in direct contact with the
dry ice. Guinea pig pestles are packaged in three- to five-packs. They are autoclaved and
packed in the cooler as described for the mouse pestles. It is important to keep the pestles
cold so they fit snuggly into the homogenization tubes; if they warm up, they slightly
expand and do not fit.
NOTE: All processing of tissues from M. tuberculosisinfected animals must be done in
the biosafety cabinet and glove box. Ensure that the blower, power, and light switches of
the biosafety cabinet are in proper working order before starting any laboratory techniques
in this instrument.
Materials
70% ethanol
Mice or guinea pigs
5% Lysol solution
Necropsy board
Absorbent paper
250-ml plastic beakers
Surgical kits containing scissors and forceps
Small biohazard bags
Tube rack
Homogenization tubes (Glas-Col cat. no. 099C S31 for mouse or cat. no. 099C S37
for guinea pig)
Prepare for necropsy
1. Place a necropsy board into the hood in a central location without blocking any of
the air vents. Cover the board with absorbent paper.
2. Fill a 250-ml plastic beaker with 70% ethanol and place it inside the hood. Place the
scissors and forceps into the beaker.
3. Place a small biohazard bag inside the hood.
4. Place one rack containing labeled homogenization tubes inside the hood.
Several racks may be required for the entire count but only one rack should be in the hood
at any given time.
5. Place any additional supplies inside the hood (PCR tubes, histology cassettes,
syringes, etc.).
Collect sample
6. Euthanize animals usually four to five at a time according to Basic Protocol 5 or 6.
Using a wash bottle, thoroughly wet the fur with 70% ethanol to sterilize the area.
Actinobacteria
(High G+C
Gram Positive)
10A.5.13
Current Protocols in Microbiology
Supplement 7
7. Transfer the animals to the hood and place them on clean, dry, absorbent paper on
the necropsy board. Place the animals on their backs and pin them to the board using
syringe needles.
8. Make a midline incision(s) using the scissors.
9. Retract the skin above the head and below the thighs by pulling it with gloved fingers
to expose the peritoneum.
10. Cut the peritoneum using scissors and retract it to expose the internal cavity.
Remove spleen
The spleen is attached to the greater curvature of the stomach by connective tissue.
11a. To remove the spleen all at one time: Take hold of as much of the spleen as possible
with forceps. Gently pull the spleen free of the peritoneum, tearing the connective
tissue behind the spleen.
11b. To remove the spleen in sections: Grab hold of the top and bottom portions of the
spleen and tear from the connective tissue one part at a time.
It is usually more efficient to remove the spleen all at once, particularly if large numbers
of animals will be used. Avoid cutting into the intestine.
12. Uncap the appropriate tube and place the spleen into the tube. Place the spleen as
far down the tube as possible to avoid any tissue touching the cap once it is replaced.
Cap the tube.
Remove liver
13. With small forceps, grasp the right lateral lobes of the liver to a point across the
inferior vena cava.
14. With scissors, gently flip these lobes back over so that the top portion of the forceps
can grasp these lobes as well as the common bile duct.
15. When the forceps have a firm hold, make an incision with the scissors between the
liver and the diaphragm and cut the inferior vena cavathis then frees the liver.
16. With a quick but gentle upward motion, extricate the free liver as one organ.
17. Uncap appropriate tube, place the liver in designated tube, and re-cap tube.
Remove lungs
18. Make an incision through the thoracic cavity using scissors and cut back the rib
cage (the lung lobes should be readily visible on either side of the heart). With small
forceps, take hold of the bronchial tree close to the heart and cut the lung lobes away.
19. Uncap appropriate tube, place the lung in designated tube, and re-cap tube.
20. If collection of tissue for PCR is required, proceed to the glove box to homogenize
tissue.
Formalin-fixed tissue may be infused, placed in cassettes, and stored (see below).
Clean up
21. Place mouse carcasses into the biohazard bag situated in the hood.
22. Remove gloves and place them in the biohazard bag. Exit the hood and put on a fresh
pair of gloves. Proceed to the next experimental group.
Animal Models of
M. tuberculosis
Infection
To prevent the possibility of a spill, racks of completed tubes should be placed at the back
of the bench to ensure that they cannot be tipped over.
10A.5.14
Supplement 7
23. At the completion of necropsy, place all mice and trash in the small biohazard bag
and tape it closed. Place the entire bag inside another bag and re-tape. Remove the
bag from the hood and autoclave as soon as possible (never store). Rinse the necropsy
board with 5% Lysol, wipe, and place the board back into its container. Wipe the
entire hood with 5% Lysol, followed by 70% ethanol and discard the paper towel in
a biohazard bag for disposal.
Organ Homogenization
Once organs have been harvested and placed in homogenization tubes, they are ready to
be disrupted. Once this is performed, they can then be serially diluted in dilution tubes to
prepare for plating on agar. The homogenization procedure creates a significant aerosol
hazard, therefore, using a glove box married to the biosafety cabinet in which the organs
were dissected is recommended.
BASIC
PROTOCOL 8
Materials
5% Lysol solution
Organs in homogenization tubes (see Basic Protocol 7)
70% ethanol
7H11 agar plates
Saline, sterile
Glove box in biosafety cabinet
Aluminum boat
Pestles (Glas-Col cat. no. 099C S21 for mice or Biospec Products cat. no.
985370-14 for guinea pigs), sterile and chilled
Homogenizer (e.g., Glas-Col homogenizer system)
Pipet boats
24-well tissue culture plates
Automated pipettor
Multi-channel pipettor
Set up for homogenization
1. Open the outer access door of the airlock entry port for the glove box chamber and
place an aluminum boat containing 5% Lysol within the airlock chamber. Close and
secure the outer door of the airlock and then place hands in the glove ports and open
the inner access door of the airlock entry port.
2. Remove the boat from the airlock and place it within the glove box chamber with
the lid off. Close the inner access door.
3. Transfer a tube rack with ten homogenization tubes containing organs through the
airlock as described above. Transfer paper towels soaked in 70% ethanol into the
glove box.
4. With hands in the glove ports, carefully remove the foam test tube stoppers from
the homogenization tubes and place them off to the left side of the hood on a paper
towel.
5. Transfer a pack of sterile chilled pestles into the glove box. Carefully open the pestle
pack and place the chilled pestles into the homogenization tubes, taking care not to
splash or spill the tube contents.
Pestles can be placed into the tubes one at a time before homogenizing, or in groups of
ten (remember that the pestles warm up rapidly in the tubes).
Actinobacteria
(High G+C
Gram Positive)
10A.5.15
Current Protocols in Microbiology
Supplement 7
Homogenize organs
6. Remove the homogenization tubes one at a time from the test tube rack and attach
the shaft of the pestle into the chuck of the grinder.
There are various ways to do this, but the authors recommend the Glas-Col homogenizer
systeman alternative is electric power drill bits (e.g., Black and Decker), which also
fitwhich must be secured in the glove box (Fig. 10A.5.5) with an activating power pedal
that is placed outside the box. This has an appropriate chuck to attach to the top of the
pestle, plus it spins at a high speed needed for thorough homogenization.
Animal Models of
M. tuberculosis
Infection
Figure 10A.5.5 (A) Homogenizing tube for mouse organs tted tightly with a plastic pestle. (B)
Homogenizer system with pestle tted into the chuck.
10A.5.16
Supplement 7
Figure 10A.5.6 In the glove box, the pestle has been t into the homogenization tube and the
unit activated to spin the pestle, disrupting the tissue in the tube. Do not rush this step; a high
shearing force is needed to disrupt tissues and reduce the bacteria to a single cell suspension so
that subsequent plating and counting is accurate.
7. When secured, firmly hold the homogenization tube with both hands and then actuate
the grinder via the foot pedal.
It is essential that a firm grasp of the homogenization tube is maintained while performing
this technique as the pestle will rotate in the tube at a high rpm (Fig. 10A.5.6).
8. While the pestle is rotating, move the tube up and down slowly so that all the tissue
is homogenized well.
Do not rush this step. If the tissue cells are not completely disrupted and the bacterial
clumps are not completely disassociated, there may be an undercount of the bacterial load.
Clean up
9. After homogenization is complete, remove the pestle from the chuck (keeping it
inside the tube), hold the tube and pestle over the Lysol-filled aluminum pestle boat,
and gently pull the pestle out and place it into the boat.
10. Return tube to the test tube rack and repeat until all tubes with organs have been
homogenized.
11. After homogenization, recap the tubes with foam plugs and allow the chamber to
purge for 5 min to clear any residual aerosols.
12. When all homogenizations have been completed, pass paper towels and wash bottles
containing 5% Lysol and 70% ethanol into the glove box. Wipe the interior surfaces
of the glove box with 5% Lysol followed by 70% ethanol.
13. Place all used paper towels and pestle packs into the pestle boat. Cover the pestle boat
with a lid, remove from the glove box, and place into a red biohazard bag contained
within a pan. Autoclave as soon as possible.
Actinobacteria
(High G+C
Gram Positive)
10A.5.17
Current Protocols in Microbiology
Supplement 7
17. Remove the plates from their plastic bag and place them on the hood surface. Make
sure the underside is properly labeled for each dilution to be plated.
18. Dispense 800 l of sterile saline into 24-well tissue culture plates using an automated
pipettor, leaving the first row empty.
Always use some type of mechanical pipet-aid to pipet homogenates.
19. Dispense undiluted homogenate into the first row of a 24-well plate using a 1-ml
pipet. Discard the pipet into the Lysol-filled boat.
20. Using a multi-channel pipettor and sterile wide-bore tips (to prevent blockage),
carefully make serial 200-l dilutions across the plate (1:5 dilutions using 200 l
in 1000 total). Change tips with every row. Dispense used tips into the Lysol-filled
pipet boat.
21. Using a 1-ml pipet, dispense 100 l of each homogenate dilution onto the agar plates.
Use a separate pipet for every dilution; if this is not done, bacteria will carry over
from dilution to dilution and massively over-count the bacterial load.
22. Bag, label, and incubate the agar plates at 37 C as described above (see also
UNIT 10A.1). Count bacterial colonies 3 weeks later, when colony formation is clearly
visible and easy to count.
Most laboratories prefer to store incubated plates upside down. This does not influence
colony formation, but it does allow water that sometimes condenses out of the agar to
accumulate on the lid. If left upright this water can flush the individual colonies onto the
sides of the plate making counting difficult. Count colonies on quadrants on which there
20 to 100 and then calculate back from the dilution on that quadrant. Liver plates may
give nothing at zero or 1 dilutions, do not panic, this is simply due to inhibition by liver
enzymes at higher concentrations.
SUPPORT
PROTOCOL 1
Animal Models of
M. tuberculosis
Infection
10A.5.18
Supplement 7
1. When the autoclaved agar has cooled to 56 C, remove the flask from the water bath
and place it inside a laminar flow hood. Carefully loosen the foil and aseptically
transfer 100 ml of sterile OADC into 900 ml agar.
Current Protocols in Microbiology
2. Replace the foil on the flask. Gently swirl the flask to mix the OADC solution with
the agar.
3. Prime the 5-ml repeating syringe by pushing the plunger up and down until the barrel
fills with medium and there are minimal air bubbles.
4. Dispense 4.5 ml into each petri dish quadrant, or if using one without divisions, fill
with 18 ml of agar (any bubbles that form should be gently shaken off to the edge).
Carefully stack the plates as they are filled making sure to work toward the center of
the sterile hood.
5. After dispensing the agar, immediately remove the flask with the syringe and tubing
still in it from the hood and take to the sink. Set syringe aside for cleaning at a later
time.
6. Turn off the hood light. Carefully push the stacks of plates to the center back of
the hood. Do not cover with foil. Allow the plates to stand for at least 1 hr at room
temperature with the blower of the hood turned on and the light turned off.
This is sufficient time to allow the agar to solidify. Even though the agar in these plates
is light sensitive, leaving them for 1 hr or so has no effect. Always check that the plates
have solidified before proceeding to step 7.
7. When the agar has solidified, label plates on one edge of the lid with an indelible
colored marker and be sure to record method of labeling.
For example, place a blue line on the side of the lid on all the plates to make it easy to
identify this batch.
8. Re-bag the plates upside down in plastic bags, and seal with tape.
9. Remove bagged plates from the hood and place in plastic tubs. Label the outside of
the tub with a piece of tape with the batch no. and the date of the pour and your
initials written on it.
10. Place the entire tub for 3 days in a 37 C incubator.
11. After 3 days, check plates for contamination.
12. Remove two plates from the batch and place into a plastic sandwich bag. Write the
batch number and date poured on the bag and incubate for an additional 4 to 5 days
in the 37 C incubator.
This is to ensure there is no slow growing contamination on the plates.
SUPPORT
PROTOCOL 2
There are various Middlebrook agar formulations designed for growing colonies of
M. tuberculosis; the authors recommend 7H11. It is necessary to supplement this agar
with OADC to facilitate growth. A method for OADC preparation is provided here, or it
may be commercially purchased (VWR).
Actinobacteria
(High G+C
Gram Positive)
10A.5.19
Current Protocols in Microbiology
Supplement 7
Materials
6 M sodium hydroxide (NaOH)
Oleic acid (C18 H34 O2 )
Sodium chloride (NaCl)
Bovine serum albumin (BSA fraction V)
Dextrose
Antibiotic(s) (see recipe)
37 , 50 , and 56 C water baths
1-, 2-, and 4-liter Erlenmeyer flasks
500-ml sterile filter units
Sterile OADC bottles (500-ml clear glass bottles)
37 C incubator
115-ml sterile filter unit
Hand-held vacuum pump
Prepare OADC
1. Prepare the sodium oleate solution as shown in Table 10A.5.1. Warm in a 50 C water
bath until the solution is clear.
2. While the sodium oleate solution is in the water bath, dissolve the following in 950
ml water:
(a) NaCl:
(b) BSA:
(c) dextrose:
8.1 g/liter
16.2 g/2 liters
32.4 g/4 liters
50.0 g/liter
100.0 g/2 liters
200.0 g/4 liters
20.0 g/liter
40.0 g/2 liters
80.0 g/4 liters
5. Aseptically dispense 100 ml of sterile medium into sterile 500-ml OADC bottles
and place the bottles 1 hr in a 56 C water bath.
6. After 1 hr, remove bottles from the water bath and place the medium overnight in a
37 C incubator.
Table 10A.5.1 Sodium Oleate Solution
For 1 liter
Reagent
Animal Models of
M. tuberculosis
Infection
For 2 liters
For 4 liters
H2 O
30
60
120
6 M NaOH
0.6
1.2
2.4
Oleic acid
0.6
1.2
2.4
10A.5.20
Supplement 7
7. Heat the bottles 1 hr in a 56 C water bath and place back in the 37 C incubator
overnight.
8. Check the bottles for contamination (medium remains cloudy); discard medium if
this happens.
9. Assign a batch number and store upto 1 month at 4 C until needed.
Filter OADC
10. Place one bottle of OADC into a 37 C water bath for at least 20 min prior to use to
allow it to warm up.
11. Open the sterile 115-ml filter units and place in clean hood.
12. Aseptically dispense 100 ml of OADC into the filter unit.
13. Add the appropriate amount of antibiotics to the OADC.
14. Filter each bottle of OADC using a hand-held vacuum. Do not apply more than
15 lbs of pressure at one time.
SUPPORT
PROTOCOL 3
Materials
70% ethanol
7H11 agar base
Asparagine
Glycerol
2-liter Erlenmeyer flask
Aluminum foil
Autoclave tape
56 C water bath
1. Wearing a clean laboratory coat, face mask, and gloves, rinse gloves with 70%
ethanol.
2. Weigh out the following ingredients and place them into a 2-liter Erlenmeyer flask:
6. After checking the color of the agar, place the flask into a 56 C water bath and allow
the agar to equilibrate for at least 30 min
Actinobacteria
(High G+C
Gram Positive)
10A.5.21
Current Protocols in Microbiology
Supplement 7
BASIC
PROTOCOL 9
Materials
Ultraspec (Biotecx Laboratories)
95% ethanol
Lysol
Distilled water
Figure 10A.5.7 Commercial tissue tearer that can be used to rapidly disrupt samples followed
by processing for PCR techniques. The worker is holding the tissue sample in a homogenizing
tube large enough for guinea pig tissues.
Animal Models of
M. tuberculosis
Infection
10A.5.22
Supplement 7
2. Fill the tubes with 1 ml of Ultraspec (agitated and warmed up to room temperature
prior to use).
3. Prepare two 50-ml conical tubes, one with 40 ml water and the second one with
40 ml of 95% ethanol.
4. Collect infected tissue samples and place a small piece of each organ into a 5-ml
round-bottom tube containing 1 ml of Ultraspec. Immediately proceed to homogenization in the glove box using a tissue tearer (Fig. 10A.5.8).
Figure 10A.5.8 Representative results for bacterial load determinations in mice after aerosol
(A) or intravenous (B) infection. Notice the distribution in the target organs in the latter. If higher
uptakes (especially in the lungs) are observed, then the inoculum was clumped together.
Actinobacteria
(High G+C
Gram Positive)
10A.5.23
Current Protocols in Microbiology
Supplement 7
5. Rinse the tissue tearer tip in 95% ethanol for 30 sec followed by a second rinse in
distilled water. Disrupt the tissues using the tearer at full speed for 10 sec or until
the tissue is completely disrupted.
6. Clean the outside of the tubes with Lysol. At this point, disrupted tissue can either
be processed for RNA or be frozen on dry ice and kept at 70 C.
7. Clean the tearer in 95% ethanol and then water between each use.
BASIC
PROTOCOL 10
Materials
Lung
10% formalin
5% Lysol
Histology cassettes
1. Infuse the lung with 10% formalin by injection down the trachea or bronchus.
2. Place in a histology cassette in 10% formalin.
3. Keep the cassette in the BSL III laboratory for at least 48 hr.
4. Wipe the cassettes with 5% Lysol.
These can now be removed and processed for histology.
BASIC
PROTOCOL 11
Animal Models of
M. tuberculosis
Infection
10A.5.24
Supplement 7
Materials
Mice or guinea pigs
70% ethanol
30% OCT (VWR cat. no. 25608-930) in 1 PBS
Dissection board with pins
Surgical instruments
5- or 10-ml syringes with 18-G needles
Embedding capsules (flat bottom 4 4): e.g., Peel-A-Way disposable embedding
molds (22 30mm, 20-mm deep; Thermo Scientific cat. no. 1830)
Aluminum foil
Plastic lock baggies
Small biohazard bags
Procedure for mice
1a. Euthanize the animal (see Basic Protocol 5), pin to board, and rinse with 70% ethanol.
2a. Cut back the skin as described above so that the chest cavity is exposed and the lungs
can be viewed.
3a. Place a syringe needle barrel under the trachea to elevate it in relation to the chest
cavity.
4a. Fill a 5-ml syringe with 30% OCT mixture and cap with an 18-G needle.
It is very important that there are no bubbles in the syringe, this can be accomplished by
filling the syringes early and allowing the bubbles to rise to the top so they can be flushed
out.
5a. Slide the needle into the trachea and begin gently injecting the mixture into the lungs
to inflate them.
6a. While leaving the syringe in place in the trachea, remove the whole lung and place
it directly into the labeled embedding capsules, which have been coated with OCT.
7a. When all samples have been extracted, care should be taken to center the samples
(to make the sectioning procedure easier). Also, make sure the tissues are covered
with the OCT.
8a. Place the capsules directly onto liquid nitrogen (they will float).
In a few minutes the OCT will begin to appear white in color.
9a. Remove the capsules, wrap in aluminum foil, and place into plastic lock bags. Seal,
label, and place directly onto dry ice. Transfer to a freezer if you intend to store for
any time.
2b. Place the infused lobe in a Peel-A-Way mold that is 1/4 full of OCT. Begin to freeze
immediately by dipping the mold into liquid nitrogen.
3b. As soon as the bottom starts to freeze fill the rest of the mold with OCT and continue
freezing.
This is done in two steps to prevent the lung from floating to the top of the mold.
4b. Wrap frozen samples individually in aluminum foil and label for storage as needed.
Actinobacteria
(High G+C
Gram Positive)
10A.5.25
Current Protocols in Microbiology
Supplement 7
BASIC
PROTOCOL 12
Materials
Mice
10-ml syringes filled with ice-cold heparin solution fitted with 26-G needles
Tissue culture medium (e.g., RPMI or DMEM), cold
Collegenase/DNase (150/50 U/ml) (Sigma cat. no. C-7657 and D-5025) solution
thawed and on ice
Geys solution: 4.15 g NH4 Cl, 0.5 g KHCO3 , 500 ml ddH2 O, bring to pH 7.2
Dissection board with pins
Surgical instruments: long and small scissors, two tissue forceps
Autoclave bags
Small Petri dishes (60 15mm)
Sterile razor blades or long-sharp scissors
37 C water bath with rocking
Nylon strainers (70-m; VWR cat. no. 21008-952)
Sterile 15-ml centrifuge tubes
1. Euthanize mouse (see Basic Protocol 5). Retract skin and open thoracic cavity as
explained above. Hold the heart gently using forceps.
2. Using a 10-ml syringe filled with ice-cold heparin solution, insert the needle into the
dark spot on the right ventricle of the heart and inject the heparin until the lungs
turn white and inflate.
A concentration of 50 U/ml is used per lung.
11. Resuspend cell pellet and add 2 ml Geys solution. Leave for 5 min at room temperature.
10A.5.26
Supplement 7
Antibiotics
10 mg cycloheximide
50 mg carbenicillin
2.0 ml ddH2 O
Weigh out each antibiotic, using an analytical balance, into separate 5-ml snap-cap
tubes. Add 1.0 ml of ddH2 O to each tube and mix to dissolve. When dissolved,
combine antibiotics into one tube.
Filter sterilize the antibiotic mixture prior to adding to a bottle of OADC. Prepare
fresh.
COMMENTARY
Background Information
A large number of laboratories have now
developed reproducible models of tuberculosis infection in the mouse or guinea pig. This
has resulted in major advances in the understanding of the host response to M. tuberculosis at both the immunological and pathological
levels.
In the mouse, early studies, primarily using
the technique of passive cell transfer (Orme
and Collins, 1983; Lefford, 1984; Orme,
1987), demonstrated the role of T cells and
their subsets, but this approach has now been
largely replaced by the use of gene-disrupted
mouse models, which have been used to show
the importance, or lack of importance, of various T cell subsets (DSouza et al., 1997;
Flynn and Chan, 2001; Turner et al., 2001),
the role of key cytokines, such as gamma interferon, IL-12, IL-23, and IL-27 (Cooper et al.,
1993; Flynn et al., 1993; Pearl et al., 2004;
Holscher et al., 2005), and chemokines, particularly CCR2 (Peters et al., 2001; Kipnis
et al., 2003). The pathologic process in the
lungs of mice and guinea pigs (Rhoades et al.,
1997; Turner et al., 2003) is now also routinely
reported.
This model is also very useful for the more
practical issue of identifying new drugs and
vaccines. Drugs that are active in the mouse
invariably give similar efficacy in humans, and
thus can be evaluated in intravenously or aerogenically infected mice both as full-term reg-
Critical Parameters
A factor critical to the success of any model
of mycobacterial infection in mice or guinea
pigs is the condition of the inoculum. Large
Actinobacteria
(High G+C
Gram Positive)
10A.5.27
Current Protocols in Microbiology
Supplement 7
Anticipated Results
The growth of M. tuberculosis in the mouse
is influenced by the virulence of the inoculum,
the infection route, and the degree of resistance of the mouse strain used. In the case
of immunocompromised mice, such as those
with specific gene deletions, or those devoid
of lymphocytes (SCID, Rag/, etc.), the infection may be progressive in the lungs.
Representative growth curves for M. tuberculosis H37Rv in 8- to 10-week-old female
Animal Models of
M. tuberculosis
Infection
Figure 10A.5.9 Survival times of guinea pigs infected with M. tuberculosis H37Rv. Note that if
more virulent infections (such as Erdman K01) are used, expect the curves to move to the left.
Carefully begin monitoring animals 8 to 10 weeks after exposure so that animals in distress can
be euthanized.
10A.5.28
Supplement 7
Literature Cited
Cooper, A.M., Dalton, D.K., Stewart, T.A., Griffin,
J.P., Russell, D.G., and Orme, I.M. 1993. Disseminated tuberculosis in interferon gamma
gene-disrupted mice. J. Exp. Med. 178:22432247.
DSouza, C.D., Cooper, A.M., Frank, A.A.,
Mazzaccaro, R.J., Bloom, B.R., and Orme, I.M.
1997. An anti-inflammatory role for gamma
delta T lymphocytes in acquired immunity
to Mycobacterium tuberculosis. J. Immunol.
158:1217-1221.
Orme, I.M. 2006. Preclinical testing of new vaccines for tuberculosis: A comprehensive review.
Vaccine 9:2-19.
Actinobacteria
(High G+C
Gram Positive)
10A.5.29
Current Protocols in Microbiology
Supplement 7
Laboratory Maintenance of
Mycobacterium marinum
UNIT 10B.1
This unit describes methods for laboratory culture, storage, and maintenance of
Mycobacterium marinum, and for processing this organism for infection assays. M. marinum is a natural pathogen for sh and amphibians, and the disease symptoms that
it causes in those animals are very similar to human infections by M. tuberculosis.
M. marinum is also closely related to M. tuberculosis at the genetic and biochemical
levels.
Although the methods for culturing, handling, and maintenance of M. marinum have some
aspects in common with those for M. tuberculosis (UNIT 10A.1), there are many others that
apply strictly to M. marinum. M. marinum grows optimally in vitro in bacterial culture
medium at 30 to 32 C, with a generation time of 6 to 8 hr at the mid-log phase of growth,
and its biosafety requirement is BSL-2 (UNIT 1A.1). Hence, M. marinum grows signicantly
faster (4 to 6 times) and can be handled in the laboratory more easily (BSL-2, meaning
that it can be manipulated on the benchtop) than M. tuberculosis (BSL-3, meaning that
it must be kept strictly in a biosafety hood). For these reasons, M. marinum has been
increasingly used as a model for studying the pathogenesis of M. tuberculosis.
In this unit, methods are described for culturing M. marinum in liquid medium (see Basic
Protocol 1) and on solid agar medium (see Basic Protocol 2), and for processing the
organism to be used in infection assays (see Basic Protocol 3).
CAUTION: Mycobacterium marinum is a Biosafety Level 2 (BSL-2) pathogen. Follow
all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: It is important to use a refrigerated centrifuge when performing centrifugations
at 25 C to prevent the sample from exceeding 32 C.
BASIC
PROTOCOL 1
Materials
7H9 liquid culture medium (see recipe) or Sautons liquid culture medium
(see recipe)
M. marinum: ATCC #BAA-535 (human isolate) or ATCC #927 (sh isolate)
M. marinum freezing medium (see recipe)
Liquid nitrogen tank with canes to accommodate cryovials
Contributed by Lian-Yong Gao and Joanna Manoranjan
Current Protocols in Microbiology (2005) 10B.1.1-10B.1.8
C 2005 by John Wiley & Sons, Inc.
Copyright
Actinobacteria
(High G+C
Gram Positive)
10B.1.1
2. Thaw a vial of M. marinum frozen stock (as obtained from supplier, or see step 5b)
on ice for 15 to 20 min, and inoculate 10 l of the stock into the prewarmed culture
container from step 1. Tightly seal the container and culture M. marinum in the
shaking incubator at 30 to 32 C with continuous shaking at 100 rpm for 4 days in
the dark, to reach mid- to late-log phase of growth (OD600 1.2 to 1.5, corresponding
to a density of 12 109 cells/ml).
To make the M. marinum culture reach mid-to late-log phase faster, use a higher inoculation volume. For example, add 200 l of the thawed frozen stock to 50 ml of the culture
medium to reach mid-to late-log phase in 2 days. The disadvantages of heavier inoculation are: (1) the culture will contain a higher percentage of bacteria originating from
the frozen stock, some of which may not be viable, and this may affect subsequent assays;
and (2) the frozen stock will be used up more quickly, which may be an issue with certain
strains that are not available in large quantities, e.g., low-passage clinical isolates.
M. marinum grows mostly as clumps/aggregates, and the optical density needs to be
measured immediately after addition of the culture to the spectrophotometer cuvette.
Evaluation of M. marinum density in relationship to OD600 is achieved by measuring
OD600 of the M. marinum single-cell suspension and enumerating CFU of the single-cell
suspension on 7H10 agar medium (see Basic Protocol 2).
5a. For short-term storage: Seal the culture container and store it at room temperature
in a dark place.
Laboratory
Maintenance of
Mycobacterium
marinum
M. marinum can remain viable for 2 months at room temperature in the dark.
10B.1.2
Current Protocols in Microbiology
5b. For long-term storage: Transfer 50 ml of the conuent culture from step 3 to a
50-ml conical tube and centrifuge 10 min at 2700 g, 25 C. Remove and discard
the supernatant using a pipet, then resuspend the pellet in 10 ml freezing medium at
1 1010 cells/ml. Divide into 1-ml aliquots in cryovials and store at 80 C.
M. marinum remains viable for over 20 years under these conditions. Storing M. marinum
in liquid nitrogen can preserve viability for an even longer period of time.
BASIC
PROTOCOL 2
Materials
M. marinum: ATCC #BAA-535 (human isolate) or ATCC #927 (sh isolate)
growing in liquid culture (see Basic Protocol 1)
100-mm 7H10 agar plates (see recipe)
Ethanol
15- and 50-ml conical centrifuge tubes
10-ml syringes with 26-G needles
Tabletop centrifuge
Hemacytometer
Microscope with 40 objective
30 to 32 C incubator
Spreader
Sealable plastic bags to accommodate petri dishes (Fisher)
Inoculating loop
Additional reagents and equipment for counting cells using a hemacytometer
(Strober, 2003)
1. Transfer 10 ml of liquid M. marinum culture into a 50-ml conical centrifuge tube.
2. Draw the cell suspension into a 10-ml syringe and pass it through a 26-G needle
three times to break the bacterial clumps. Transfer the suspension to a 15-ml conical
centrifuge tube.
3. Centrifuge 2 min at 1000 g, 25 C, to separate single-bacteria suspension from
clumps. Transfer the upper, clearer single-bacteria suspension to a new 15-ml conical
tube.
4. Repeat centrifugation as in step 4, then transfer the upper, single-bacteria suspension
to a new 15-ml conical tube. Count the density of M. marinum using a hemacytometer
under a microscope with a 40 objective (Strober, 2003). Make appropriate dilutions
for the next step of plating.
5. Transfer 100 l from each of the above M. marinum dilutions of varying densities to
the center of an individual 100-mm 7H10 agar plate that has been prewarmed to 30
to 32 C in the incubator.
6. Dip the triangular part of the spreader in ethanol, then ame to sterilize the spreader.
Use the spreader to spread the bacterial suspension evenly on the agar surface.
Actinobacteria
(High G+C
Gram Positive)
10B.1.3
Current Protocols in Microbiology
Materials
M. marinum: ATCC #BAA-535 (human isolate) or ATCC #927 (sh isolate)
growing in liquid culture (see Basic Protocol 1)
Phosphate-buffered saline (PBS; APPENDIX 2A)
15- and 50-ml conical centrifuge tubes
10-ml syringes with 26-G needles
Tabletop centrifuge
Hemacytometer
Microscope with 40 objective
1. Transfer 50 ml of liquid M. marinum culture into a 50-ml conical tube. Centrifuge
10 min at 2700 g, 25 C. Remove and discard the supernatant using a pipet, then
resuspend the pellet in 30 ml of PBS prewarmed to room temperature.
2. Repeat step 1 twice to wash away the bacterial culture medium, and nally resuspend
the pellet in 10 ml PBS.
3. Draw the cell suspension into a 10-ml syringe and pass it through a 26-G needle three
times to break the bacterial clumps. Transfer the suspension to a 15-ml conical tube.
Laboratory
Maintenance of
Mycobacterium
marinum
This procedure can be performed on the benchtop. Aerosols are generally not a
health/safety issue with this organism; however, safety glasses are strongly recommended
to prevent splashing into the eyes.
10B.1.4
Current Protocols in Microbiology
ADC supplement
Add the following components to 400 ml water with constant stirring at room
temperature and continue stirring for 40 min:
4.25 g NaCl
25 g bovine serum albumin (BSA) fraction V
10 g dextrose (glucose)
15 mg catalase (Sigma; store up to 1 month at 4 C)
Adjust the nal volume to 500 ml with H2 O
Adjust pH to 6.8 to 7.0 using 1 N NaOH
Pass through 0.22-m lter to sterilize
Store up to 1 month at 4 C
Catalase is added to degrade oxygen intermediates such as H2 O2 , produced by mycobacteria
during growth.
10B.1.5
Current Protocols in Microbiology
OADC supplement
Add 15 ml oleic acid solution (see recipe) to 485 ml ADC supplement (see recipe)
and mix well on a magnetic stirrer. Adjust pH to 7.0 with 1 N NaOH. Pass through
a 0.22-m lter to sterilize. Store up to 1 month at 4 C.
Oleic acid solution
Add 2.4 ml oleic acid and 2.4 ml of 6 M NaOH to 120 ml H2 O. Mix well. Store up
to 1 year at 4 C
Sautons liquid culture medium
Add the following components to 925 ml water with constant stirring on a warm
(50 C) hot plate and continue stirring for 40 min:
0.5 g KH2 PO4
0.5 g MgSO4 7H2 O
4.0 g L-asparagine
10 ml 0.5% (w/v) ferric ammonium citrate solution
2.0 g citric acid
0.1 ml 1% (w/v) ZnSO4 solution
60 ml glycerol
5 ml 10% (v/v) Tween 80
2.0 g dextrose (glucose)
Adjust pH to 7.0 with 1 N NaOH
Cool to room temperature
Pass through a 0.22-m lter to sterilize. Alternatively, autoclave the above medium
without Tween 80, then supplement the cooled medium (<50 C) with the abovementioned amount of Tween 80. Store up to 1 month at 4 C.
COMMENTARY
Background Information
Laboratory
Maintenance of
Mycobacterium
marinum
Because of some of the unique properties of M. marinum, the methods for its laboratory culture and maintenance are different
from those for M. tuberculosis in many aspects. For example, M. marinum is cultured
at 30 to 32 C, due to its lower temperature requirement for optimal growth (Clark
and Shepard, 1963). Also, M. marinum displays somewhat less clumpy growth than
M. tuberculosis and can be cultured in liq-
10B.1.6
Current Protocols in Microbiology
should be kept sealed. Keeping the liquid culture volume at one-fth the full volume of the
container should provide adequate oxygen to
allow optimal growth of M. marinum (Gao
et al., 2003). However, oxygen requirements
may differ among strains of M. marinum. If
the culture does not proceed to mid-log phase,
oxygen may be a limiting factor. Try increasing the size of the culture container to achieve
optimal growth.
Agitation conditions
M. marinum grows optimally with gentle
agitation. Shaking at 100 rpm on an orbital
shaker is ideal (Gao et al., 2003). However,
as agitation is more intense for larger culture
containers (e.g., bottles or asks >1 liter), use
slower shaking speeds (<100 rpm).
Temperature requirements
The optimal growth temperature for
M. marinum is 30 to 32 C. Below 30 C, M.
marinum grows at lower rates. M. marinum
remains static at 35 to 37 C and loses viability at higher temperatures (Clark and Shepard,
1963). Pay attention to the incubating temperature. If the culture is accidentally exposed to
37 C or higher for an extended period of time
(>1 day), start a new culture.
Light requirements
M. marinum grows at an optimal rate under
dark conditions (Gao et al., 2003). Strong light
conditions decrease the M. marinum growth
rate while inducing production of golden pigments. However, the light effect is reversible.
If the growth of M. marinum is halted by exposure to strong light, transfer the culture back
to dark conditions to resume bacterial growth.
Anticipated Results
For M. marinum growth in liquid culture
medium, the anticipated doubling time is 6 to
8 hr in 7H9 and 8 to 10 hr in Sautons medium.
For M. marinum growth on agar-based culture
medium, the anticipated time for colony formation is 6 days. In the presence of antibiotics or certain chemicals, slower growth rates
are anticipated. The human (ATCC #BAA535) and the sh (ATCC #927) isolates grow
at similar rates, and both can infect cells (of
sh and mammalian origins) and animals (sh)
with similar virulence potential (Talaat et al.,
1998).
Time Considerations
Starting with the inoculation of M. marinum
frozen stock into the liquid culture medium
Actinobacteria
(High G+C
Gram Positive)
10B.1.7
Current Protocols in Microbiology
Literature Cited
Andersen P, Askgaard D, Ljungqvist L, Bennedsen J, Heron I. 1991. Proteins released from Mycobacterium tuberculosis during growth. Infect.
Immun. 59:1905-1910.
Clark, H. F. and Shepard, C. C. 1963. Effect of environmental temperatures on infection with Mycobacterium marinum (Balnei) of mice and a
number of poikilothermic species. J. Bacteriol.
86:1057-1069.
Gao, L. Y., Groger, R., Cox, J. S., Beverley, S. M.,
Lawson, E. H., and Brown, E. J. 2003. Transposon mutagenesis of Mycobacterium marinum
identies a locus linking pigmentation and intracellular survival. Infect. Immun. 71:922-929.
Key References
Laboratory
Maintenance of
Mycobacterium
marinum
10B.1.8
Current Protocols in Microbiology
UNIT 10B.2
STRATEGIC PLANNING
Safety Considerations
M. marinum is a BSL-2 organism, and an occasional human pathogen, causing a superficial granulomatous infection of the skin (Wolinsky, 1979). Users should comply with
their institutions requirements for BSL-2 pathogens. The authors recommend gloves,
laboratory coats, and face shields as personal protective equipment. Furthermore, as a
fish pathogen, extreme care should be taken to disinfect all M. marinum cultures with
Amphyl or Lysol IC at a final concentration of 1%, or by sterilization in an autoclave
prior to disposal into sewage systems. Water used for housing of fish and frogs should
be decontaminated with 10% bleach prior to sewage disposal. Any equipment should be
wiped down with 70% ethanol, 0.004% NPD (Steris Corporation), or 1% Lysol after use.
Carcasses of infected animals should be treated as biohazardous waste and disposed of
according to institutional guidelines. Accidental splashes to the skin or eyes, or accidental inoculations (i.e., needle sticks) should be washed immediately and thoroughly with
warm water. Soap should be used on the skin. A qualified physician should be consulted
for consideration of preemptive treatment.
Actinobacteria
(High G+C
Gram Positive)
Contributed by Christine L. Cosma, J. Muse Davis, Laura E. Swaim, Hannah Volkman, and Lalita
Ramakrishnan
10B.2.1
Supplement 3
Zebrash and
Frog Models of M.
marinum Infection
10B.2.2
Supplement 3
Zebrafish embryos, juveniles, and adults can be purchased from the Zebrafish International Resource Center (ZIRC), University of Oregon, Eugene (http://www.zfin.org).
Adults are $7.00 each while 100 embryos can be purchased for about $40 to $80 (higher
prices for commercial organizations). Again the cost of the animals is insignificant relative to the cost of housing (a typical system to house 1000 fish, or 1 rack, costs about
$10,000, see Systems for Housing Frogs and Fish), care (fish food costs $100/month for
1000 fish), supplies, and personnel costs.
Recirculating systems
Recirculating systems are used to house uninfected juvenile and adult zebrafish. They
are designed to support multiple tanks situated on the shelves of a rack that can stand
alone on a laboratory bench or the floor, or can be combined to create a multi-rack
system. There are several manufacturers of such systems, namely Aquatic Habitats
(http://www.aquatichabitats.com); Aquaneering, Inc. (http://www.aquaneer.com), Allentown Caging Equipment Company, and Thoren Caging Company. The principle of the
recirculating system is that water flowing to the tanks is reused or recirculated continuously. The water is filtered in steps comprised of mechanical, biological, and chemical
disinfection. A pad mechanically filters the solids, such as uneaten food and bioflocculants, and then the water flows into a sump. A pump forcibly pushes water first through a
50-m cartridge filter and then through a biofilter containing a substrate such as sponge
pads, or plastic or ceramic balls or beads, which supply a large surface area onto which
denitrifying bacteria colonize and metabolize fish waste. For a comprehensive description
of biofiltration, see Malone and Rusch (1997). Water then flows into an activated carbon
canister, which chemically adsorbs the organic molecules from soluble fish waste (Moe,
1992). An ultraviolet (UV) light sterilizer disinfects the water before returning it to the
fish tanks. Recirculating systems are water efficient and exchange rates vary depending
on the density of fish (3% to 10% per day).
Current Protocols in Microbiology
Actinobacteria
(High G+C
Gram Positive)
10B.2.3
Supplement 3
Flow-through systems
The flow-through system is ideal for housing experimentally infected fish and uses large
amounts of water to provide a continuous supply for the animals at precise environmental
conditions. Flow-through systems are single pass, meaning the water flows into the tanks
only once and is then discharged. Because large amounts of water are required for the
flow-through system, tap water is used as a water source. The only filtration required is
a two-step pretreatment process involving mechanical removal of small particles via a
50-m cartridge filter followed by dechlorination using large tanks of carbon (US Filter
Corp, http://www.usfilter.com). The filtered water is mixed in a large tank by means of a
continuous environmental monitor (YSI 5200, YSI Incorporated), which automatically
doses sea salt solution to maintain conductivity and sodium bicarbonate solution to
maintain pH, before flowing to the fish tanks. The discharged water is UV sterilized or
chlorinated to kill the mycobacteria before draining to the sewer.
Frog Care
Materials needed for storing the frogs, proper housing maintenance, and proper handling
include: tanks, plastic buckets, a mesh screen, nets and net soak, and bleach. Crickets
are needed for feeding the frogs and can be kept in a 50-gallon plastic garbage container
with screen-paneled lid.
Maintaining frog environment
House five or fewer frogs per static tank (described above) in 1 frog water (see recipe).
Move frogs to fresh water in a clean tank every other day (e.g., Monday/Wednesday/
Friday), using nets to catch and transfer them. Latex gloves irritate their skin so minimize
handling, and never handle infected frogs or their water without personal protective
equipment (see Safety Considerations). Disinfect nets between uses by soaking in Net
Soak (Jungle Laboratories Corporation; http://www.junglelabs.com).
Change water, tank, and haul-out platform every other day after feeding day. Performing
tasks as specified here ensures that frogs are not sitting in excess waste products on the off
day (Saturday in the authors laboratory). However, animals should be monitored daily.
Zebrash and
Frog Models of M.
marinum Infection
Fish Care
Synthetic sea salt mix (Instant Ocean, Aquarium Systems., Inc.) should be used to prepare
the water in the tank. Other materials necessary to maintain a suitable living environment
are: bleach (used for disinfection), sodium thiosulfate (removes chlorine from the water),
crushed coral (calcium chloride), and sodium bicarbonate (helps maintain pH at 7.4).
Conductivity and pH meters will be needed to monitor the temperature and pH of the
tank, respectively. An aquaculture water chemistry kit (model FF-1A, Hach Company;
http://www.hach.com) will be needed to test for ammonia. Also, a mesh bag will be
needed for proper handling of the fish.
10B.2.4
Supplement 3
Fish Feeding
At the larval stage, the feeding materials needed will be: TetraMin flake food (Drs. Foster
and Smith; http://www.drsfostersmith.com), Paramecium Multimicronucleatum starter
culture, larval powder food consisting of 1:1:1 Spirulina (Argent Chemical)/Paenaeid
shrimp diet (Argent Chemical)/OSI microfood (Drs. Foster and Smith), and Artemia
(brine shrimp cysts; Argent Chemical). At the adult stage, the fish will continue to be
fed TetraMin flake food and Artemia along with Aquatox food (Aquatic Eco-systems;
http://www.aquaticeco.com). Two brine shrimp hatchers (Aquatic Ecosystems) along
with an air pump will be needed during both the larval and adult stages. To culture the
paramecia, 5-ml syringes, 500-ml clear, sterile glass bottles, and a 5-liter rectangular
plastic container, with secure lid (e.g., Rubbermaid), will be needed.
Table 10B.2.1 Fish Tank Filtration Replacement Schedule
Filter
Change frequency
Cartridge filter
Carbon
Every 2 weeks
UV lamps
Biofilter
Every 6 monthsa
a Set aside 1/8 of (seed) substrate in fish water (see Reagents and Solutions). Clean remaining
Actinobacteria
(High G+C
Gram Positive)
substrate and place into filter with seed substrate loaded nearest to incoming flow of water.
10B.2.5
Current Protocols in Microbiology
Supplement 3
Zebrash and
Frog Models of M.
marinum Infection
10B.2.6
Supplement 3
BASIC
PROTOCOL 1
This protocol describes a method for infecting adult frogs, and subsequent enumeration
of bacterial colony forming units (cfu). At the desired times following infection, frogs
may be euthanized and tissues harvested for analysis. See Background Information and
Anticipated Results and references therein for guidance on the course of infection in
frogs. The three-lobed liver is large (Fig. 10B.2.1), and as such, provides abundant
material for analysisthat is, cfu determination as well as ample material for sectioning
and histology. In addition, liver granulomas are discrete and well formed, while those of
the spleen are sometimes more diffuse. The spleen is much smaller and usually serves
as an additional source of cfu counts. However, any tissue may be harvested depending
on the interests of the investigator. Furthermore, all procedures described here have been
developed for M. marinum strain M (ATCC # BAA-535) and its derivatives. Other strains
may be utilized depending on the interests of the investigator.
Materials
Frogs (see Strategic Planning)
0.2% (w/v) tricaine (ethyl 3-aminobenzoate, methanesulfonate salt), pH 7.0
70% ethanol
7H9 liquid culture medium (see recipe)
PBST: PBS (APPENDIX 2A) supplemented with 0.05% (v/v) Tween 80, sterile
Bacteriological plates: usually 7H10 agar plates (see recipe) supplemented with
10 mg/liter amphotericin B and antibiotics (see recipe for supplements to
7H10 agar plates) if appropriate
Actinobacteria
(High G+C
Gram Positive)
10B.2.7
Current Protocols in Microbiology
Supplement 3
2. Place frog in a plastic bag and lay it on the bench surface, ventral side up. Place hand
over frog, as shown in Figure 10B.2.2, to restrain.
The plastic bag merely aids in restraint of the frog. To avoid suffocation, leave enough
air in the bag and do not restrain frog for more than 1 min. Frogs must be healthy and
acclimated before beginning the experiment.
3. Select an intraperitoneal injection site just left or right of midline and above the hind
legs, but avoiding organs (see Fig. 10B.2.2).
Zebrash and
Frog Models of M.
marinum Infection
Figure 10B.2.1 Internal organs of Rana pipiens. The large tri-lobed liver (A) can be removed to
reveal lungs (*) and spleen (arrow) shown in B. For color version of this gure, see http://www.
currentprotocols.com
10B.2.8
Supplement 3
Figure 10B.2.2 To inject frogs, place in a clear plastic bag and restrain as shown. Injection site
should be at the base of the abdomen to either the right or left of the midline. Insert needle far
enough to penetrate musculature, but not deep enough to pierce organs. For color version of this
gure, see http://www.currentprotocols.com
4. Using a tuberculin syringe fitted with a 27-G needle, insert needle through the
plastic bag and into the frog at the injection site, and dispense 0.1 ml culture into the
peritoneal cavity.
5. Quickly return frog to its tank and resume normal care schedule (see Strategic
Planning).
Perform dissection
6. At the desired time after infection (see Time Considerations), anesthetize frogs in a
2-liter beaker with 200 ml of 0.2% tricaine, pH 7.0, or in another suitable container
with sufficient tricaine solution to reach a depth of 0.5 in.
Tricaine dissolved in water is highly acidic (pH 2 to 3) and must be neutralized (e.g., with
NaOH or sodium bicarbonate) prior to placing animals in the solution. Failure to do so
will result in undue suffering for the animals, and poor absorption of the tricaine.
7. Remove frog from beaker and pinch legs with considerable pressure. If any responsiveness is observed, return frog to the anesthetic and try again after several minutes.
Frogs should be completely unresponsive in 10 min.
8. Pin frog ventral side up and spray with 70% ethanol to disinfect skin.
9. Dissect frog using sterile instruments to remove skin in the following manner.
a. Using dissecting forceps (with teeth), grasp the skin midline at the base of the
abdomen and make a small incision in the skin.
b. Inserting one prong of a pair of scissors at the incision site, cut in a straight
diagonal line from the incision site up to the axilla.
c. Repeat on the other side.
Actinobacteria
(High G+C
Gram Positive)
10B.2.9
Current Protocols in Microbiology
Supplement 3
d. When both cuts have been made, fold the skin up over the head.
Some cutting of connective tissue between the skin and the underlying musculature may
be required.
10. Switch to new, sterile instruments and cut open the underlying musculature in a
similar manner to expose body cavity (Fig. 10B.2.1). To avoid damaging tissues, use
toothless dissecting forceps.
11. First, make a cut to remove the ventricle of the beating heart to ensure death.
Some bleeding may occur, but this does not pose a problem for subsequent manipulations.
13. If desired, remove underlying organs (Fig. 10B.2.1B), revealed after removal of the
liver, in a similar fashion.
Two additional organs often used for bacterial enumeration are the spleen and lungs.
The spleen is small and round, typically 2 to 5 mm in diameter. Lungs are unilobular and
may be punctured prior to removal. Since these organs are small, they are not typically
weighed, and cfu are represented on a per-organ basis. Avoid cutting gastrointestinal
organs, doing so will contaminate the body cavity and organs with intestinal flora.
For tissues to be analyzed by microscopy, refer to Basic Protocol 5.
14. Weigh the liver and add 5 ml sterile 7H9 liquid culture medium supplemented with
OADC and 0.05% Tween 80.
Use 2 ml for spleen or 3 ml for lungs. When processing many frogs, tissues may sit in
closed tubes for up to 2 hr at room temperature before adding medium.
16. Prepare serial dilutions of homogenate in PBST and plate desired dilutions on bacteriological plates.
Typically, a frog infected with 1 106 bacteria will have 1 104 to 1 107 cfu/ml of
liver homogenate at 6 weeks post-infection. Refer to Anticipated Results and references
therein for more guidance on estimating the bacterial load.
Zebrash and
Frog Models of M.
marinum Infection
10B.2.10
Supplement 3
BASIC
PROTOCOL 2
Materials
M. marinium (ATTC #BAA-535 or other strain of interest)
PBS (APPENDIX 2A), sterile
Bacteriological plates: usually 7H10 agar plates (see recipe) supplemented with 10
mg/liter amphotericin B and antibiotics (see recipe for supplements to 7H10
agar plates) if appropriate
0.02% (w/v) tricaine in fish water (see recipe for fish water)
Fish (see Strategic Planning)
50-ml vaccine bottles
1-ml syringes with 21-G (or larger) needles
1
/2 in., 30-G needles
33 C incubator
250-ml beakers
Plastic spoon
Petri dish
Additional reagents and equipment for culture of M. marinum for inoculation (see
Support Protocol 1)
NOTE: After inoculation, fish must be housed in a flow-through system (see Strategic
Planning). For most consistent results, use age-matched fish of the same family, and
acclimate fish prior to injection to ensure maximal health. See Strategic Planning for
details.
1. Prepare log-phase cultures of desired M. marinum strains as described in Support
Protocol 1. Prepare serial dilutions in PBS, with the final dilution made in a 50-ml
vaccine bottle.
2. To eliminate shearing of the mycobacteria, fill each 1-ml syringe using a 21-G (or
larger) needle, and then carefully exchange for a 30-G needle.
Dosage volume should be 50 l per fish, thus allowing 20 doses per 1-ml syringe. A single
syringe will be sufficient to inject a group of fifteen fish, with enough culture remaining in
the syringe for plating in triplicate. Depending upon the virulence of the strain, inoculates
of 5 to 500 cfu/fish are appropriate. Make dilutions of the culture accordingly to achieve
this number.
A 30-G needle is used to minimize trauma to the fish. Otherwise, avoid passing bacterial
stocks through such small needles to minimize lysis.
3. Plate two 50-l aliquots from this needle onto bacteriological plates to enumerate
the average number of bacteria per injection. Incubate injection plates at 33 C until
colonies can be counted.
Since the needle may become contaminated with intestinal flora, microorganisms in
the water, or those on the surface of the skin upon inoculation of the fish, the authors
recommend plating two aliquots prior to inoculation of the fish (step 5, below). A final
Actinobacteria
(High G+C
Gram Positive)
10B.2.11
Current Protocols in Microbiology
Supplement 3
aliquot may be plated after the last fish has been injected (step 7, below) to help determine
whether the dose remains constant for all fish injected with that needle, although this plate
may become contaminated. Cells should be spread on the plate using a spreader (see
APPENDIX 4A for common microbiological techniques, and UNIT 10B.1 for those specific to
M. marinum).
4. Prepare anesthetic bath of 0.02% tricaine in fish water (100 ml) in a 250-ml beaker,
and a recovery bath of fish water (containing no tricaine) in a spare tank.
5. Put fish to be injected, five at a time, into the anesthetic bath. When the fish roll onto
their dorsal sides, immediately spoon from the beaker into a petri dish and orient
ventral side inward, gently holding fish in place (Fig. 10B.2.3).
IMPORTANT NOTE: Fish should roll within 1 min. If fish remain in the anesthetic bath
too long, they will not recover. It is recommended that the fish not remain in anesthetic
any longer than necessary.
6. Insert needle above the pelvic fin and left of the midline, holding the needle at a 45
angle (see Fig. 10B.2.3). Insert needle just enough to penetrate peritoneal cavity and
inject 50 l bacterial suspension.
A good guideline is to insert the needle until the bevel tip is completely inside the fish.
7. When all five fish are injected, transfer to recovery bath and begin another group
until fifteen fish have been injected. Wait to see that all fish have recovered and then
transfer back into holding tank.
Working rapidly helps to reduce immediate mortalities; however, some loss should be
expected. An inexperienced researcher may lose a significant number of fish due to injury,
anesthesia overdose, or keeping fish out of the water for too long. With experience,
mortality may be kept fairly low (<5% loss). A novice may want to anesthetize only one
or two fish at a time to start.
It is not necessary to sterilize the injection site; in addition to the potentially toxic effects
of antiseptic, keeping fish out of water for a prolonged period of time increases the chance
of immediate mortality.
8. Check infected fish twice daily and record mortalities for determination of a survival
curve.
Alternatively, fish may be removed and killed according to a desired time course for
bacterial enumeration (see Basic Protocol 3) and/or histology (see Basic Protocol 4),
depending on the goals of the experiment and the scientific question(s) being addressed.
Zebrash and
Frog Models of M.
marinum Infection
10B.2.12
Supplement 3
Figure 10B.2.3 Intraperitoneal injection of adult zebrash. For color version of this gure, see
http://www.currentprotocols.com
Current Protocols in Microbiology
BASIC
PROTOCOL 3
Materials
Infected fish (Basic Protocol 2)
PBS (APPENDIX 2A), sterile
Fish water (see recipe) supplemented with 1.5 mg/ml kanamycin sulfate
Fish water (see recipe) supplemented with 1.5 mg/ml kanamycin sulfate and 0.05%
(w/v) tricaine
70% ethanol
7H10 agar plates (see recipe), 10 mg/liter amphotericin B, 25 mg/liter polymyxin B
sulfate, 20 mg/liter trimethoprim, and 50 mg/liter carbenicillin disodium salt
(see recipe for supplements to 7H10 agar plates)
17 100mm sterile polystyrene tubes
Petri dish
Omni homogenizer (model TH-115; Omni International) with sterile Omni tips
(disposable generator probes)
1-ml serological pipet with filter plug
Sterile glass test tubes
33 C incubator
1. For each fish, pipet 1.5 ml PBS into a sterile 17 100mm polystyrene tube and
weigh each tube to an accuracy of 0.1 g.
2. Bathe all fish to be processed in fish water supplemented with 1.5 mg/ml kanamycin
for 1 hr.
3. To euthanize fish, transfer them to fish water containing 1.5 mg/ml kanamycin and
0.05% tricaine for 10 to 15 min to ensure death.
Do not leave dead fish floating in tricaine solution longer than is necessary.
4. Transfer fish to sterile petri dish and spray with 70% ethanol to disinfect. Allow
ethanol to air dry then transfer fish individually to the polystyrene tubes (one fish
per tube).
Actinobacteria
(High G+C
Gram Positive)
10B.2.13
Current Protocols in Microbiology
Supplement 3
Materials
Infected fish (Basic Protocol 2)
Dietrichs fixative (see recipe)
70% ethanol
Embedding cassettes (e.g., Tru-Flow, Fisher Scientific)
1. Fix fish whole by submerging in Dietrichs fixative for a minimum of 3 days.
Before fixing fish, they must either be dead of natural causes, or must first be euthanized
as described in Basic Protocol 3, step 3, or by another humane method consistent with
local institutional or governmental guidelines.
2. Transfer fish to embedding cassette in 70% ethanol for storage and process for
histology by standard techniques.
Zebrash and
Frog Models of M.
marinum Infection
Always use pencil to label the cassettes. Ink will be erased by the chemicals used for
processing.
10B.2.14
Supplement 3
BASIC
PROTOCOL 5
The use of fluorescent bacteria allows for efficient visualization in infected tissues. Fluorescence makes possible the use of multiple, differentially marked strains. Also, promoter
fusions to fluorescent proteins can be used as reporters of bacterial gene expression in
vivo. Bacteria expressing GFPmut3 (Cormack et al., 1996), dsRed (Clontech), and ECFP
(Clontech) can be easily visualized in tissues using appropriate filter sets on a standard fluorescence microscope (see references for spectral properties of the fluorescent
proteins). Frog liver granulomas can be visualized as dense foci of DAPI-stained nuclei, which may contain bacteria. Frogs produce a dark pigment, which is taken up by
macrophages, and at times may obscure some bacteria. Adult zebrafish granulomas often
show a nuclear-free area of central necrosis (caseum), surrounded by a dense area of
flattened, spindle-shaped nuclei and an outer epithelioid layer (Gauthier et al., 2003;
Cosma et al., 2004).
Similar procedures are described for analysis of frogs and adult zebrafish.
Materials
Infected frog tissues (see Basic Protocol 1, step 12), or infected whole fish (see
Basic Protocol 2)
4% (w/v) paraformaldehyde in 1 PBS (see APPENDIX 2A for PBS)
5%, 15%, and 30% (w/v) sucrose in 1 PBS (see APPENDIX 2A for PBS)
OCT compound (Tissue-Tek)
Antifade reagent with DAPI (e.g., Slow-Fade, Molecular Probes; or similar
antiquenching reagent)
Nail polish
17 60mm (8-ml) glass vials with screw caps (VWR)
Razor blade
Aluminum foil boats for freezing of tissue sections (Figure 10B.2.4)
Microtome/cryostat
Fluorescence microscope
Figure 10B.2.4
10B.2.15
Current Protocols in Microbiology
Supplement 3
Dissect tissue
1a. For frogs: While dissecting the animal (see Basic Protocol 1), remove a piece of liver
5-mm 5-mm 5-mm and place it in a 17 60mm glass vial.
This tissue should be removed first, taking care not to mangle it or in any other way
compromise its integrity.
1b. For fish: Cut whole zebrafish transversally into two or three sections using a sharp
razor blade.
Before processing fish, they must either be dead of natural causes, or must first be
euthanized as described in Basic Protocol 3, step 3, or by another humane method
consistent with local institutional or governmental guidelines.
6. Prepare an aluminum foil boat (double layer) with a 1-cm deep 1-cm wide well
and fill with OCT compound.
The glass vials make useful molds for making foil boats (Fig. 10B.2.4).
7. Place the tissue in the well, submerging it in OCT, with the surface to be cut facing
the bottom.
For fish, this should be the cut surface so that when sections are cut, whole body transverse
sections are obtained.
8. Place boat in a dry ice/ethanol bath to freeze tissue. Carefully remove the boat and
wipe off excess ethanol. Store tissue block at 80 C until use.
Ethanol interferes with the freezing of OCT, so it is important to avoid getting ethanol on
the tissue block or in the OCT.
Perform cryosectioning
9. Prepare frozen sections using a microtome/cryostat using standard techniques, or
according to the manufacturers instructions.
Zebrash and
Frog Models of M.
marinum Infection
10B.2.16
Supplement 3
Alternative tissue preparation and cutting protocols may be found in Watkins (1989).
It is ideal to use 5- to 10-m sections for examination of fluorescent bacteria in granulomas.
Current Protocols in Microbiology
12. Seal with nail polish, and examine using a fluorescence microscope (see
Commentary).
Typically 400 total magnification allows for careful examination of bacteria in the
tissue. Store slides indefinitely at 20 C or colder.
SUPPORT
PROTOCOL 1
This support protocol describes the preparation of bacterial stocks to be used for infection of adult frogs and zebrafish. Due to the clumping nature of this organism,
cfu do not necessarily represent the number of organisms present unless steps are
taken to isolate single-cell suspensions. The procedures described here do not utilize
single-cell suspensions, yet are fairly reproducible if cultures of similar density are prepared as described each time. Several methods exist to isolate single-cell suspensions of
M. marinum, including passage of cultures through a 5-m filter, sonication, or passing
cultures through a syringe fitted with a needle to break up clumps. However, the impact of
these procedures has not been evaluated in the adult animal models of infection described
here. It is possible that planktonic bacteria isolated by filtration may possess different
infectious properties than bacteria growing in clumps (akin to biofilms). Similarly, injection of excessive amounts of cell debris generated by sonication or passage through a
syringe may impact infection in ways that have not been evaluated to date. See UNIT 10B.1
for further information on the culture and characteristics of M. marinum grown in vitro.
Materials
7H9 liquid culture medium (see recipe)
M. marinum (ATCC # BAA-535 or other strain of interest), frozen stock (UNIT 10B.1)
Antibiotics as required
LB agar plate
25-cm2 tissue culture flasks
33 C incubator
1. Add 5 ml fresh 7H9 liquid culture medium supplemented with OADC, Tween 80,
and antibiotics (if required) to a 25-cm2 tissue culture flask, and inoculate with a
small scraping of M. marinum frozen stock or a bacterial colony. Incubate flask at
33 C in a horizontal position until it reaches an OD600 between 0.3 and 0.6.
2. After inoculating cultures, transfer a few microliters of each culture, as well as
uninoculated medium onto an LB agar plate. Incubate this plate alongside the cultures
and check for the presence of any contaminating growth.
Some M. marinum may grow on the LB plate, however, if growth of any other bacteria or
molds is observed, this is an indication that the culture and/or medium are contaminated.
Do not use cultures that are contaminated.
There is no need to spread the inoculum.
Actinobacteria
(High G+C
Gram Positive)
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4. Vortex culture, then maintain static in a vertical position on the benchtop for 10 to
15 min to allow the largest clumps to settle. Pull suspension of dispersed cells from
the top and place in a separate sterile tube. Measure OD600 .
While this step removes the largest of clumps, most bacteria will still be found growing
in aggregates.
Materials
M. marinum (ATCC # BAA-535 or other strain of interest, usually marked with a
fluorescent marker)
1.5 EMPT (see recipe)
Dechorionated zebrafish embryos at 30 hr post-fertilization or later (see Support
Protocol 4 for dechorionation details)
Sterile zebrafish embryo medium (see recipe)
Micropipettor with microloader tips for loading needles (Eppendorf)
Injection needles: needles made from aluminosilicate capillaries (SM100F-10,
Harvard Apparatus) pulled on a Sutter P-2000 micropipet puller, with the
following settings: Heat = 350, FIL = 4, VEL = 50, DEL = 225, PUL = 150
Microscope with Microinjector (e.g., Eppendorf 5246) and Micromanipulator (e.g.,
Narishige GJ-1)
Deep-well microscope slides (VWR)
Extra fine jewelers forceps (Miltex no. 17-305 or Dumont no. 5)
Slide warmer, optional (e.g., Barstead no.26005 or 26020)
Microprobe (2 to 3 cm of 32-G platinum wire mounted on the end of a Pasteur
pipet)
Multi-well culture plates, or small petri dishes
Large-bore glass pipets (Chase Scientific Glass, Inc. #63AS3WT, distributed by
Fisher #XC-9993639, note that these are not typical Pasteur pipets)
Zebrash and
Frog Models of M.
marinum Infection
10B.2.18
Supplement 3
Figure 10B.2.5 Zebrash embryo anatomy: The most-used injection site for infection has been
the caudal vein (cv). Other useful sites have been the fourth ventricle of the brain (4v), the
pericardium (pc) and the yolk ball (yb). The yolk circulation valley (ycv) is an excellent place
to view macrophages both infected and uninfected during the early hours of blood circulation
(Herbomel et al., 1999). Adapted from Westereld (2000).
NOTE: Standard borosilicate capillaries may also be used, but the authors find clumping
of mycobacteria to be less problematic when using aluminosilicate capillaries. If only
borosilicate is available, note that the settings on a Sutter P-2000 micropipet puller
are different for producing the same shape needle. Use: Heat=300, FIL=4, VEL=40,
DEL=150, PUL=125.
6. Using jewelers forceps, break off the very tip of the needle while applying injection
pressure to the needle (Fig. 10B.2.7A).
Break off enough to allow free but not excessive flow. It is better to break off too little than
too much.
7. Adjust the constant pressure on the needle to produce a faint, continuous stream out
of the needle.
Constant pressure at 5 to 20 hPa is usual.
Because of the tendency for mycobacteria to clump and clog the needle, it is necessary
to break off a bit more of the needle than is typically done when injecting other liquids.
If excessive clogging is encountered, resyringe the culture (see Support Protocol 2), and
load a new needle.
Actinobacteria
(High G+C
Gram Positive)
10B.2.19
Current Protocols in Microbiology
Supplement 3
Figure 10B.2.6 Standard injection setup. Right-handed operators prefer to use the micromanipulator on the left, since more delicate work must be done with the microprobe.
9. Transfer one embryo to the microscope slide and, using the microprobe, position it
gently so that the needle points to the intended injection target.
For caudal vein injections, try lowering the needle onto the flank near the urogenital
opening until it begins to press down on the embryo. With the embryo thus partially
immobilized, tap the dorsal edge of the tail opposite the injection site to impale the
embryo. See Figure 10B.2.7B and C.
Zebrash and
Frog Models of M.
marinum Infection
10B.2.20
Supplement 3
For caudal vein injections, three useful signs of injection volumes are vein only (Fig.
10B.2.7), blush (a slight red color in the yolk circulation valley), and full red (obvious
red dye in the circulation valley). These, in combination with the concentration of the
injection mix, can help to control the injection dose. Once appropriate settings have been
determined, use them consistently for all embryos and when plating the injection dose
(see Support Protocol 3).
Current Protocols in Microbiology
Figure 10B.2.7 Injection procedures. (A) Opening the end of the needle with jewelers forceps
(use a different set than those used for dechorionation). The higher the concentration of bacteria
in the needle, the greater the length of needle that must be broken off to prevent clogging. (B)
Caudal vein injectioninserting the needle. The platinum wire probe provides the force necessary
to penetrate the periderm. Most operators nd that moving the needle up and down, and setting
its initial position, are the only times the micromanipulator is moved. (C) Successful caudal vein
injectionphenol red dye is visible in the caudal vein, owing toward the heart. With practice, a
skilled operator can perform 100 to 150 such injections per hour. For color version of this gure,
see http://www.currentprotocols.com
11. Transfer the injected embryo to an appropriate container or multi-well plate containing sterile zebrafish embryo medium using a large-bore pipet.
For holding embryos after injection, wells of 6-well tissue culture plates, or single 60
15mm petri dishes, hold 10 to 25 embryos each. Long-term survival of embryos is
enhanced by lower densities (10 per well/dish).
To avoid contaminating embryos with a different bacterial strain, use a separate largebore pipet for each infection group, and also separate pipets for injected and uninjected
embryos.
12. At a time point 1 to 2 hr after injection, place the embryos in fresh sterile zebrafish
embryo medium.
BASIC
PROTOCOL 7
Materials
Sterile zebrafish embryo medium (see recipe) supplemented with 20 g/ml
kanamycin
Infected embryos (see Basic Protocol 6)
Fish water (see recipe) supplemented with 200 g/ml tricaine
Trypsin-EDTA (Invitrogen)
PBS (APPENDIX 2A)
1% (v/v) Triton X-100
7H10 agar plates supplemented with OADC (see recipe) and antimicrobial agents
(see recipe for supplements to 7H10 agar; see Basic Protocol 2)
30 and 33 C incubators
Water bath ultrasonicator (Bransonic, model no. 1510R-NT)
Current Protocols in Microbiology
Actinobacteria
(High G+C
Gram Positive)
10B.2.21
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3. Remove most of the zebrafish embryo medium with a pipet tip, taking care that the
embryo remains in the tube. Add 500 l of 200 g/ml tricaine, and place tubes for
1 hr on ice.
The minimum time required to kill the embryo is 1 hr. Shorter incubations may result in
the embryos reviving when brought back to room temperature.
Materials
M. marinum (ATCC #BAA-535 or other strain of interest) frozen stock (UNIT 10B.1)
Zebrafish embryo medium (see recipe), sterile, or PBS (APPENDIX 2A)
20% (w/v) phenol red dye stock solution in H2 O
1-ml tuberculin syringes with 27-G needles
Additional reagents and equipment for preparing M. marimum (see Support
Protocol 1, steps 1 to 3) and loading the microinjector needle (see Basic Protocol
6, steps 1 and 2)
1. Prepare M. marinum as described in Support Protocol 1, steps 1 to 3. Grow to an
OD600 between 0.3 and 0.9.
Zebrash and
Frog Models of M.
marinum Infection
2. No more than 1 hr before injection, vortex the culture vigorously and pipet 200 l
into a sterile 1.7-ml microcentrifuge tube. Microcentrifuge 2 min at maximum
speed.
10B.2.22
Supplement 3
3. Remove supernatant and resuspend the pellet in the appropriate volume of zebrafish
embryo medium or PBS.
IMPORTANT NOTE: Bacterial viability appears to decrease with extended time in
zebrafish embryo medium, therefore, prepare the suspensions as close to injection time
as practical and leave in zebrafish embryo medium for no more than 1 hr.
Resuspending in the original volume (200 l) produces a 1 solution, which will yield
inoculates of roughly 100 to 300 bacteria per full red injection. A 1:10 dilution of this
resuspension produces a 0.1 bacterial stock. A full red injection with a 0.1 stock
yields roughly 20 to 100 bacteria per full red injection. These numbers should only be
taken as a recommended starting point. Each researcher develops a sense for how much
injection time, and how thick a bacterial suspension will be required to achieve a desired
dose. See Support Protocol 3 and Commentary for additional discussions of dosing.
4. Just before loading the microinjection needle, pass the bacterial suspension through
a 27-G needle attached to a 1-ml syringe 5 to 20 times to reduce clumping.
An appropriate balance must be struck between reducing clumps and lysing bacteria.
Ideally, the suspension should be passed through the needle only enough to reduce
clumping. The bacterial suspension will become clear with increased passages through
the needle. See Support Protocol 3 for discussion on estimating doses.
SUPPORT
PROTOCOL 3
Materials
PBS (APPENDIX 2A)
Middlebrook 7H10 agar plates (see recipe)
Injection needles loaded with bacterial suspension (see Basic Protocol 6 steps 1 to
10)
Micromanipulator (e.g., Narishige GJ-1) and microinjector (e.g., Eppendorf 5246)
33 C incubator
1. Apply a 20-l drop of PBS to the middle of a Middlebrook 7H10 agar plate.
2. Elevate the injection needle containing the bacterial suspension with the micromanipulator, slide the 7H10 agar plate under it, and lower the needle near the top of the
PBS droplet.
Extreme caution must be taken so as not to break the needle tip during this manipulation.
3. Lower the needle into the PBS and immediately apply the standard amount of
injection time, then immediately lift the needle out of the PBS.
Actinobacteria
(High G+C
Gram Positive)
10B.2.23
Current Protocols in Microbiology
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4. Spread the PBS and bacteria evenly onto the plate and incubate for 5 to 7 days at
33 C, or until colonies can be counted.
If using fluorescent bacteria, an alternative method involves injecting the dosage
amount onto a slide containing 20 l distilled water. The droplet of liquid is allowed to
dry and the number of fluorescent bacteria can be counted directly using a fluorescence
microscope. Using a slide inscribed with a microscopic grid can be helpful. Hemacytometer slides work well. Of course, it is essential to back this method by plating for viable
cfu, but the numbers have been found to correlate fairly well.
SUPPORT
PROTOCOL 4
Materials
Zebrafish embryos (see Strategic Planning)
Zebrafish embryo medium (see recipe), sterile
100 15mm petri dishes (or similar size)
Two pair extra-fine jewelers forceps (Miltex #17-305 or Dumont #5)
Dissecting microscope
NOTE: Keep forceps for dechorionation separate from those used for injections.
1. Place embryos in 100 15mm (or similar size) petri dishes with just enough
zebrafish embryo medium (with or without PTU, see Critical Parameters) to cover
the bottom.
Zebrash and
Frog Models of M.
marinum Infection
Figure 10B.2.8
10B.2.24
Supplement 3
2. Gently swirl the medium inside the petri dish, allowing the embryos to collect in a
cluster at the center.
3. Manually remove chorions one embryo at a time.
Many techniques exist, but start by pinching a small portion of the chorion with one pair
of extra-fine jewelers forceps while steadying the embryo with the other pair of forceps.
Next, pinch the chorion near the first pinch with the second pair of forceps, and pull apart
(see Fig. 10B.2.8). It may help to pull the torn chorion up to the air/water interface, and
gently tug the chorion upward, leaving the embryo in the water.
Dietrichs fixative
150 ml 100% ethanol
50 ml formalin
10 ml acetic acid
290 ml H2 O
Store indefinitely at room temperature
EMP
7.5 ml PTU stock solution (see recipe)
100 ml zebrafish embryo medium (see recipe)
Store up to 1 week at room temperature
Actinobacteria
(High G+C
Gram Positive)
10B.2.25
Current Protocols in Microbiology
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Fish water
Prepare water for fish by adding sea salt to a conductivity of 1500 S. To a mesh
bag, add 1 teaspoon (4.9 cm3 ) crushed coral (CaCl2 ) for every 10 gallons (38 liters)
of water to be treated. Place in the spillway (plastic receptacle which receives all
discharge water and channels it into the sump). Measure conductivity, pH, and
temperature twice daily to be certain conditions are within range. Ideal operating
parameters are: temperature = 27 to 28 C, pH = 7.4 0.2, and conductivity =
1500 100 S). For tricaine solutions, tricaine is simply added to this solution in
the desired amount.
If difficulties in anesthetizing occur, check the pH and correct to 7.2 as needed with sodium
bicarbonate.
All of Hanks stock solutions should be stored indefinitely at 4 C. Adapted from Westerfield
(2000).
10B.2.26
Supplement 3
OADC supplement
Dissolve the following in 700 ml dd H2 O:
50 g BSA, fraction V
50 ml 1% oleic acid (see recipe)
20 g dextrose
8.5 g NaCl
Bring to 1 liter final volume
Filter sterilize using a 0.22-m filter
Store up to 6 months at 4 C
Oleic acid, 1%
Add 5 g oleic acid to 500 ml 0.2 N NaOH. Heat solution to 55 C to dissolve
completely. Store at 20 C in 50-ml aliquots indefinitely. Thaw at 65 C prior to
use with OADC.
10B.2.27
Current Protocols in Microbiology
Supplement 3
COMMENTARY
Background Information
Mycobacteria are intracellular pathogens
known for their ability to establish persistent
infections. Mycobacterium marinum, a natural
pathogen of poikilothermic species such as the
frog and fish models described here, mediates
infection in much the same way that species
of the Mycobacterium tuberculosis complex
cause infection in mammalian hosts (Cosma
et al., 2003). M. marinum infection of ectotherms has gained popularity as a model for
tuberculosis infections for a variety of reasons (Ramakrishnan, 2004). First, the lower
growth temperature of this organism makes it
less hazardous for human health (see Strategic
Planning, Safety Considerations). Second, its
faster generation time allows work to proceed
more quickly than with M. tuberculosis complex organisms. For example, colonies appear
on plates in 5 to 7 days as compared to 3 weeks
for M. tuberculosis. Finally, M. marinum infection of zebrafish and frogs allows the study
of two important aspects of mycobacterial
infection that are not well replicated in the
M. tuberculosis-mouse model. The first aspect
is that infection of frogs leads to a chronic
and asymptomatic infection for the lifetime
of the animal, modeling the long latency seen
in some humans infected with M. tuberculosis (Ramakrishnan et al., 1997). The second
aspect is that the zebrafish model allows the
study of caseating granulomas (Cosma et al.,
2004), another important feature of human disease not replicated by the mouse model.
Zebrash and
Frog Models of M.
marinum Infection
Figure 10B.2.9 Sources of macrophages in the developing zebrash. (A) Early macrophages
arise in the rostral blood island around the 5-somite stage and begin to migrate into the future
yolk circulation valley around the 14-somite stage. (B) There is a nest of likely hematopoiesis
near the caudal vein during early larval stages. It is suspected but not known if a distinct wave of
macrophages is produced here. (C) The main site of myelopoiesis in the adult is the head kidney.
Adapted from Westereld (2000) and Davidson and Zon (2004).
10B.2.28
Supplement 3
Critical Parameters
Adult frog infections
Rana pipiens used for experimental infection are wild-caught and outbred animals and,
as such, exhibit variable responses to infection
as measured by tissue cfu burden (Ramakrishnan et al., 1997). Therefore, a sufficient number of animals should be used to obtain statistically significant results. Immunosuppression
using corticosteroids will result in fulminant
infection and death of the animals.
Adult zebrafish infections
Mycobacteriosis in fish is a chronic, longterm infection relative to other fish diseases.
It is characterized by histological observation of acid-fast bacilli and caseous granulomas occurring within the hematopoietic organs, namely the liver, kidney, thymus, spleen,
and pancreas (Gauthier et al., 2003). Yet, M.
marinum is clearly more pathogenic for zebrafish than for frogs, causing a slow but progressive illness that results in the death of the
animal. The acute phase response to the formation of thousands of granulomas and internal organ remodeling causes several gross
outcomes. Some fish may develop a large focalized lesion in the peritoneum, while others display a diffuse hemorrhagic condition
that progresses into systemic ascites where the
scales of the fish stand on end, like a pine
cone. This condition is typical in fish and
results from a generalized bacteremia and kidney failure. Sick fish behave differently than
healthy fish, sitting on the bottom of the tank or
hiding in a corner to ward off attacks by healthier tankmates. Pecking at the weaker fish by
healthier ones may operate as a mode of transmission among fish within a group. Mortalities
are best collected immediately because healthier tankmates eat the gut of infected, fallen
fish.
To minimize in-tank effects, each tank
should be statistically evaluated in an independent manner and therefore, tank replication
is essential. In-tank effects include group behavioral differences, sex ratio differences, and
transmission variability.
Actinobacteria
(High G+C
Gram Positive)
10B.2.29
Current Protocols in Microbiology
Supplement 3
injection, producing the need for operator consistency with injection time and periodic plating of the injection volume. The authors find
that a given needle will give gradually decreasing doses, so for maximum reproducibility, it
is best to plate the same needle before and
after injecting the embryos. While consistent
culture densities, resuspension volumes, and
injection amounts (vein only, blush, full
red) can help control the applied dose, enough
variety is still seen in doses to require constant
assessment of the number of bacteria applied
to each fish. Plating needles (on agar for cfu
determination, and on microscope slides for
visual inspection) and lysing fish to count bacteria soon after infection are the main ways to
assess dosage (see Basic Protocol 4 and Support Protocols 2 and 3 for details). However,
most researchers find that using fluorescent
bacteria allows direct observation in each embryo of the relative level of infection within a
few hours of injection. If a particular level of
infection is desired, it is helpful to perform a
range of injections and screen them afterwards
for those individuals with the desired level of
infection.
Visualizing the infection by microscopy
A typical light microscope fitted with
DIC (differential interference contrast; see
UNIT 2A.1) optics and capable of fluorescence
microscopy is useful for many kinds of observations in zebrafish embryos and larvae.
For screening purposes, individual embryos
can be held in glass 10-well plates (Aquatic
Eco-systems, Inc.) and viewed with a 10
Zebrash and
Frog Models of M.
marinum Infection
Figure 10B.2.10 Mounting an embryo on a deep-well slide, by a left-handed operator. (A) Using
a ne paintbrush to manipulate the embryo, slide it under the edge of the coverslip, then gently
slide the coverslip to cover the well. (B) Finish moving the embryo under the coverslip before it
is forced too high up the side of the well. Embryos and larvae of various sizes can be held in
place by adjusting how close to the edge of the well they are mounted. (C) Blotting away excess
zebrash embryo medium clamps the coverslip down securely onto the slide. Because of the
shape of the embryo at its thickest point, a side view will either favor the dorsal or ventral view. The
mount shown gives a better view of the ventral aspect of the left side, allowing a good view of the
heart and pericardium, common locations for infected macrophages. This view also maintains the
traditional posedorsal up, head to the leftwhen viewed under a compound microscope. Note
that DIC microscopy is compromised when using deep-well slidessee text.
10B.2.30
Supplement 3
Anticipated Results
Adult frog infections
Experimental inoculation of adult Rana
pipiens with M. marinum results in a life-long,
but asymptomatic infection (Ramakrishnan
et al., 1997). Inoculation with as few as 10
to 100 bacteria will result in a chronic infection; however, bacteria may not be detectable
in all animals until 4 to 6 weeks post-infection.
Granulomas are reliably observed at doses
of >104 cfu. Fairly low levels of infection
(102 cfu/g tissue) can be detected by cfu determination, although 105 to 106 cfu/g tissue are
needed for consistent visualization in sections
(Cosma et al., 2004).
Adult zebrafish infections
Injecting bacteria is the most effective route
of experimental inoculation and is most reliable for replicating consistent infections.
There appears to be a range of survival responses depending on the initial dose administered. When a low number of bacteria
is delivered (e.g., 5 cfu), the acute phase
of pathogenesis may take 6 to 8 weeks to
develop. When >200 cfu are delivered, the
acute phase results in 1 week. An intermediate inoculum strikes an optimal pathogenesis
for experimental observation. At moderate to
high doses, hallmark clinical signs of disease
manifest within 3 weeks, and culminate in
100% mortality of the tank population in
3 months time. At low doses, some fish may
Actinobacteria
(High G+C
Gram Positive)
10B.2.31
Current Protocols in Microbiology
Supplement 3
clear the infection while others go on to develop acute disease. For further details, consult
Swaim et al. (2006).
Zebrafish embryo
In general, the infection proceeds as follows (Davis et al., 2002; Volkman et al., 2004):
free bacteria can be seen in the blood for as
long as 3 hr post-injection, but the majority
are taken into macrophages quickly, a process
that can be viewed in the yolk circulation valley. After 3 to 4 hr, infected macrophages are
seen in various tissues. Over the next 2 to 5
days (depending on infection dose) the bacteria multiply within the macrophages, and
aggregates of infected cells will form. Likely
sites for aggregation are ventral to the caudal
vein (a site of early hematopoiesis), in muscle near the injection site, in the pericardium,
and in the brain. Bacterial dose contributes to
the rate of aggregate formation. The authors
have produced aggregation in as few as 2 to
3 days with heavy doses, while lightly infected
embryos may not form aggregates for 5 to
7 days. In general, fish that form aggregates
early in larval development die of the infection more quickly, but the vast majority will
die within 2 weeks regardless of dose.
Time Considerations
Frog infections
Acclimatization requires 2 to 4 weeks and
culture preparation will take 1 week. Injection time for 60 frogs is 2 hr. The duration of
infection depends on the experimental goals.
The authors have observed mycobacteria in
the liver as early as 3 days following infection,
and have isolated mycobacteria from livers of
frogs infected for as long as 17 months. Up
to 60 frogs can be killed, tissues harvested,
and homogenates plated by a team of three researchers in a (grueling) 12-hr day. Allow 7 to
14 days for complete colony counts, depending on the vigor of the bacterial strains being
used. To freeze tissues, allow 4 to 24 hr for
fixation and rehydration, and 10 to 15 min to
freeze each tissue.
Zebrash and
Frog Models of M.
marinum Infection
Literature Cited
Aubry, A., Jarlier, V., Escolano, S., TruffotPernot, C., and Cambau, E. 2000. Antibiotic
susceptibility pattern of Mycobacterium marinum. Antimicrob. Agents Chemother. 44:31333136.
Cody, S.H. and Williams, D.A. 1997. A novel organ
bath design enables the use of saline-immersible
lenses on inverted microscopes. J. Microscopy
185:94-97.
Cormack, B.P., Valdiva, R.H., and Falkow, S. 1996.
FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.
Cosma, C.L., Sherman, D.R., and Ramakrishnan,
L. 2003. The secret lives of the pathogenic mycobacteria. Ann. Rev. Microbiol. 57:641-76.
Cosma, C.L., Humbert, O., and Ramakrishnan,
L. 2004. Superinfecting mycobacteria home to
established tuberculous granulomas. Nat. Immunol. 5:828-835.
Cosma, C.L., Klein, K., Kim, R., Beery, D., and
Ramakrishnan, L. 2006. Mycobacterium marinum Erp is a virulence determinant required
for cell wall integrity and intracellular survival.
Infect. Immun. 74:3125-3133.
Danilova, N., Hohman, V.S., Sacher, F., Ota, T.,
Willett, C.E., and Steiner, L.A. 2004. T cells and
the thymus in developing zebrafish. Dev. Comp.
Immunol. 28:755-767.
Davidson, A.J. and Zon, L.I. 2004. The definitive (and primitive) guide to zebrafish
hematopoiesis. Oncogene 23:7233-7246.
Gauthier, D.T., Rhodes, M.W., Vogelbein, W.K.,
Kator, H., and Ottinger, C.A. 2003. Experimental mycobacteriosis in striped bass Morone saxatilis. Dis. Aquat. Organ 54:105-117.
Herbomel, P., Thisse, B., and Thisse, C. 1999. Ontogeny and behaviour of early macrophages in
the zebrafish embryo. Development 126:37353745.
10B.2.32
Supplement 3
Key References
Davis, J.M., Clay, H., Lewis, J.L., Ghori, N.,
Herbomel, P., and Ramakrishnan, L. 2002.
Real-time visualization of Mycobacteriummacrophage interactions leading to initiation of
granuloma formation in zebrafish embryos. Immunity 17:693-702.
This paper presents a characterization of the zebrafish embryo infection model, including typical
course of infection, cell behaviors, and bacterial
responses.
Ramakrishnan, L., Valdiva, R.H., McKerrow, J.H.,
and Falkow, S. 1997. Mycobacterium marinum
causes both long term subclinical infection and
acute disease in the leopard frog (Rana pipiens).
Infect. Immun. 65:767-773.
This paper describes the experimental inoculation
of R. pipiens and subsequent progression of the infection.
Swaim et al., 2006. See above.
This paper describes the experimental inoculation
of adult zebrafish and subsequent disease progression.
Westerfield, 2000. See above.
Available online at http://www.zfin.org. This work
covers most basic aspects of zebrafish methodology
and maintenance.
Actinobacteria
(High G+C
Gram Positive)
10B.2.33
Current Protocols in Microbiology
Supplement 3
UNIT 11A.1
Chlamydia species are obligate intracellular bacteria that require growth inside a mammalian host cell for propagation and survival. As a result, Chlamydia cannot be grown on
conventional bacteriological medium. This property makes Chlamydia difcult organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sack of
the embryonated hen egg was the only way to isolate and propagate the organism. Since
then, a tissue culture system has been available that allows easier laboratory culture and
maintenance of the Chlamydia species. The advent of tissue culture growth systems have
allowed researchers to isolate, culture, and purify large quantities of chlamydial organisms from both clinical specimens and laboratory stock cultures, and has made possible
the tremendous advancements in chlamydial research that have occurred during the last
30 years.
With the exception of lymphogranuloma venereum (LGV) serovars, most Chlamydia
trachomatis strains do not readily infect tissue culture cells. Infectivity can be enhanced
by either chemical or mechanical assistance. Basic Protocol 2 describes the procedure
used for infecting monolayer tissue culture cells with C. trachomatis by pretreating tissue
culture cells with diethylaminoethyl (DEAE)-dextran, whereas the Alternate Protocol
describes the procedure for infecting monolayer tissue culture cells by centrifugation of
the bacterial inoculum onto the host cell monolayer. Although Chlamydia can be easily
passaged in tissue culture without direct purication, most researchers purify the bacteria
away from host cell components for almost all research applications. Basic Protocol 3
describes a large-scale density gradient purication of chlamydial elementary bodies
(EBs) that have been propagated in monolayer tissue culture cells. By altering the time
that Chlamydia are harvested and the homogenization method performed, the replicative
form of Chlamydia (reticulate bodies or RBs) can also be puried away from host cell
components. Basic Protocol 4 describes the procedure for density gradient purication
of RBs. Chlamydia can be obtained from the American Type Tissue Culture (ATCC),
individual chlamydial researchers, or directly from clinical specimens. The isolation of
C. trachomatis from clinical specimens is described in Basic Protocol 1 and the procedure
for calculating the number of infectious forming units of chlamydial stocks is described
in Support Protocol 2. The procedure for determining the sterility of chlamydial stocks
is described in Support Protocol 1. Finally, Support Protocol 3 describes the procedure
for culture and passaging mammalian monolayer tissue culture cells that are needed for
growth of C. trachomatis. Monolayer tissue culture cell lines can be continually passaged
in culture and are the most common cell type used for propagating C. trachomatis in the
laboratory. HeLa 229, McCoy, and L929 are the most common cell lines used because
they are susceptible to all C. trachomatis strains. Other cell lines may be used, but the
susceptibility to C. trachomatis infection and incubation times must be determined by
each researcher.
CAUTION: When working with C. trachomatis, appropriate safeguards must be utilized in situations where aerosols can be generated (e.g., sonication and centrifugation
steps), as these procedures may pose a greater risk for laboratory infections. During
these procedures, wear protective clothing, including lab coat, gloves, safety glasses, and
mask. All procedures should be performed in a BSL-2 biosafety cabinet unless otherwise
indicated.
Chlamydiae
Contributed by Marci A. Scidmore
Current Protocols in Microbiology (2005) 11A.1.1-11A.1.25
C 2005 by John Wiley & Sons, Inc.
Copyright
11A.1.1
Materials
Freshly trypsinized McCoy cells (see Support Protocol 3)
Clinical specimens stored in chlamydial transport buffer (see recipe for chlamydial
transport buffer), 4 C
Complete medium supplemented with 10% FBS (see Critical Parameters)
supplemented with 1 g/ml cycloheximide (see recipe), 37 C
Sucrose/phosphate/glutamate buffer (SPG; see recipe)
12-mm shell vials
48-well tissue culture plates
Sonicator
5-ml sterile tubes
Ten Broek tissue homogenizers (for tissue samples only)
Low-speed bench-top centrifuge with tissue plate adaptors
Additional reagents and equipment for xing and staining cells (see Support
Protocol 1)
1. Seed freshly trypsinized McCoy cells into 12-mm shell vials and 48-well tissue
culture plates (1 vial and 1 well/sample). Add 1 ml of at least 1 105 cells/ml to
each vial. Incubate cells 24 hr or until a monolayer is formed.
Isolation and
Laboratory
Maintenance of
C. trachomatis
2a. For samples containing heavy debris or from rectal swabs: Sonicate specimens for
10 sec at a frequency that does not cause cavitation or bubble formation. Centrifuge
10 min at 175 g, 4 C. Transfer supernatant to a 5-ml sterile tube and discard pellet,
which contains cellular debris.
2b. For tissue specimens: Homogenize in a Ten Broeck tissue homogenizer and sonicate
for 20 sec at a frequency that does not cause cavitation or bubble formation.
11A.1.2
Current Protocols in Microbiology
5. Fix and stain infected cell samples in 48-well tissue culture plates with Chlamydiaspecic antisera by following Support Protocol 2, steps 7 to 13, except increase
volumes of primary and secondary antibodies to 400 l. If inclusions are present
precede to step 6.
This is done to check for inclusion formation to determine whether infectious Chlamydia
were present in the specimen.
If no inclusions are present, sonicate cells in shell vial for 20 sec, and repeat steps 3 to 5,
except inoculate entire sample onto fresh McCoy monolayer seeded in 12-mm vials. This
step is needed if original samples contain few organisms.
6. If specimen is positive, aspirate culture medium and add 1 ml SPG to each 12-mm
vial. Sonicate for 20 sec.
To maintain a stock of each clinical isolate, Chlamydia may be stored at 80 C at this
point.
At this point in the procedure, the sonicate, which contains a mixture of EBs and lysed cells,
can be either stored at 80o C for several years (see Background Information) or used to
reinfect fresh monolayers to propagate the organism. If large numbers of organisms are
needed, continue passaging the infected tissue sonicate in tissue culture cells. Increase
numbers of cells to be infected as needed.
The number of passages needed to obtain high titers is dependent on numerous factors,
including the chlamydial strain and the number of organisms in the original specimen.
BASIC
PROTOCOL 2
C. trachomatis are human pathogens and in vivo epithelial cells are the primary target cells
of Chlamydia. However, in vitro, Chlamydia can infect and replicate within both epithelial
and broblast cell lines of both human and nonhuman origin. Although numerous cell lines
are capable of supporting chlamydial growth and differentiation, those most commonly
used for culture and maintenance of C. trachomatis are McCoy, L929, and HeLa 229
cell lines. Other cell lines may be used, but should be tested for their susceptibility to
C. trachomatis infection prior to any large-scale experiment. This protocol can be used
for tissue culture cells grown in a wide variety of plastic tissue culture vessels, including
all sizes of tissue culture plates and asks. Refer to Table 11A.1.1 for guidelines to help
determine appropriate cell concentrations, and inoculation and culture volumes for each
of the commonly used tissue culture plates and asks. In this protocol, infectivity of
the non-LGV serovars is enhanced by pretreatment with DEAE-dextran. For large-scale
preparations of puried organisms, carry out this protocol prior to proceeding with Basic
Protocol 3 or Basic Protocol 4. For small-scale experiments in which tissue culture cells
are seeded onto tissue culture plates (with or without coverslips), carry out this protocol
or the Alternate Protocol.
Chlamydiae
11A.1.3
Current Protocols in Microbiology
Table 11A.1.1 Culture Conditions for Commonly Used Tissue Culture Plates and Flasks
Size of plate or
ask
Cell concentration
(cells/ml)
DEAE-dextran
(ml)
Inoculum volume
(ml)
Final culture
volume (ml)
6-well plate
4.0 105
0.5
12-well plate
2.0 10
0.35
24-well plate
1.0 10
0.5
0.2
48-well plate
4.0 10
0.35
0.15
0.81.0
1.0 10
10
2.0
4050
150-cm ask
5
5
Materials
Freshly trypsinized monolayer tissue culture cells (see Support Protocol 3)
Complete tissue culture medium (see Critical Parameters) supplemented with 10%
FBS and 10 g/ml gentamycin, 37 C
10 DEAE-dextran (see recipe)
HBSS (APPENDIX 2A)
Stock of titered C. trachomatis EBs, 80 C (see Support Protocol 2)
SPG (see recipe)
Complete medium (see Critical Parameters) supplemented with 10% FBS,
10 g/ml gentamycin, and 1 g/ml cycloheximide
Platform rocker at 37 C
Inverted phase-contrast microscope
NOTE: DEAE-dextran pretreatment of host cells (step 2) can be omitted with C. trachomatis LGV strains.
1. Dilute freshly trypsinized cells into complete medium supplemented with 10% FBS
and 10 g/ml gentamycin, and transfer into an appropriately sized plastic tissue
culture vessel. Incubate cells 24 hr.
Use culture medium that is recommended for each specic cell line.
Seed cells in an appropriately sized tissue culture vessel for each experiment. See Table
11A.1.1 for the most common tissue culture vessels used and the cell concentration needed
for each size.
11A.1.4
Current Protocols in Microbiology
3. During the DEAE-dextran treatment of the tissue culture cells, remove the frozen
stock of Chlamydia EBs from the 80 C freezer and transfer immediately to a 37 C
water bath. Gently agitate the tube continuously until EBs are thawed. To maintain
sterility, wipe the tube with 70% ethanol and store on ice until ready to use.
EBs should be thawed in <60 sec.
EBs rapidly lose viability, therefore, thaw stock immediately before use and store on ice. In
addition, do not thaw and reuse chlamydial stocks. Discard unused Chlamydia according
to appropriate biosafety protocols dictated by each institution.
5. Wash cells with 1 to 5 ml HBSS. Remove HBSS by decanting or pipetting and add
desired amount of diluted EBs to each ask. Rock the ask back and forth several
times to ensure that the inoculum covers the entire monolayer. Incubate asks on a
large platform rocker 2 hr at 37 C.
Tissue culture plates do not need to be rocked.
To prevent contamination, ensure that the caps of the tissue culture asks are tightly
closed.
If unable to rock cultures at 37 C, rock cultures 3 hr at room temperature. Inoculate LGV
serovars for 60 min at room temperature or 30 min at 37 C.
See Table 11A.1.1 for appropriate volumes of EBs used to inoculate each type of tissue
culture vessel.
6. Add appropriate volume of complete medium supplemented with 10% FBS, 10 g/ml
gentamycin, and 1 g/ml cycloheximide to each plate or ask and incubate for desired
time (Table 11A.1.2).
See Table 11A.1.1 for specic volumes of culture medium to be used for each type of tissue
culture vessel.
Incubation time varies among different C. trachomatis serovars and can range from 36 to
72 hr post infection. Infection times are only suggestions, and may need to be altered by
individual researchers depending on the cell culture system used and the rates of infections
that are achieved by each individual laboratory.
Cycloheximide inhibits host protein synthesis and gives C. trachomatis a metabolic advantage, thus resulting in higher yields of infectious organisms. The genital and ocular
serovars of C. trachomatis require cycloheximide in the culture medium, while the LGV
serovars do not. Therefore, cycloheximide can be eliminated from the growth medium
when using LGV serovars.
Because Chlamydia are bacteria, antibiotics such as penicillin and streptomycin commonly used to inhibit bacterial contamination in tissue culture cells cannot be used since
they will inhibit chlamydial development. However, Chlamydia replicate within a unique
vacuole (the inclusion) that does not fuse with endocytic or lysosomal organelles. Therefore, although gentamycin is routinely used to inhibit extracellular bacterial growth,
gentamycin-supplemented culture medium can be used when growing Chlamydia, because gentamycin cannot cross mammalian membranes and does not come into contact
with internalized Chlamydia. Although gentamycin may be internalized into mammalian
cells by pinocytosis, pinocytotic vesicles also do not fuse with chlamydial-containing
vacuoles.
Chlamydiae
11A.1.5
Current Protocols in Microbiology
Table 11A.1.2 Culture Conditions for Chlamydia trachomatis Infections of Monolayer Tissue
Culture Cells
Centrifugation or
DEAE-dextran
LGV
No
18
44
Trachoma
Yes
24
72
Biovar
a Temperature is at 37 C.
Figure 11A.1.1 Inclusion formation by C. trachomatis serovar L2. HeLa 229 monolayer cells
were infected with C. trachomatis, serovar L2 at an MOI of 1 and incubated at 37 C. At 18 and
44 hr post-infection, infected cells were xed in methanol and stained sequentially with mouse
anti-chlamydial LPS anti-sera and uorescein isothiocyanate (FITC)conjugated goat anti-mouse
antisera. Stained cells were viewed by Nomarski microscopy (A and B) and epi-uorescence
(C and D) to visualize inclusion formation. Inclusions (I) are indicated by arrows. Nucleus (N). Bar
equals 10 m.
7. Check for the infection efciency by examining inclusion formation by phasecontrast microscopy.
Isolation and
Laboratory
Maintenance of
C. trachomatis
11A.1.6
Current Protocols in Microbiology
ALTERNATE
PROTOCOL
A second method of enhancing infectivity of non-LGV C. trachomatis serovars is accomplished by centrifuging the bacterial inoculum onto the host cell monolayers. DEAEdextran pretreatment of cells as in Basic Protocol 2 and centrifugation are equivalent
methods for increasing the infectivity of non-LGV strains, and can be used interchangeably. This procedure can only be used for cells grown in shell vials or tissue culture
plates because most centrifuges cannot accommodate tissue culture asks. If using tissue
culture asks, carry out Basic Protocol 2.
2. Remove the frozen stock of Chlamydia EBs from 80 C freezer and transfer immediately to a 37 C water bath. Gently agitate the tube continuously until EBs are
thawed. To maintain sterility, wipe the tube with 70% ethanol and store on ice until
ready to use.
EBs thaw in <60 sec.
EBs rapidly lose viability, therefore, thaw stock immediately before use and store on ice. In
addition, do not thaw and reuse chlamydial stocks. Discard unused Chlamydia according
to appropriate biosafety protocols.
4. Remove culture medium from tissue culture cells and add desired amount of diluted
EBs to each well. Centrifuge tissue culture plate 60 min at 900 g, room temperature.
See Table 11A.1.I for appropriate volumes of EBs used to inoculate each type of tissue
culture plate.
To prevent laboratory contamination, use a biohazard rotor when centrifuging
C. trachomatis.
Tissue culture cells are not easily infected with the ocular and genital serovars of
C. trachomatis. To improve infection of tissue cultures with these serovars, tissue culture cells are either pretreated with DEAE-dextran (see Basic Protocol 2) or the inoculum
is centrifuged onto the monolayer.
Chlamydiae
11A.1.7
Current Protocols in Microbiology
6. Check for the infection efciency by examining inclusion formation by phasecontrast microscopy.
With experience, the percentage of infected cells can be determined by phase-contrast
microscopy. By phase-contrast or Nomarski microscopy, inclusions will appear as large
refractile vacuoles lled with numerous EBs, which sometimes can be seen moving caused
by Brownian motion (Figure 11A.1.1). LGV serovars grow much faster than trachoma
serovars. Depending on the serovar being used, inclusions will be visible 18 to 24 hr
post-infection.
BASIC
PROTOCOL 3
Materials
C. trachomatisinfected monolayer tissue culture cells (20 infected 150-cm2 asks;
see Basic Protocol 2)
HBSS (APPENDIX 2A)
70% and 95% (v/v) ethanol
95% (v/v) ethanol-soaked gauze strips
30%, 40%, 44%, and 54% (v/v) Renogran solutions (see recipe)
Disposable large cell scrapers, 3.0-cm blade width
10-ml disposable pipets, sterile
250- and 40-ml centrifuge tubes with sealing caps
Sonicator (e.g., Labsonic U Ultrasonic homogenizer with needle probe 40T,
Ultrasonic Power)
Low-speed refrigerated centrifuge (Beckman JA-14 and JA-21 rotors or equivalent)
18-G metal cannulae
10-ml disposable syringes
25 89mm ultraclear ultracentrifuge tubes (Beckman cat. no. 344058)
Ultracentrifuge with swinging bucket SW28 rotor
1.5-ml screw-cap microcentrifuge tubes
NOTE: For large-scale preparations of density gradient puried EBs, use approximately
twenty 150-cm2 asks. Although volumes are given per 150-cm2 ask, this procedure
may be scaled up or down accordingly, depending on the numbers of infectious EBs
desired.
Isolation and
Laboratory
Maintenance of
C. trachomatis
NOTE: All solutions and centrifuge tubes should be pre-chilled on ice prior to use and
kept on ice during use.
11A.1.8
Current Protocols in Microbiology
Prepare cells
1. Carefully decant culture medium from C. trachomatisinfected monolayer tissure
culture cells and add 5 ml HBSS to each 150-cm2 ask.
CAUTION: At this point in the procedure all culture medium, subsequent washes, and
plasticware will be contaminated with infectious Chlamydia. All centrifuge tubes, pipets,
and tissue culture plates will also be contaminated with infectious Chlamydia, and must
be decontaminated or disposed of appropriately according to each institutions biohazard
waste disposal procedures. Decontaminate nondisposable plasticware by soaking in a
dilute bleach solution for 1 to 2 days. Wash and autoclave for re-use.
Large-scale purications of chlamydial EBs are expensive given the large quantities of
plasticware and FBS-supplemented media required. Rather than purifying Chlamydia
from tissue culture cells grown in 150-cm2 asks, less expensive alternatives include
propagating Chlamydia in tissue culture systems, such as roller bottles or suspension
culture systems. If purifying EBs from infected cells grown in either of these two systems,
begin this procedure with step 3.
Decant infected culture medium into an appropriate waste container containing a dilute
Roccal solution to kill any Chlamydia that may be present in the culture medium. Discard
Roccal waste solution appropriately according to biohazard waste disposal protocols
specic for each institution.
2. Dislodge infected cells from each ask with a large disposable sterile cell scraper.
Pipet the cell suspension up and down several times with a sterile 10-ml pipet,
ushing the bottom of ask to ensure that all cells have been displaced from ask.
Combine and transfer cell suspension to a sterile 250-ml centrifuge tube that has
been pre-chilled on ice.
To prevent contamination of the centrifuge, use tubes with sealing caps.
Depending on the size of the preparation, different-sized centrifuge tubes may be required.
3. Sonicate the cell suspension two times for 20 sec at 50 W each. Sterilize the probe
by aming and wiping with 95% ethanol before and after each sonication.
CAUTION: To prevent aerosol contamination that may occur during the sonication process, cover the opening of the centrifuge tube with three overlapping ethanol-soaked gauze
strips.
Keep cell suspension on ice during sonication steps.
4. Centrifuge the sonicated cell suspension 15 min at 500 g (e.g., 3000 rpm in a
Beckman JA-14 rotor), 4 C. Decant the supernatant into a sterile 250-ml centrifuge
tube and store on ice.
5. Resuspend the pellet in 5 ml HBSS/150-cm2 ask. Repeat sonication and centrifugation as in steps 3 and 4. Pool the centrifuged supernatants and discard the pellet
appropriately.
The discarded pellet contains unbroken cells and nuclei.
11A.1.9
Current Protocols in Microbiology
9. Ultracentrifuge 30 min at 40,000 g (18,000 rpm in Beckman SW-28 rotor with the
brakes off), 4 C.
10. While the samples are in the ultracentrifuge, prepare discontinuous Renogran gradients containing 4 ml of 40% Renogran solution, 12 ml of 44% Renogran Solution,
and 8 ml of 54% Renogran solution in clear ultracentrifuge tubes (one gradient/about
ve 150-cm2 asks).
Make sure to carefully layer each Renogran solution over the previous layer such that
each layer is not disturbed. Visualization of a well-dened step in the gradient at each
interphase should be observed.
11. Carefully decant supernatant leaving the pellet undisturbed at the bottom of the
ultracentrifuge tube.
The pellet should be whitish in color. If it is yellowish in color and appears stringy, then
it is contaminated with cellular material and DNA.
12. Resuspend each pellet in 1 ml SPG/150-cm2 ask with an 18-G metal cannula attached to a sterile 10-ml disposable syringe. Make sure that the suspension is homogenous.
The EB pellet can be difcult to resuspend; therefore, always use a cannula to resuspend
EBs. Make sure the suspension is homogenous before proceeding.
The pellet contains partially puried EBs and is termed 30% Renogranpuried EBs or
seed stock. It can be used for passage of chlamydial stocks in culture and for isolation of
additional density gradientpuried EBs. For seed stock storage, proceed to step 17.
For most other applications, proceed with density gradient purication.
13. Carefully layer equal volumes of resuspended 30% Renogranpuried EBs over
the preformed Renogran gradients in the ultracentrifuge tube from step 10. Add
more SPG containing the 30% Renogranpuried EBs into the ultracentrifuge tube
and continue to ll ultracentrifuge tubes until 1/8 in. (3.175 mm) from the top.
Ultracentrifuge 60 min at 40,000 g (18,000 rpm in Beckman SW-28 rotor with the
brakes off), 4 C.
14. Carefully collect the white elementary body band at the 44%/54% Renogran interphase using an 18-G metal cannula attached to a sterile 10-ml syringe. Transfer
collected EBs to a sterile 40-ml centrifuge tube with sealing cap and place on ice.
Based upon the density of EBs (1.2 g/ml), EBs equilibrate at the 44%/54% interphase and
appear as a cloudy white band.
Isolation and
Laboratory
Maintenance of
C. trachomatis
Collect EBs from the top of gradient by inserting the 18-G metal cannula attached to a
sterile 10-ml syringe directly into the gradient and withdrawing the white EB band. It
may be easier to remove and discard the top part of the gradient, and then insert a clean
cannula to withdraw the EB band.
11A.1.10
Current Protocols in Microbiology
15. Dilute the density gradientpuried EBs with 10 vol sterile SPG and centrifuge
30 min at 30,000 g (15,000 rpm in JA-20 rotor), 4 C, to remove the residual
Renogran.
16. Carefully decant the supernatant and resuspend the pellet in 1 ml SPG/150-cm2 ask.
Keep on ice.
The puried EBs can be directly passaged and used to infect fresh monolayer cultures or
stored at 80 C for long-term storage.
Store EBs
17. Dispense aliquots of density puried EBs into 1.5-ml screw-cap microcentrifuge
tubes and store at 80 C.
Frozen stocks cannot be refrozen and thawed without substantial loss in viability. Therefore, depending on the intended application, storage aliquot sizes may vary from 50l to
1 ml.
Stocks kept at 80 C will retain titers for several years.
EBs will become less infectious after storage. Therefore, to ensure an accurate titer, titer
EBs after they have been stored at 80 C (see Support Protocol 2).
SUPPORT
PROTOCOL 1
Materials
BHI blood agar plates (see recipe), 37 C
Puried chlamydial organisms
1. Prepare BHI blood agar plates and pre-warm to 37 C.
Blood agar plates permit the growth of most bacterial species.
2. Streak an aliquot of puried chlamydial organisms onto blood agar plates and incubate
24 hr at 37 C.
3. Check for bacterial growth on plates.
Chlamydia will not grow on blood agar plates. Therefore, if the preparation is sterile, there
will be no microbial growth on plates. If the preparation is contaminated, then bacterial
growth, either individual colonies or swarming, will be present on plates. If contaminated,
discard EBs.
SUPPORT
PROTOCOL 2
For most applications, the precise number of infectious forming units (IFU) is required.
The most common way to calculate IFU is by quantifying the number of inclusions
formed from a given amount of starting material. This method is accurate if cells are
infected at an MOI of 1, since a single infectious EB gives rise to a single inclusion.
The easiest method to quantify inclusions is by indirect immunouorescence using an
antibody specic to C. trachomatis. This procedure is used to quantitate the number of
IFUs present in density gradientpuried organisms (see Basic Protocol 3) or in amplied
Chlamydiae
11A.1.11
Current Protocols in Microbiology
stocks of clinical specimens (see Basic Protocol 1). It is also used before proceeding with
Basic Protocol 2 or the Alternate Protocol in order to accurately infect cells with a desired
MOI. If desired, this procedure, starting at step 7, can also be used to count inclusions
formed upon completion of Basic Protocol 1, Basic Protocol 2, or the Alternate Protocol.
Since RBs are noninfectious, puried RBs (see Basic Protocol 4) cannot be counted using
this procedure.
Materials
Freshly trypsinized monolayer tissue culture cells (see Support Protocol 3)
Complete tissue culture medium (see Critical Parameters) supplemented with 10%
FBS, 10 g/ml gentamycin, and 1 g/ml cycloheximide, 37 C
Frozen stocks of C. trachomatis EBs (see Basic Protocol 3) or infected cells (see
Basic Protocol 2 or Alternate Protocol)
HBSS (APPENDIX 2A)
70% (v/v) ethanol
SPG (see recipe)
100% methanol
PBS (APPENDIX 2A)
PBS supplemented with 3% (w/v) BSA (see recipe)
Primary antibody specic to C. trachomatis (e.g., Abcam or Chemicon)
Fluorophore-conjugated secondary antibody
24-well tissue culture plates
Biohazard swinging-bucket rotor adapted for microtiter plates (e.g., Sorvall
biohazard microtiter rotor, cat. no. 75006-444)
12-mm-diameter coverslips (no. 1 thickness), optional
37 C water bath
Inverted epi-uorescence microscope with appropriate lter sets
Hand-held counter
Prepare cells
1. Seed monolayer tissue culture cells in 24-well tissue culture plates containing 1 ml
complete tissue culture medium supplemented with 10% FBS, 10 g/ml gentamycin,
and 1 g/ml cycloheximide, at a density of 13 105 cells per well. Incubate 24 hr.
To ensure accurate results, perform titer in triplicate. For each EB stock preparation to
be titered, seed eighteen wells.
HeLa 229 or L929 broblasts are commonly used to titer C. trachomatis EBs.
If an inverted epi-uorescence microscope equipped with long-working distance objectives
is unavailable, culture cells on 12-mm diameter glass coverslips (no. 1 thickness) in 24well tissue culture plates.
2a. For isolated EBs: Thaw an aliquot of EBs in a 37 C water bath. Agitate continuously
until EBs are completely thawed. Keep EBs on ice.
EBs should be completely thawed in <60 sec.
To maintain sterility, wipe the storage tube with 70% ethanol before proceeding.
EBs rapidly lose viability, therefore, thaw stock immediately before use and store on ice. In
addition, do not thaw and reuse chlamydial stocks. Discard unused Chlamydia according
to appropriate biosafety protocols dictated by each institution.
Isolation and
Laboratory
Maintenance of
C. trachomatis
2b. For infected cell monolayers: Grow infected cells in 24-well plates. After 44-hr postinfection, wash cells one time in HBSS, and then lyse each well with 250 l sterile
water. Incubate cells 5 min at room temperature and pipet up and down several times
to ensure complete lysis.
11A.1.12
Current Protocols in Microbiology
3. Make ten-fold serial dilutions of EBs (step 2a) or infected cell lysates (step 2b) in
SPG or HBSS (101 , 102 , 103 , 104 , 105 , and 106 ). Make at least 1 ml of each
dilution. Keep on ice.
SPG or HBSS may be used interchangeably in this step.
4. Remove culture medium from monolayers and add 200 l of each dilution to three
separate wells.
5. Centrifuge tissue culture plates 60 min at 900 g, room temperature, in a biohazard
swinging bucket rotor adapted for microtiter plates.
A biohazard rotor (e.g., Sorvall) is used to prevent bacterial contamination of the
centrifuge.
If a centrifuge or rotor for microtiter plates is unavailable, cells can be pretreated with
45 g/ml DEAE-dextran for 15 min prior to infection (see Basic Protocol 2).
If titering LGV strains, remove culture medium and inoculate 200 l of each dilution
directly onto monolayers. Incubate cells for 30 to 60 min and proceed to step 6.
7. Remove infected culture medium and wash cells with HBSS two times.
Dispose of infected culture medium appropriately.
8. Remove the wash solution and add 1 ml of 100% methanol to each well. Incubate
10 min at room temperature. Remove methanol and wash the cells three times in
PBS. For each wash, add 1 ml PBS, incubate 5 min at room temperature, and then
remove PBS. Add 1 ml PBS containing 3% BSA to xed cells and incubate for 30 to
60 min to block nonspecic sites of antibody binding. Store up to several days at 4 C.
Do not let cells dry out at any time during the procedure.
Fixation conditions differ between different antibodies. If using a commercial antibody,
follow manufacturers recommendations for xation conditions.
Once infected cells are xed, they are no longer considered infectious. Therefore, the
remaining procedures can be performed on the bench top.
Chlamydiae
11A.1.13
Current Protocols in Microbiology
11. Remove primary antibody from each sample and wash with PBS three times. For
each wash, add 1 ml PBS, incubate 5 min, and remove PBS.
12. Add 200 l of diluted secondary antibody to each well and incubate for at least
60 min at room temperature with rocking.
Keep samples away from direct light to prevent uorophore from bleaching.
13. Remove secondary antibody and wash with PBS three times. For each wash, add
1 ml PBS, incubate 5 min, and remove nal PBS wash. Store xed and stained cells
in PBS at 4 C.
Fixed and washed cells can be stored in PBS at 4 C for several days without loss of
uorescence.
If cells were cultured on glass coverslips (see step 1), remove coverslips from tissue
culture plate with forceps and 18-G needle, rinse by submerging in beaker of water,
and dry coverslip by wicking with a Kimwipe. Place coverslip, cell-side down, on drop
of antifading agent on a glass slide. Slides can be viewed with an inverted or upright
epi-uorescence microscope.
14. To calculate the number of infectious forming units (IFU), view xed and stained
cells with an inverted conventional epi-uorescence microscope using appropriate
uorescence lter set for the specic uorophore-conjugated secondary antibody
that was used.
15. Using a hand-held counter, count all inclusions, which will appear as large uorescent vacuoles, in each eld (see Figure 11A.1.1). Count at least 15 elds per each
dilution.
Count appropriate dilution so that there are 30 to 100 inclusions in each eld.
16a. To determine the infectious forming units: Calculate the IFUs using the following
equation:
where
and where
and where
16b. To determine the titer: Calculate the titer using the following equation:
Isolation and
Laboratory
Maintenance of
C. trachomatis
11A.1.14
Current Protocols in Microbiology
BASIC
PROTOCOL 4
Materials
Infected cells in 150-cm2 asks (see Basic Protocol 2)
10 K-36 buffer (see recipe)
Large cell scraper
250-ml centrifuge tube
40-ml Dounce homogenizer with a tight-tting pestle
Glass microscope slides and coverslips
Phase-contrast microscope
Beckman JA-14 rotor or equivalent
Additional reagents and equipment for density gradient purication (see Basic
Protocol 3)
1. Harvest infected cells in 150-cm asks 18 to 24 hr post-infection. Dilute 10 K-36
buffer 1:10 in sterile water to make a nal concentration of 1. Add 5 ml of 1 K-36
buffer to each 150-cm2 ask. Dislodge cells using a large cell scraper and transfer to
a 250-ml centrifuge tube. Store on ice.
Infected cells need to be harvested before infectious EBs are produced. Since the length
of the developmental cycle varies between strains, harvest times should be determined
empirically for each different chlamydial strain. Typically, LGV RBs are harvested at
18 hr post-infection while non-LGV RBs are harvested 24 hr post-infection.
2. On ice, transfer 30 to 40 ml of lysate into a 40-ml Dounce homogenizer and homogenize the lysate with 15 to 30 strokes using a tight-tting pestle. Every ve to ten
strokes, transfer a small drop of lysate onto a glass slide and cover with a coverslip.
View cellular lysate under a phase-contrast microscope to monitor cell lysis. Continue homogenizing until >90% of cells are lysed with the nuclei still intact. Transfer
lysate to a 250-ml centrifuge tube. Repeat homogenization step with remaining lysate
until it is homogenized.
CAUTION: Do not create any aerosols during homogenization procedure.
3. Combine all homogenized lysate into a 250-ml centrifuge tube with sealing cap and
centrifuge 15 min at 500 g (3000 rpm in a Beckman JA-14 rotor), 4 C. Transfer
supernatant into a clean 250-ml centrifuge and store on ice. Resuspend pellet in
30 ml K-36 buffer and transfer into Dounce homogenizer. Repeat homogenization
with another 20 strokes. Transfer into a new 250-ml centrifuge tube and centrifuge
15 min at 500 g, 4 C.
Chlamydiae
11A.1.15
Current Protocols in Microbiology
4. Pool the supernatants and centrifuge 30 min at 30,000 g (14,000 rpm in Beckman
JA-14 rotor), 4 C. Discard supernatant and resuspend pellets in 30 ml K-36 buffer.
5. Continue with Basic Protocol 3, step 8, and layer homogenate onto 30% Renogran
cushions. Proceed with remainder of Basic Protocol 3 except substitute K-36 buffer
for SPG.
6. Check purity of RB preparation by transmission electron microscopy (UNIT 2B.1).
Remember that RBs are not infectious and cannot be used to infect cells. If the preparation
is a pure population of RBs with no EB contamination, no inclusions should be formed if
the puried RBs were used to infect host cells.
RBs can be distinguished from EBs based upon the following parameters: (1) RBs are
1 m, while EBs are 0.5 m and (2) RBs have a decondensed nucleoid structure
as opposed to EBs, which contain a highly condensed nucleoid structure that appears
electron dense.
SUPPORT
PROTOCOL 3
Materials
Frozen immortalized tissue culture cell lines in liquid nitrogen: HeLa 229 (ATCC
no. CCL-2.1), McCoy (ATCC no. CRL-1696), or L929 cell lines (ATCC no.
CCL-1)
70% (v/v) ethanol
Complete medium (see Critical Parameters) supplemented with 10% FBS and
10 g/ml gentamycin, 37 C
HBSS (APPENDIX 2A) without Ca2+ and Mg2+
Trypsin/EDTA solution (see recipe), 37 C
37 C water bath
15-ml sterile centrifuge tubes
75-cm2 tissue culture asks
Inverted phase-contrast microscope
Hemacytometer slide with coverslip
Light microscope
Hand-held cell counter
Thaw and recover frozen monolayer tissue culture cell lines
1. Remove frozen tissue culture cells from liquid nitrogen freezer and immediately
place vial into a 37 C water bath. Agitate continuously until vial is thawed.
Cells will usually thaw in <60 sec.
Thaw cells as quickly as possible to prevent lysis of cells that may occur due to ice crystal
formation.
Isolation and
Laboratory
Maintenance of
C. trachomatis
To maintain sterility of cells, avoid water droplets near the vial top.
11A.1.16
Current Protocols in Microbiology
2. To ensure sterility of cells, wipe top of the vial with 70% ethanol before opening.
Transfer the thawed cell suspension into a sterile 15-ml centrifuge tube containing 10 ml prewarmed complete medium containing 10% FBS supplemented with
10 g/ml gentamycin. Centrifuge 5 min at 200 g, room temperature. Carefully
remove supernatant with pipet, resuspend cell pellet in 1 ml complete medium containing 10% FBS supplemented with 10 g/ml gentamycin, and transfer to 75-cm2
tissue culture ask containing 25 ml of complete medium containing 10% FBS supplemented with 10 g/ml gentamycin.
Cells must be pelleted and fresh medium must be added to remove DMSO, which is present
in the freezing medium, before cells can be plated and cultured.
7. Incubate ask 5 to 10 min. Tap bottom of ask on countertop to detach cells. Check
cultures with an inverted phase-contrast microscope to make sure that all cells are
rounded up and detached from the surface. If not all of the cells are detached, continue
incubating ask an additional 1 or 2 min.
8. Add 5 ml prewarmed complete medium containing 10% FBS and 10 g/ml gentamycin to each ask containing trypsinized cells. Draw up cell suspension into
a sterile pipet and rinse cell layer two to three times to dissociate any remaining
adherent cells.
Determine cell number
9. Carefully clean the surface of a hemacytometer slide and coverslip with 70% ethanol.
Use only a specially designed hemacytometer coverslip.
A hemacytometer slide is needed to accurately count the number of cells. Each hemacytometer slide contains two separate counting chambers with etched 3 3mm grids.
Each grid is divided into nine 1-mm squares. The four corner and the central squares are
used for counting.
10. Use a sterile pipet to transfer cell suspension (from step 8) to the edge of each
hemacytometer counting chamber by holding the tip of the pipet under the coverslip
and dispersing one drop of suspension. Allow cells to settle for several minutes before
counting.
Chlamydiae
11A.1.17
Current Protocols in Microbiology
11. View hemacytometer slide under a light microscope. Use an appropriate objective
lens such that the central area of the grid lls the entire microscope eld.
12. Count the cells in all four corners and the central square with a hand-held cell counter.
Count the cells that are touching the middle line of the triple line on the top and left of
each square. Do not count the cells touching the middle line of the bottom or right side of
each square.
where 104 is the correction factor for the volume of cells counted.
Each square is 1 1 mm, with a depth of 0.1 mm.
Isolation and
Laboratory
Maintenance of
C. trachomatis
DEAE-dextran, 10
180 mg DEAE-dextran (450 g/ml nal concentration)
Adjust volume to 400 ml with H2 O
Filter sterilize through a 0.2-m lter
Dilute 1:10 for pretreatment of tissue culture monolayers (45 g/ml nal concentration)
Store up to 1 year at room temperature
11A.1.18
Current Protocols in Microbiology
K-36 buffer, 10
87.1 g K2 HPO4
68.0 g KH2 PO4
74.5 g KCl
8.7 g NaCl
H2 O to 1 liter
Adjust pH to 7.0 with 1 M KOH
Autoclave 20 min
Store up to 1 year at 4 C
PBS supplemented with 3% (w/v) BSA
Dissolve 3 g BSA into 100 ml PBS (APPENDIX 2A). Filter sterilize using a low-protein
binding 0.22-m lter. Store indenitely at 20 C.
Renogran solutions, 30%, 40%, 44%, 54%
Prepare the following in a biological safety cabinet to maintain sterility:
30, 40, 44, or 54 ml Renocal-76 or Hypaque-76 (for 30%, 40%, 44%, or 54%
solutions, respectively)
10 ml 10 K-36 buffer (see recipe)
Adjust volume to 100 ml with sterile H2 O
Store up to 6 months at 4 C
RenoCal-76 (E.R. Squibb and Sons) or Hypaque-76 (Nycomed) are the two most common
sources of Renogran and they are supplied as sterile 76% solutions containing a 66%
solution of diatrizoate meglumine and a 10% solution of diatrizoate sodium. Use directly
from bottle when making dilutions.
Sucrose-phosphate-glutamate buffer
75 g sucrose
87 ml 0.2 M Na2 HPO4 (dibasic sodium phosphate)
13 ml 0.2 M NaH2 PO4 (monobasic sodium phosphate)
0.72 g L-glutamic acid
H2 O to 1 liter
Adjust pH to 7.4 with 2 N NaOH if necessary
Aliquot to 100-ml bottles and autoclave for 20 min
Store up to 1 year at 4 C
Trypsin/EDTA solution
0.25 g trypsin
0.2 g EDTA (disodium ethylenediamine tetraacetic acid)
H2 O to 100 ml
Filter sterilize
Store indenitely at 20 C
Various concentrations of Trypsin/EDTA solutions are readily available from commercial
manufacturers. The two most common sources are Invitrogen (GIBCO) and Mediatech.
COMMENTARY
Background Information
Maintenance of C. trachomatis
C. trachomatis are obligate intracellular
bacteria that replicate within a nonacidied
membrane-bound vacuole termed an inclusion (Moulder, 1991). Within the inclusion, all
Chlamydiae
11A.1.19
Current Protocols in Microbiology
Isolation and
Laboratory
Maintenance of
C. trachomatis
11A.1.20
Current Protocols in Microbiology
Chlamydiae
11A.1.21
Current Protocols in Microbiology
Lymphogranuloma
venereum
(LGV,
Serovars L1, L2, and L3). The LGV serovars
also infect the mucosal surfaces of the
genital tract, but in contrast to the trachoma
biovars, which remain localized to mucosal
surfaces, the LGV serovars are more invasive
and inltrate the submucosa resulting in
lymphatic involvement (Schachter, 1999).
Although infections by the LGV serovars
have been virtually eliminated in the U.S.,
they are still prevalent in tropical areas of
Africa, Central America, and Asia. The
primary lesions develop on the penis, labia,
vagina, and cervix, and may result in a mild
proctitis. Left untreated, secondary lesions
consisting of lymphoadenopathy may form.
Within the nodes, abscesses form and may
rupture resulting in chronic draining stulas.
At this stage, the abscesses may disappear or
the disease may become systemic, resulting
in fever, myalgia, and headache. In some
cases, chronic inammation and scarring
may occur. Diagnosis is made following
isolation of the organism from the primary
lesion or from aspirated pus, or by serological
assays such as the microimmunouorescence
tests.
Critical Parameters
Handling organisms
If beginning with clinical specimens, make
sure that specimens are appropriately collected
in chlamydial transport buffer and stored at
4 C. Chlamydia must be isolated from specimens within 24 hr post-collection. If beginning
with frozen stocks of puried organisms, make
sure to store organisms at 80 C immediately
upon receipt and only thaw organisms immediately before use, otherwise, a loss in viability will result. C. trachomatis are considered
BSL-2 organisms and must be handled according to appropriate federal and local biosafety
standards (UNIT 1A.1). Wear appropriate protective clothing (gloves, laboratory coat, protective eye glasses) when handling the organism.
In addition, wear a mask when sonicating infected cell lysates.
Isolation and
Laboratory
Maintenance of
C. trachomatis
propagation of C. trachomatis species are McCoy and L929 (mouse broblast), and HeLa
229 (human epithelial) cell lines. Monolayer
cell cultures are the most common cell type
used, however, L929 and HeLa S3 suspension tissue culture cells have been used successfully for at least some non-LGV C. trachomatis strains. For initial isolation of clinical specimens, McCoy cells are almost exclusively used. Use culture medium appropriate
for each cell type. Common cell culture media include RPMI 1640 and minimal essential
medium (MEM), both supplemented with 10%
FBS. Media are most commonly purchased as
either a liquid or a powder.
Culture vessel selection
Chlamydia can be cultured in cells grown in
almost any type of plastic tissue culture vessel
and the choice of vessel depends entirely on
the application and the numbers of organisms
needed. For initial isolation of clinical specimens, shell vials are used. No matter what
type of vessel is used, the protocols are identical except for cell numbers and volumes. Refer
to Table 11A.1.1 for appropriate cell numbers,
inoculation, and culture volumes used for the
different types of vessels.
Infection times
The infection or incubation period is not
an absolute number and depends on numerous parameters, including the specic C. trachomatis serovar or strain being used, the MOI
being used to infect the cells, the efciency
of infection, and the specic application. Approximate incubation times for large-scale purication of EBs and RBs are given in Table
11A.1.2. Note that these are just approximate
times and the actual times may need to be experimentally determined by each researcher.
To passage Chlamydia, they must have completed their developmental cycle so that sufcient numbers of infectious EBs will have been
formed. Remember, RBs, which are formed
during the middle of the developmental cycle,
are not infectious and cannot be used to propagate the organism. When growing Chlamydia
to be passaged into fresh monolayers or to be
puried as stock EBs, carefully monitor the
infected cells by phase-contrast microscopy.
Collect the cells when the inclusions are large
and are practically lling the entire cytoplasm
of the host cell. At this stage in the developmental cycle, the inclusions will be easily visible by phase-contrast microscopy. Do not let
the infection continue too long as this may result in premature host cell lysis.
11A.1.22
Current Protocols in Microbiology
Problem
Possible cause
Solution
No inclusion formation
Incorrect titer
Incorrect titer
Chlamydiae
11A.1.23
Current Protocols in Microbiology
Table 11A.1.3 Troubleshooting Guide for Laboratory Maintenance of Chlamydia trachomatis, continued
Problem
Possible cause
Solution
No inclusions detected by
indirect
immunouorescence
Troubleshooting
Table 11A.1.3 provides many of the common problems encountered with the methods
detailed in this unit, as well as their possible
causes and solutions.
Anticipated Results
From a large-scale density gradient preparation of C. trachomatis EBs, titers ranging
from 1 108 to 1 1010 IFU/ml are usually obtained. Generally, higher titers of C. trachomatis LGV EBs are obtained compared to
non-LGV EBs.
Time Considerations
Isolation and
Laboratory
Maintenance of
C. trachomatis
Literature Cited
Caldwell, H.D., Kromhout, J., and Schachter, J.
1981. Purication and partial characterization of
the major outer membrane protein of Chlamydia
trachomatis. Infect. Immun. 31:1161-1176.
Moulder, J.W. 1991. Interaction of chlamydiae and
host cells in vitro. Microbiol. Rev. 55:143-190.
Schachter, J. 1999. Infection and disease epidemiology. In Chlamydia: Intracellular Biology, Pathogenesis, and Immunity (R.S. Stephens, ed.)
pp. 139-169. American Society for Microbiology Press, Washington, D.C.
Key References
Phelan, M.C. 1998. Basic techniques for mammalian cell tissue culture. In Current Protocols
in Cell Biology (J.S. Bonifacino, M. Dasso, J.
Lippincott-Schwartz, and K.M. Yamada, eds.)
Unit 1.1. John Wiley and Sons, Inc., New York.
This is an excellent resource for understanding the
basic techniques of mammalian cell tissue culture.
11A.1.24
Current Protocols in Microbiology
Internet Resources
http://www.chlamydiae.com
Chlamydiae
11A.1.25
Current Protocols in Microbiology
UNIT 12.1
ABSTRACT
Hydrophobic antigen tissue Triton extraction (HATTREX) provides a method to extract
and identify hydrophobic bacterial membrane proteins from host-infected tissues. The
non-ionic detergent Triton X-114 is used to solubilize host-infected tissues at 4 C.
Subsequent phase partitioning of Triton X-114 extracted material at 37 C results in a
detergent poor aqueous phase and a detergent rich detergent phase. While soluble
proteins partition to the aqueous phase, hydrophobic proteins, such as proteins of the
outer and inner membranes, can be found in the detergent phase. Characterization of
the detergent phase sample provides insights into the proteome of bacterial membranes
C 2008 by John Wiley &
during infection. Curr. Protoc. Microbiol. 9:12.1.1-12.1.5.
Sons, Inc.
Keywords: Triton X-114 r in vivo r hydrophobic r membrane r antigen
Although several pathogenic species of spirochetes can be cultured in vitro, the proteome
of in vitro cultivated spirochetes often differs significantly from the proteome expressed
during the course of an infection. This unit describes a methodology to provide samples
enriched for the membrane proteomes of spirochetes during infection. Membrane proteins
of the infecting spirochete can be preferentially extracted from infected mammalian
tissues due to the hydrophobic nature of most outer membrane proteins and the phase
partitioning properties of the non-ionic detergent Triton X-114 at 37 C. Infected host
tissues are triturated into a suitable buffer containing Triton X-114 at 4 C. After extraction
for a suitable length of time and removal of nonsoluble material, extracted samples
are incubated at 37 C to induce phase partitioning and generate a detergent rich
detergent phase and detergent poor aqueous phase. The more hydrophobic proteins
of the bacterial membranes are recovered in the detergent phase, which can then be
processed for further analyses, including separation by 1-D or 2-D SDS-PAGE and
immunoblotting (Crother et al., 2003, 2004; Nally et al., 2007). The HATTREX procedure
could potentially be adapted for analysis of other types of bacteria.
BASIC
PROTOCOL
CAUTION: Follow all biosafety requirements relevant to the microorganism under investigation. Refer to UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for instructions
on safe handling of microorganisms.
Materials
Tissue to be processed
Tissue suspension buffer (see recipe)
Triton X-114 (protein grade, Calbiochem)
Spirochetes
Current Protocols in Microbiology 12.1.1-12.1.5, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc1201s9
C 2008 John Wiley & Sons, Inc.
Copyright
12.1.1
Supplement 9
Trichloroacetic acid
Acetone
1-D SDS-PAGE solubilization buffer (see recipe)
2-D SDS-PAGE solubilization buffer (see recipe)
PowerGen 125 tissue grinder (Fisher Scientific) or similar device
Vortex
Shaking incubator
37 C water bath
Day 1
Tissue solubilization
1. Weigh tissue to be processed.
2. In an appropriately sized tube, add tissue suspension buffer to a concentration of
50 mg tissue per ml of buffer. Using a tissue grinder, triturate the tissue as rapidly as
possible according to the manufacturers instructions.
If tissue type contains connective tissues, or skin, dicing into small pieces will aid in the
grinding process. Ideally, fresh tissue should be used but similar results are generated
using tissue which is snap frozen in a dry ice-ethanol bath at time of procurement and
stored at 80 C until use.
3. Grind tissue until a homogeneous mixture is achieved, using caution not to cause
foaming of the sample.
CAUTION: If working with BSL-2 or higher organisms, appropriate precautions should
be taken. Always perform grinding in a Biosafety hood and wear protective outerwear,
including gloves, labcoat, a mask, and safety glasses. For more information refer to
UNIT 1A.1.
Do not overheat the sample. Incubate on ice between grindings.
Different tissue types will vary in their ability to be triturated. Tissues such as skin can form
large clumps during the grinding process. In addition to dicing the tissue into small pieces,
increasing the volume of the tissue suspension buffer can aid in the grinding process.
6. Incubate the sample in a 37 C water bath for 10 min to separate the detergent and
aqueous phases.
Analysis of
Bacterial
Membrane
Proteins Produced
During
Mammalian
Infection Using
HATTREX
The sample should become cloudy and the two phases will become visible with time. If
this is not the case, incubate the sample for another 10 min, as the larger the volume, the
longer the incubation time.
7. Centrifuge the sample 15 min at 3400 g, room temperature to separate the detergent
and aqueous phases. Remove aqueous phase.
The detergent phase will be the lower phase. If phases do not separate, try a longer
incubation at 37 C as described above or add more ice-cold tissue suspension buffer with
1% to 3% Triton X-114. Vortex briefly and repeat from step 6.
12.1.2
Supplement 9
8. Add ice-cold solubilization buffer (equal to the volume of aqueous phase removed)
to the detergent phase and vortex until the solution is homogeneous. This will
remove contaminating aqueous phase proteins. Proceed from step 6. Repeat this
wash procedure twice.
10. Pellet the precipitate by centrifuging 30 min at 16,000 g, 4 C. Carefully pour off
the supernatant without disturbing the pellet and wash the precipitate with 3 vol of
20 C acetone.
11. Pellet the precipitate by centrifuging 30 min at 16,000 g, 4 C. Carefully pour off
the supernatant without disturbing the pellet and allow the protein precipitate to air
dry 5 min.
Overdrying results in a pellet which is more difficult to resuspend.
12. Resuspend the protein pellet in an appropriate amount of 1-D or 2-D SDS-PAGE
solubilization buffer, depending on your needs.
The protein pellet can be difficult to solubilize and may require overnight incubation.
Spirochetes
12.1.3
Current Protocols in Microbiology
Supplement 9
Analysis of
Bacterial
Membrane
Proteins Produced
During
Mammalian
Infection Using
HATTREX
The success of the HATTREX process depends on several factors. HATTREX enriches
for the antigenic hydrophobic proteins and allows these proteins to be analyzed by SDSPAGE. However, in order to visualize these
proteins, the bacterial presence in the tissue
has to be of a sufficient quantity. The flip side
of this parameter is the relative antigenicity of
each protein. Finally, the quality of the antisera used to identify the individual antigens is
extremely important due to the fact that the
majority of proteins extracted by this process
are of host origin.
Another important factor in the HATTREX
procedure is the detergent to protein ratio. The
amounts presented in this protocol are a general guideline due to the varying nature of different tissue types. Varying the solubilization
volume or detergent concentration may be required to achieve a successful extraction and
phase separation.
While this protocol uses TCA-acetone
to precipitate the hydrophobic proteins, alternative precipitation procedures, such as
chloroform-methanol precipitation, can be
employed which can result in recovery of different proteins (Wessel and Flugge, 1984).
Identification of antigens in the sample
can be achieved using several methods. Immunoblotting with monospecific sera can be
used to look for the presence of a specific protein. Alternatively, more complex sera, such as
serum from infected patients, can be used to
identify a range of antigens. Two-dimensional
SDS-PAGE of the detergent phase sample pro-
Anticipated Results
Results will depend on several parameters
including the numbers of spirochetes found in
host tissue of interest, the relative antigenicity of individual proteins, and the quality of
the antisera. Immunoblotting provides an additional level of sensitivity. In rabbits and SCID
mice infected with the Lyme disease spirochete, Borrelia burgdorferi, antigens were detected in tissues which harbored as few as 104
to 105 B. burgdorferi per microgram of host
DNA (Crother et al., 2003, 2004).
In addition, this protocol can be used to detect antigenic proteins in the aqueous phase of
the sample preparation generated as described.
However, these samples often contain large
amounts of host proteins, making detection of
spirochete proteins more difficult.
Time Considerations
This protocol involves an overnight incubation. Day 1 will involve minimal time of up to
30 min. Day 2 can vary but will ideally require
4 to 5 hr. Precipitated protein pellets can be
stored at 20 C indefinitely.
Literature Cited
Barbour, A.G. and Hayes, S.F. 1986. Biology of
Borrelia species. Microbiol. Rev. 50:381-400.
Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol.
Chem. 256:1604-1607.
Brandt, M.E., Riley, B.S., Radolf, J.D., and
Norgard, M.V. 1990. Immunogenic integral
membrane proteins of Borrelia burgdorferi are
lipoproteins. Infect. Immun. 58:983-991.
Brusca, J.S. and Radolf, J.D. 1994. Isolation of integral membrane proteins by phase partitioning
with Triton X-114. Methods Enzymol. 228:182193.
Cox, D.L., Chang, P., McDowall, A.W., and Radolf,
J.D. 1992. The outer membrane, not a coat
of host proteins, limits antigenicity of virulent
Treponema pallidum. Infect. Immun. 60:10761083.
Crother, T.R., Champion, C.I., Wu, X.Y., Blanco,
D.R., Miller, J.N., and Lovett, M.A. 2003.
Antigenic composition of Borrelia burgdorferi
during infection of SCID mice. Infect. Immun.
71:3419-3428.
12.1.4
Supplement 9
Spirochetes
12.1.5
Current Protocols in Microbiology
Supplement 9
UNIT 12A.1
ABSTRACT
The spirochetal bacteria that cause syphilis, yaws, and bejel cannot be cultivated in vitro.
This unit describes methods for the isolation of subspecies of Treponema pallidum and
other pathogenic treponemes from clinical specimens, the propagation of these isolates
in rabbits, isolation of clonal populations of T. pallidum, and techniques for maintenance
C
of frozen stocks of these treponemes. Curr. Protoc. Microbiol. 7:12A.1.1-12A.1.18.
2007 by John Wiley & Sons, Inc.
Keywords: syphilis r treponeme r rabbit r chancre r cerebrospinal fluid
INTRODUCTION
Treponema pallidum is comprised of three subspecies: T. pallidum subsp. pallidum,
which causes syphilis; T. pallidum subsp. pertenue, which causes yaws; and T. pallidum
subsp. endemicum, which causes bejel or endemic syphilis. These organisms all cause
human infection and cannot be propagated in vitro; experimental infection of rabbits is
the preferred method for propagation of these subspecies. In addition to these treponemes,
T. paraluiscuniculi causes a naturally occurring infection in rabbits, is not considered
to be infectious for humans (Graves and Downes, 1981), and is closely related to the
human treponemes described above. Another closely related treponeme, referred to as
the Fribourg-Blanc (or simian) treponeme, was isolated from a baboon in Guinea and is
propagated in rabbits (Fribourg-Blanc et al., 1963).
Because of the importance of syphilis to human health, considerable effort has been placed
on the cultivation of T. pallidum subsp. pallidum in vitro. Although limited replication
was accomplished in a tissue culture system 25 years ago (Fieldsteel et al., 1981; Norris, 1982), continuous culture has not been accomplished, and isolates must still be
propagated in the rabbit. Routine propagation is accomplished by intratesticular passage, although rabbits can also be infected by intradermal, intravenous, and intracisternal
routes. Similarly, isolation of new strains of pathogenic treponemes is accomplished by
inoculation of clinical material (e.g., chancre exudates, cerebrospinal fluid, and blood)
intratesticularly into rabbits (Turner et al., 1969). This unit describes methods for isolation, propagation, and maintenance (Basic Protocols 1, 2, 3, and 4) of these species of
treponemes, hereafter referred to collectively as noncultivable treponemes. A Support
Protocol provides a dark-field microscopy method for quantifying the organisms. Additionally, this unit describes a method for the isolation of individual clones of treponemes
in the rabbit (Basic Protocol 5).
CAUTION: Treponema pallidum and related treponemes are Biosafety Level 2 (BSL-2)
pathogens. Follow all appropriate guidelines and regulations for the use and handling of
pathogenic microorganisms. Review the Biosafety portion of Strategic Planning carefully
prior to undertaking these procedures. Also see UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
CAUTION: These experiments require Animal Biosafety Level 2 (ABSL-2) conditions.
Follow all appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1
Current Protocols in Microbiology 12A.1.1-12A.1.18, November 2007
Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc12a01s7
C 2007 John Wiley & Sons, Inc. All rights reserved.
Copyright
Spirochetes
12A.1.1
Supplement 7
1B)
CAUTION: Personal protective equipment (gown, mask, eye protection, gloves) should
be worn whenever there is risk of coming in contact with infected materials. See Biosafety
(below) for more details.
IMPORTANT NOTE: T. pallidum does not survive for long periods outside of a host.
Specimen preparations must be injected into the rabbit as rapidly as possible, at a
maximum of 1 hour after collection. See Materials (below) for more details.
NOTE: Reagents and equipment should be ready in advance because rapid harvesting
and re-inoculation of treponemes is essential for optimal survival and, thus, yield of
organisms. See Materials (below) for more details.
NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) and conform to governmental
regulations regarding the care and use of laboratory animals.
NOTE: All solutions and reagents must be sterile.
STRATEGIC PLANNING
Biosafety
The noncultivable treponemes require BSL-2 laboratory and animal care facilities. All
personnel handling T. pallidum or related organisms, including personnel caring for
infected animals, should be carefully trained in such precautions. All personnel should
wear appropriate personal protective equipment (PPE) including laboratory coat or gown,
gloves, and eye splash protection. Masks are suggested for minimization of allergen
exposure during handling of rabbits. Spontaneous aerosolization of T. pallidum from
suspensions does not occur, although spraying and splashing of organisms from syringes
is possible if a needle disengages from a syringe during injection. Disposable Luer-lok
needles and syringes must be used for all injections, and recapping or removal of needles
must be avoided.
T. pallidum infections of laboratory personnel can occur by accidental parenteral inoculation (needlestick) or by spillage or splashing of suspensions of treponemes (or infected
rabbit or human blood and cerebrospinal fluid) on mucosal surfaces or open skin; these
infections are not transmitted through intact skin. In case of suspected or known accidental exposure, laboratory personnel should be treated prophylactically with an antibiotic
regimen known to be effective for incubating syphilis, and the individual should be
monitored serologically (RPR or VDRL test; Larsen, 1999) at the time of treatment and
every three months for one year. A prophylactic treatment protocol should be developed
in advance of any such occurrence so that treatment is available immediately and at all
hours. All persons handling infectious treponemes, even without known or suspected
exposure, should be monitored serologically (by RPR or VDRL test) at 6 month intervals to identify inapparent infection. This testing should also be offered to animal care
personnel. Whenever possible, all personnel who are isolating T. pallidum from human
samples should be immunized for hepatitis.
Isolation and
Laboratory
Maintenance of
T. pallidum
Infected rabbits are not infectious during routine handling unless they have open skin
lesions (resulting from intradermal or intravenous inoculation). Nonetheless, personnel
handling all infected rabbits should wear the PPE as described above. Saliva, semen,
urine, and feces from infected rabbits are not infectious. Therefore, bites or scratches
should be washed and treated as they would for any injury caused by a rabbit, but specific
treatment for syphilis is not warranted.
12A.1.2
Supplement 7
Tissues and carcasses, lesion exudates, blood, and cerebrospinal fluid from infected rabbits are potentially infectious and should be handled with care. All wastes should be
disposed of in a well labeled biohazard container so that housekeeping or laboratory support personnel are not at risk of inadvertent exposure. Tissue wastes, including carcasses
from infected rabbits, must be disposed of in a manner that ensures decontamination
(e.g., autoclave, incineration). Suspensions of treponemes can be disinfected (using one
of the agents listed below) or autoclaved prior to disposal in a sink drain. Reusable glass
tubes containing blood or CSF from infected rabbits should be disinfected first before
being subjected to normal glassware washing. T. pallidum is inactivated by 70% ethanol,
Clidox (chlorine dioxide), phenol-based disinfectants (e.g., Vesphene), and 1% bleach
(sodium hypochlorite). Spills or splashes should be contained immediately and the area
disinfected with one of the above agents.
Materials
Propagation and maintenance of noncultivable treponemes requires several uncommon
items and methods, including rabbits, a rabbit restraint board, and a specialized microscope for quantification of treponemes. In some instances, sedative agents or anesthetic
agents are desirable or required (e.g., rabbits should be anesthetized before inoculation
of HIV+ fluids to decrease the likelihood of a needle stick injury to the technologist).
Such materials are described below.
In addition, T. pallidum are extremely fragile and do not survive for long periods outside
of a host. Thus, inoculation of clinical samples or strains should be performed within
1 hr of sample collection. This requires careful planning of all procedures and advance
preparation of required reagents. In the case of propagation of treponemal strains, organisms must be harvested from tissues, quantified, and injected into rabbits within the
specified time frame.
Rabbits
Noncultivable treponemes have been propagated in several rabbit strains, although most
modern laboratories use adult male New Zealand White (NZW) rabbits. For optimal propagation of treponemes, rabbits should be healthy, adult (3 months old, 2.5 to 3.0 kg
for NZW), and have well developed testes. The rabbits should receive no antibiotics
(either therapeutic or in their feed) before or after infection. Because of the possibility
of inapparent infection of rabbits with T. paraluiscuniculi, all rabbits should be screened
serologically for evidence of treponemal infection using both a nontreponemal (VDRL or
RPR) test and a treponemal (e.g., FTA-ABS) test. The RPR and VDRL tests are available
commercially and can be used directly with rabbit serum according to the manufacturers
directions. The FTA-ABS test must be adapted for use with rabbit serum by substitution
of FITC-labeled anti-rabbit IgG for the FITC anti-human IgG in indirect immunofluorescence. Detailed instructions for these tests have been published (Larsen, 1999), including
the adaptation of the FTA-ABS for rabbit serum (Lukehart, 1982). Although one could
use a commercial hemagglutination or particle agglutination treponemal test for rabbit
serum, these tests have not been used extensively for this purpose and their sensitivity
for detecting T. paraluiscuniculi infection is unknown. Reactivity in either treponemal or
nontreponemal test excludes a rabbit from use for treponemal infection or strain isolation.
Rabbits should be housed in an 18 C to 20 C temperature-controlled room to facilitate
treponemal growth and lesion development. Despite careful temperature control, many
laboratories experience less robust treponemal growth and lesion development during the
summer months, perhaps due to hormonal changes in the rabbits. Note that all procedures
involving rabbits must be approved in advance by the Institutional Animal Care and Use
Committee (IACUC).
Spirochetes
12A.1.3
Current Protocols in Microbiology
Supplement 7
Isolation and
Laboratory
Maintenance of
T. pallidum
Figure 12A.1.1 Rabbit restraining board. (A) Photograph of a stainless steel board and leather
ties. (B) Diagram with dimensions. Bend a 1/2 -in. lip around the bottom of the board (indicated
by the pale gray lines and the word "bend"). Weld metal at corners to avoid sharp edges. The
dimensions of the leather straps are shown at right.
12A.1.4
Supplement 7
BASIC
PROTOCOL 1
The noncultivable treponemes are propagated most easily by serial passage in rabbit
testes. The Nichols strain is the most widely used strain of T. pallidum. Since its isolation
in 1912 (Nichols and Hough, 1913), this strain has been passaged in rabbit testes by
injecting a suspension of treponemes into the body of the rabbit testis. The strain is
now highly adapted for rapid growth and the resulting local cellular events have been
well documented (Lukehart et al., 1980). The bacteria divide, increasing significantly in
number in the interstitial spaces (between the seminiferous tubules) where they trigger
a significant inflammatory response that is clinically evident as orchitis (firm, indurated
testes). Histologically, this reaction is marked by evidence of edema and T lymphocyte
infiltration. Within days of T cell infiltration, macrophages also infiltrate the interstitial
spaces and the numbers of bacteria decline precipitously due to phagocytosis and destruction by macrophages. Resolution of the inflammatory response occurs and the testes
clinically return to a normal appearance.
The goal in propagation is to harvest the organisms at peak bacterial numbers, before
immune clearance occurs. This same series of events occurs following intratesticular
infection with all of the noncultivable treponemes; however, the maximal bacterial numbers, time to development of orchitis, and severity of orchitis differ by strain and inoculum
size. With the Nichols strain, routine propagation is accomplished by inoculation of 15
107 T. pallidum per testis, in a volume of 1 to 1.5 ml per testis. Using this inoculum
size, peak bacterial numbers for the Nichols strain are obtained 9 to 11 days following
infection. Clinically the rabbits testes will be enlarged and firm at this time. The animal
is euthanized, and the testes are aseptically removed. The testes are sliced and scored,
placed into a saline/rabbit serum mixture, and agitated gently on a rotator. The numbers
of organisms extracted into the saline can be monitored by dark-field microscopy (Support Protocol). When the desired treponemal concentration is reached, the suspension is
inoculated intratesticularly into the nave, seronegative recipient rabbit. The harvesting,
extraction, and reinoculation of treponemes must be accomplished as rapidly as possible
because these organisms do not survive well outside of an animal host. It is recommended
that treponemes be reinoculated into the recipient rabbit within 1 hr of harvesting the
donor testes. Strict aseptic techniques are required to prevent contamination of the treponemal inoculum with other bacteria.
Depending upon the frequency with which treponemes are needed for experiments,
laboratories may develop different propagation schedules. For example, new rabbits may
be infected on Monday and Friday of one week, then on Wednesday of the next week,
then Monday and Friday of the following week, and so on. This is shown schematically
in Figure 12A.1.2. The generation time for the Nichols strain in rabbit testes has been
estimated to be 30 to 33 hr (Magnuson et al., 1948), and approximately 1010 treponemes
can be harvested from the testes of a single rabbit. Other strains and subspecies may grow
Figure 12A.1.2 Schematic showing a propagation schedule for the Nichols strain of Treponema
pallidum, with passage to a new rabbit every 8 to 10 days.
Spirochetes
12A.1.5
Current Protocols in Microbiology
Supplement 7
at significantly slower rates, achieve substantially lower yields, and induce less dramatic
orchitis (Turner and Hollander, 1957). When propagating strains other than the Nichols
strain, the rabbits testes should be palpated at least three times per week to identify
the time of optimal orchitis. For propagation of unfamiliar strains, some investigators
recommend infection of only one testis, so that the uninfected testis can serve as a control
for assessing orchitis. The optimal passage time for individual treponemal strains ranges
from 9 to 40 days. At the end of this protocol, the researcher will have isolated treponemes
from rabbit testes, which are then immediately inoculated into another rabbit or frozen
for storage (Basic Protocol 4).
Materials
T. pallidum sample, frozen (e.g., obtained from a colleague): up to 2 to 3 ml per
injection
Rabbits: seronegative in VDRL/RPR or FTA-ABS tests, healthy, adult males with
well developed testes and no history of antibiotic treatment: e.g., New Zealand
White (NZW), 3 months old (2.5 to 3.0 kg)
7.5% Betadine solution
70% (v/v) ethanol
Saline (0.14 M NaCl; see recipe)
Normal rabbit serum (NRS; see recipe)
Disinfectant (e.g., phenol based)
Rabbit restraining board (see Strategic Planning and Fig. 12 A.1.1)
3-cc sterile, disposable Luer-lok syringe
23-G 1-in. sterile, disposable Luer-hub needle
Cotton balls or gauze
Sharps disposal unit
18 C to 20 C BSL-2 rabbit facility
5-in. surgical scissors and 6-in. Kelly hemostat forceps, sterile: packaged as a set
prior to autoclaving; two sets required for each procedure
100 15mm petri dishes, sterile
Closed transport container (e.g., plastic box with lid)
Scalpel, sterile
6- to 7-in. forceps, sterile
5- and 10-ml sterile disposable pipets and pipetting aid
Platform rotator with disposable adsorbent material on platform
Additional reagents and equipment for sedating and euthanizing rabbits (e.g., see
http://depts.washington.edu/auts/rabbit handout 2007.pdf) and quantifying
treponemes by dark-field microscopy (Support Protocol)
Inoculate testes
1. Transport frozen sample to the vivarium on dry ice.
2. Sedate the rabbit (e.g., acepromazine by intramuscular or subcutaneous injection at
1 to 3 mg/kg body weight) according to an IACUC-approved protocol.
3. Place the rabbit securely on restraining board, using soft leather thongs to secure
each leg.
4. Thaw the treponemal suspension gently by warming with the hands.
5. Draw the suspension immediately into 3-cc Luer-lok syringe with a 23-G 1-in.
needle.
Isolation and
Laboratory
Maintenance of
T. pallidum
12A.1.6
Supplement 7
6. Press gently on the lower abdomen of the rabbit to cause the testes to descend; grasp
above the body of one testis to prevent retraction.
7. Disinfect the scrotum area well with a cotton ball or gauze saturated with 7.5%
Betadine solution.
8. Insert the needle near the distal end of the testis so that it reaches the middle of
the testis and inject approximately one-quarter of the treponemal volume. Move
the needle to another part of the testis and inject approximately one quarter of the
volume. Remove the needle.
Minor bleeding may occur at injection site. If so, squeeze rabbits scrotum until bleeding
stops.
9. Repeat steps 5 to 7 with the other testis. Discard the syringe in sharps discard
container.
10. Place rabbit back in the cage and house in an 18 C to 20 C BSL-2 rabbit facility and
observe three times per week for evidence of orchitis by palpating testes.
12. If necessary, press the lower abdomen of rabbit to cause testes to descend.
13. Thoroughly wet the scrotal area with 70% ethanol to control fur and to disinfect the
area.
14. Grasp scrotum over one testis with Kelly forceps, and with sterile scissors cut
longitudinally through scrotum along the length of the testis to expose the tunica
albuginea. Repeat for the other testis.
15. Using a new set of instruments, grasp the tunica of one testis with the Kelly forceps
and cut the tunica longitudinally with sterile scissors to expose the testis.
16. Grasp the testis with the Kelly forceps and lift the testis out of the tunica. Cut the testis
free of the surrounding tissue by cutting the surrounding fat and spermatic vessels.
Minimal fatty tissue should be left on the testis.
17. Place the testis in a sterile petri dish and replace lid. Repeat for the other testis.
Maintain aseptic technique throughout this procedure
18. Clean all surfaces and the rabbit board with disinfectant.
Recover treponemes
19. Transport the petri dish containing testes to the laboratory using appropriate biosafety
precautions (e.g., separate closed carrier).
20. If there is significant blood in the petri dish, move the testes to a clean dish before
proceeding.
Spirochetes
12A.1.7
Current Protocols in Microbiology
Supplement 7
21. Grasp the testis with sterile forceps and slice with a scalpel longitudinally, but not
completely through the testis (to form a butterfly shape).
22. Score the cut surfaces of the testis multiple times with the scalpel, then turn the testis
cut side down in petri dish.
23. Repeat for the second the testis and place cut side down in the same petri dish.
24. Add 10 ml saline and 1 to 2 ml NRS to the petri dish. Squeeze the testes several
times in the saline/NRS using forceps.
25. Place the covered petri dish on a platform rotator. Place a disposable adsorbent pad
on the rotator (in case of spills) and rotate gently at room temperature.
As an alternate approach, some investigators cut the testes into small pieces and place
them into a 50-ml conical tube for extraction of treponemes.
Quantify treponemes
26. After rotating for 10 min, collect a sample (e.g., 10 l) of the extraction and quantify
by dark-field microscopy (see Support Protocol).
If treponemes are present at 15 107 /ml, the suspension is ready to be inoculated into
a new recipient rabbit. If the concentration of treponemes is lower than 1 107 /ml, the
testes should be rescored and squeezed, then rotated for an additional 10 min.
In some cases, particularly if using a strain other than T. pallidum Nichols, the number
of organisms that can be extracted from the testes may be low. In such cases, it may be
necessary to score the testes more vigorously, lengthen the extraction time (not more than
30 to 45 min), or decrease the extraction volume to maximize treponeme concentration.
Reinoculate treponemes
27. Draw 2 to 3 ml of suspension into a 3-cc Luer-lok syringe with 23-G 1-in. needle.
It may be necessary to move small pieces of tissue so that they do not clog the needle tip.
Some investigators move the treponemal suspension to a tube using a disposable 10-ml
pipet (remote pipetting) and allow the tissue to settle briefly to facilitate filling of syringe.
In some instances with non-Nichols strains, where the testes must be vigorously manipulated to obtain organisms, the suspension is centrifuged 10 min at 250 g and the
supernatant used for subsequent inoculation.
28. Transport the syringe (or the treponemal suspension) to vivarium using appropriate
biosafety precautions (e.g., separate closed container).
29. Infect both testes of a rabbit as described in steps 2 to 8.
30. Clean all surfaces, including the restraining board, with disinfectant.
CAUTION: All contaminated material must be decontaminated or disposed as biohazardous waste.
SUPPORT
PROTOCOL
Isolation and
Laboratory
Maintenance of
T. pallidum
12A.1.8
Supplement 7
Materials
Treponemes harvested from rabbit testes (Basic Protocol 1)
Dark-field microscope and condenser oil
Slide micrometer
Microscope slide and 22 22mm coverslip
1. Measure the radius (r) of the 40 microscope field using a slide micrometer and
calculate the area (r2 ).
2. Place a measured amount (e.g., 10 l) of treponemal suspension on a microscope
slide and cover with a 22 22mm coverslip, being careful to avoid bubbles.
Calculate the fluid depth by dividing the volume (ml or cm3 ) by the area of the
coverslip (cm2 ):
0.010 cm3 /4.84 cm2 = 0.0207 mm deep
Other suspension volumes and coverslips sizes may also be used.
3. Calculate the volume of fluid (ml) viewed in a microscope field by multiplying the
field area (step 1) by the fluid depth (step 2).
4. Count the number of treponemes in 100 microscope fields, being certain to count
organisms in all planes of focus in each field.
5. Calculate the number of treponemes per ml of suspension:
treponemes/ml = (total treponemes counted in 100 fields)/(100 fields
field volume in ml)
BASIC
PROTOCOL 2
Materials
Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal
rabbit serum (NRS; see recipe)
1-ml tuberculin syringe
100 15mm petri dishes, sterile
Scalpel, sterile
Platform rotator
3-cc sterile, disposable Luer-lok syringe
23-G 1-in. needle
Additional reagents and equipment for performing venipuncture, lumbar puncture,
and punch biopsy (qualified personnel), anesthetizing rabbits (e.g., see
http://depts.washington.edu/auts/rabbit handout 2007.pdf), inoculating rabbits
and harvesting treponemes (Basic Protocol 1), and performing VDRL/RPR and
FTA-ABS screening (Lukehart, 1982; Larson, 1999)
Current Protocols in Microbiology
Spirochetes
12A.1.9
Supplement 7
1a. To collect blood samples: Collect aseptically by venipuncture into vacutainer tubes
containing EDTA as an anticoagulant, per clinical protocol.
The blood of patients with early (primary or secondary) syphilis has the highest likelihood
of successful T. pallidum isolation, although treponemes can be isolated from blood of
persons with latent syphilis.
1b. To collect cerebrospinal fluid (CSF): Collect aseptically by lumbar puncture into a
sterile tube, per clinical protocol.
The highest likelihood of isolation is from CSF of patients with primary or secondary
syphilis, although treponemes can be isolated from CSF of persons with latent syphilis.
1c. To collect chancre exudate: Squeeze the chancre gently so that serous exudate
accumulates in the ulcer; avoid blood contamination of the exudate. Collect the
exudate using a tuberculin syringe (without needle). Place the tip of syringe into a
tube containing 2 ml sterile saline/20% NRS and wash the exudate into the tube by
drawing and ejecting 1 ml of saline/20% NRS into the tube several times.
1d. To take a lesion biopsy: Collect a 4-mm punch biopsy from a secondary lesion (rash
or condylomata lata) or the leading edge of a primary chancre, per clinical protocol.
Place the biopsy into a sterile petri dish containing 1 to 2 ml saline/20% NRS. Using
a sterile scalpel, mince the tissue into very small pieces and squeeze the tissue into
the liquid using forceps. Rotate 10 min on a platform rotator at room temperature.
2. Recommended: Anesthetize the rabbit (serving as the recipient of human samples)
and wear double gloves to minimize the risk of needlestick injury.
Anesthesia is recommended for short but potentially painful procedures.
For example, administer 3 to 5 mg/kg xylazine (0.45 to 0.75 ml of 20 mg/ml for 3-kg
rabbit), wait 5 to 10 min, and then administer 35 to 50 mg/kg ketamine (1.0 to 1.5 ml of
100 mg/ml for a 3-kg rabbit), both given intramuscularly.
Alternatively, mix 10 ml of 100 mg/ml ketamine, 5 ml of 20 mg/ml xylazine, and 2 ml of
10 mg/ml acetylpromazine. Administer 0.5 ml/kg intramuscularly.
Additional useful information concerning rabbit use and handling may be obtained at
http://depts.washington.edu/auts/rabbit handout 2007.pdf.
3. Draw 2 to 3 ml of sample (blood, CSF, lesion exudate suspension, or biopsy suspension) into a 3-cc Luer-lok syringe with 23-G 1-in. needle.
4. Place rabbit securely on restraining board, using soft leather thongs to secure each
leg, and inject up to 1.5 ml of sample per testis, as described in the Basic Protocol 1,
steps 6 to 9.
5. Observe and palpate testes three times per week to detect orchitis.
6a. If orchitis develops: Harvest treponemes from testes and transfer them to a new rabbit
as described in Basic Protocol 1.
Orchitis is uncommon with the first passage of clinical samples. It is likely that there will
be a low number of organisms in this first passage.
6b. If no orchitis develops by 30 days: Collect blood from the rabbit for VDRL/RPR and
FTA-ABS screening and proceed to step 7.
7a. If seronegative in both tests: Continue to observe for orchitis and conduct monthly
serological testing until 3 months post-inoculation.
Isolation and
Laboratory
Maintenance of
T. pallidum
If, after 3 months, the rabbit has not seroconverted in both tests, longer observation is
unlikely to be fruitful.
12A.1.10
Supplement 7
7b. If the rabbit becomes seropositive in both VDRL/RPR and FTA-ABS tests: Harvest treponemes from testes (and ideally from popliteal lymph nodes; see Basic
Protocol 3) and transfer them to a new rabbit as described in Basic Protocol 1.
It is possible that no treponemes will be visible in the suspension (called blind passage),
but transfer of infection is likely to occur. Sometimes several blind or low-yield passages
are required before the strain is growing well.
Orchitis does not always develop when the rabbits become seropositive (often the case in
blind transfers).
BASIC
PROTOCOL 3
Sometimes infected rabbits will fail to develop orchitis, or one may wish to isolate
treponemes from a rabbit with a latent infection. This is best accomplished by transfer of
material obtained from the popliteal lymph nodes, because T. pallidum resides for years
after infection in many tissues, including lymph nodes. This assay is sometimes used to
assess the infection status of a rabbit (e.g., after infectious challenge of an immunized
rabbit).
Materials
Infected rabbit (Basic Protocol 1)
70% (v/v) alcohol
Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal
rabbit serum (NRS; see recipe)
Rabbit restraining board (see Strategic Planning and Fig. 12A.1.1)
4- to 5-in. curved scissors, sterile
100 15mm petri dishes, sterile
Forceps, sterile
Stainless steel mesh screen (2 2 in; e.g., from hardware store), sterile
3-cc sterile, disposable Luer-lok syringe
23-G 1-in. needle
Additional reagents and equipment for euthanizing rabbits (e.g., see
http://depts.washington.edu/auts/rabbit handout 2007.pdf) and passaging
treponemes in rabbits (Basic Protocol 1)
1. Euthanize the infected rabbit (e.g., pentobarbital by intravenous or intraperitoneal
injection at 90 mg/kg body weight) and secure it on a restraining board at both
forelegs and one hind leg. Secure the other hind leg to one of the brackets designed
for the front legs so that the flexor side of the leg is exposed.
2. Saturate the fur on the back of the leg with 70% ethanol.
3. Using the sharp point of 4- to 5-in. sterile curved scissors, puncture the skin just
behind the knee joint and extend the incision in both directions along the flexor side
in each direction for approximately 2 to 3 in.
4. Grasp the popliteal node (see Miller, 1971), contained in a fatty mass just behind the
knee joint, with forceps and cut it free. Place the node in a sterile petri dish.
5. Using sterile forceps and small curved scissors, trim the fat away from the node and
remove node to a clean petri dish.
6. Repeat steps 2 to 5 for the other popliteal node, placing both nodes in the same petri
dish.
Spirochetes
12A.1.11
Current Protocols in Microbiology
Supplement 7
7. Add 5 ml saline/20% NRS to the dish. Holding the sterile mesh screen securely with
hemostat forceps and the nodes with regular forceps, scrape the nodes on the mesh
so that the cells disperse into the saline/20% NRS.
8. Collect the fluid in a 3-cc Luer-lok syringe with a 23-G 1-in. needle, and infect a
seronegative recipient rabbit as per Basic Protocol 1, steps 1 to 10.
9. Observe the rabbit clinically and serologically as described in Basic Protocol 2, steps
5 to 7.
10. Passage organisms as described in Basic Protocol 1 or freeze cultures as in Basic
Protocol 4.
BASIC
PROTOCOL 4
Materials
Saline/50% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 10 ml normal
rabbit serum (NRS; see recipe)
Sterile glycerol or DMSO
Dry ice
95% (v/v) ethanol
Sterile 2-ml cryovials, labeled with strain name, concentration, and date.
Liquid nitrogen freezer
Additional reagents and equipment for harvesting T. pallidum suspension (Basic
Protocol 1)
1. Harvest a T. pallidum suspension as described in Basic Protocol 1, steps 11 to 26,
except use saline/50% NRS for extraction.
Ideally, the concentration of treponemes should be >107 /ml.
3. Allow suspensions to remain in the dry ice/ethanol slurry for 30 min, then move
immediately to liquid nitrogen storage.
T. pallidum can also be maintained frozen at 70 C for many years.
12A.1.12
Supplement 7
BASIC
PROTOCOL 5
In T. pallidum, the tprK gene sequence is variable (even within a strain) and undergoes
sequence variation, particularly under immune pressure in vivo (Centurion-Lara et al.,
2000; LaFond et al., 2003; Centurion-Lara et al., 2004). This sequence can be used as
a marker for clonality of T. pallidum populations and is used in this cloning protocol to
assess the clonal characteristics of treponemal populations.
Materials
Rabbits: seronegative in VDRL/RPR or FTA-ABS tests, healthy, adult males with
well developed testes and no history of antibiotic treatment: e.g., New Zealand
White (NZW), 3 months old, 2.5 to 3.0 kg
70% (v/v) ethanol or Betadine
Saline/20% NRS: mix 10 ml saline (0.14 M NaCl; see recipe) and 1 to 2 ml normal
rabbit serum (NRS; see recipe)
15-ml sterile, conical, screw cap tubes
Low speed centrifuge with BSL-2 containment
Rabbit restraining box (restraining board can be used, but commercially available
restraining box preferred)
Closed transport container (e.g., plastic box with lid)
3-cc sterile, disposable Luer-lok syringe
23-G 1-in. needle
Animal clippers for keeping rabbit back free of fur (size 40 blade, optimal)
4-mm disposable biopsy punches (e.g., Miltex)
100 15mm petri dishes, sterile
4- to 5-in. small curved scissors, sterile
Additional reagents and equipment for preparing T. pallidum suspensions (Basic
Protocol 1), determining suspension concentration by dark-field microscopy
(Support Protocol), sedating or anesthetizing and euthanizing rabbits (e.g., see
http://depts.washington.edu/auts/rabbit handout 2007.pdf), propagating
organisms in rabbits (Basic Protocol 1), maintaining treponemes as frozen stocks
(Basic Protocol 4), and sequencing DNA (e.g., see Centurion-Lara et al., 2000)
Prepare T. pallidum suspensions
1. Prepare a suspension of T. pallidum (as concentrated as possible but ideally at least
1 108 /ml) in saline/20% NRS, as described in Basic Protocol 1, steps 11 to 26.
2. Place the suspension in sterile 15-ml conical, screw-cap tube. Centrifuge 10 min at
250 g, room temperature to pellet gross cellular debris.
3. Pipet the supernatant into a sterile 15-ml conical, screw-cap tube.
4. Determine final concentration of T. pallidum suspension by dark-field microscopy
(see Support Protocol).
5. Transport suspension to the vivarium using appropriate biosafety containment
(closed container).
Spirochetes
12A.1.13
Current Protocols in Microbiology
Supplement 7
Inoculate rabbits
6. Withdraw sufficient volume of suspension into disposable Luer-lok syringe with
23-G 1-in. needle to contain 108 T. pallidum.
If needed, as much as 10 ml of suspension can be injected, although smaller volumes are
preferable.
14. Collect a 4-mm punch biopsy ideally encompassing the entire skin lesion.
15. Place the tissue in a sterile petri dish and trim off any subcutaneous fat.
16. Using sterile curved scissors, mince biopsy tissue into 2 to 4 ml saline/20% NRS.
17. Collect a 500-l sample of suspension under clean PCR conditions for DNA sequencing (see step 20).
The term "clean PCR conditions" means that the samples are prepared and amplified in
a room in which there have never been PCR amplicons or even large amounts of target
DNA. Extreme care must be taken to avoid contamination of the sample with extraneous
DNA.
When clonal treponemes are propagated from frozen stocks, however, they may undergo
sequence diversification during testicular propagation and may need to be recloned by
IV infection/biopsy again.
12A.1.14
Supplement 7
Assess clonality
20. While the organisms are being propagated in new rabbits (see Basic Protocol 1),
assess the clonality of the inoculum derived from each biopsy by sequencing the
tprK locus (e.g., see Centurion-Lara et al., 2000).
Occasionally, lesions contain multiple populations of treponemes, probably due to seeding
of the skin by two treponemes in close proximity. These cases will be detected by tprK
sequencing, and treponemes derived from them will not be clonal.
The tprK locus should be sequenced from treponemes at every stage of the cloning process:
inoculum, biopsy, and after propagation in testes. Because some strains have very high
rates of sequence change, it may be necessary to repeat the intravenous infection/biopsy
step to obtain a clonal population.
The tprK gene is comprised of 7 regions of variable sequence (V regions) with intervening
stretches of conserved sequence (Centurion-Lara et al., 2000). Although some V regions
are more likely to vary in sequence, this is not absolute, and full ORF sequencing is
necessary to assess clonality of a population. The gene is amplified by PCR, then the
amplicons are cloned into E. coli for sequencing of individual E. coli clones. In our
experience, direct sequencing of the amplicons fails to show the presence of sequence
diversity unless the diverse subpopulations are quite prevalent. We routinely sequence ten
clones from each amplification to assess clonality.
Spirochetes
12A.1.15
Current Protocols in Microbiology
Supplement 7
Isolation and
Laboratory
Maintenance of
T. pallidum
Critical Parameters
Time
Minimizing the length of time that T. pallidum organisms are outside of an animal host
12A.1.16
Supplement 7
Anticipated Results
Rabbit quality
Rabbits used for propagating T. pallidum
strains should be healthy adult males that
have not received antibiotics therapeutically
or in their food and water. The rabbit supplier should be chosen for having rabbits
that are either specifically pathogen-free for
Pasteurella multocida or with very low prevalence of symptomatic Pasteurella infection.
Immature males are not as permissive to
T. pallidum growth as fully mature rabbits.
Troubleshooting
Failure of infected rabbit to develop orchitis
Occasionally, infected rabbits will fail to
develop an orchitis. This may be due to incorrect inoculation (e.g., inoculation into the scrotal sac rather than into the body of the testis).
If replicate vials of the same strain are frozen,
it is generally preferable to begin propagation
again from a frozen stock. If no frozen stock
is available, the strain may be recoverable by
blind transfer of testes and popliteal lymph
node extracts as described in Basic Protocols
1 and 3. Even after 1 to 2 years of infection,
organisms can be recovered from testes and
nodes. Successive rounds of rapid passage of
treponemes in rabbits (short time between infection and transfer to the next rabbit) generally serves to select for more rapidly growing
populations and higher apparent adaptation.
Time Considerations
The time required to harvest treponemes
and re-inject them for routine propagation is
30 to 60 min. T. pallidum are extremely fragile and do not survive for long periods outside
of a host. Thus inoculation of clinical samples
or strains should be performed within 1 hr of
sample collection. This requires careful planning of all procedures and advance preparation
of required reagents.
Because it can take weeks to months for a
new harvest of treponemes, investigators will
have to plan very carefully for future needs.
Rabbits may need to be ordered weeks to
months in advance, time must be allowed for
serological screening of the rabbits, and 2 to
6 weeks may elapse between infection and
subsequent harvest.
Isolation of new strains will likely be more
time consuming because one or even two blind
passages are often necessary before the bacterial numbers reach concentrations that can be
determined microscopically.
Derivation and maintenance of clonal
T. pallidum populations is very time consuming. Many months of work may be required
before it is possible to confirm that a population of organisms is clonal.
Spirochetes
12A.1.17
Current Protocols in Microbiology
Supplement 7
Literature Cited
Antal, G.M., Lukehart, S.A., and Meheus, A.Z.
2002. The endemic treponematoses. Microbes
Infect. 4:83-94.
Centurion-Lara, A., Godornes, C., Castro, C., Van
Voorhis, W.C., and Lukehart, S.A. 2000. The
tprK gene is heterogeneous among Treponema
pallidum strains and has multiple alleles. Infect.
Immun. 68:824-831.
Centurion-Lara, A., LaFond, R.E., Hevner, K.,
Godornes, C., Molini, B.J., Van Voorhis, W.C.,
and Lukehart, S.A. 2004. Gene conversion: A
mechanism for generation of heterogeneity in
the tprK gene of Treponema pallidum during
infection. Mol. Microbiol. 52:1579-1596.
Nichols, H.J. and Hough, W.H. 1913. Demonstration of Spirochaeta pallida in the cerebrospinal
fluid. JAMA 60:108-110.
Norris, S.J. 1982. In vitro cultivation of Treponema
pallidum: Independent confirmation. Infect.
Immun. 36:437-439.
Radolf, J.D. and Lukehart, S.A. (eds.) 2006.
Pathogenic Treponemes: Molecular and Cellular Biology. Caister Academic Press, London.
Graves, S. and Downes, J. 1981. Experimental infection of man with rabbit-virulent Treponema
paraluis-cuniculi. Br. J. Vener. Dis. 57:7-10.
Isolation and
Laboratory
Maintenance of
T. pallidum
12A.1.18
Supplement 7
UNIT 12B.1
This unit describes methods, media, and equipment required for routine laboratory
culture and handling of the oral anaerobic spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended formulations for growth in liquid
and solid media, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. Protocols on isolation of T. denticola from clinical
samples are included. With the notable exception of specific individual nutrient requirements and media formulations, these protocols are also appropriate for working with
other species of cultivable commensal treponemes.
Basic protocols included in this chapter cover topics ranging from isolation of T. denticola
from clinical samples (including isolation of pure cultures from single colonies) through
culturing techniques applicable to biochemical analysis and genetic manipulation of
T. denticola. The isolation protocols include a relatively standard method of dispersion
and serial dilution on agar plates as well as a method using liquid medium in a 96-well
plate format. Both of these methods employ rifampicin as a selective agent to enrich for
treponemes in mixed clinical samples. Several commonly used liquid media and related
solid media formulations are described, with comments on relative characteristics and
performance of each. Methods, equipment, and conditions for inoculation, incubation,
passage, and storage of cultures are discussed. Together, this information should provide
the technical background required for routine growth and manipulation of T. denticola
in a well-equipped microbiology laboratory.
CAUTION: Treponema denticola is a Biosafety Level 1 (BSL-1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of
minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See UNIT 1A.1
and other pertinent resources (see APPENDIX 1B) for more information.
NOTE: Most strains of oral spirochetes require strictly anaerobic conditions during
transfer and growth of cultures. Unless otherwise specified, all incubations and, where
feasible, manipulations described in this unit are to be performed under strictly anaerobic
conditions (i.e., nonhumidified 85% N2 /10% H2 /5% CO2 atmosphere) in an anaerobic
glove box chamber at 37 C (see Strategic Planning).
STRATEGIC PLANNING
Media
General requirements
Several complex broth media formulations are in common use for growth of T. denticola
strains (see Reagents and Solutions). These share common features including sources
of peptides, amino acids, and trace nutrients (e.g., tryptone, brain heart infusion, yeast
extract), as well as reducing agent(s) (L-cysteine and/or thioglycolate), volatile fatty
acids, and heat-inactivated serum. All these media provide very low redox potential
(Eh = 185 to 220), which is a critical factor in growth of T. denticola (Socransky
et al., 1964). These media were originally devised for isolation of various species of oral
spirochetes, and likely contain more nutrients than are required by T. denticola. For example, T. denticola does not require glucose for growth, which is required for growth of
Spirochetes
Contributed by J. Christopher Fenno
Current Protocols in Microbiology (2005) 12B.1.1-12B.1.21
C 2005 by John Wiley & Sons, Inc.
Copyright
12B.1.1
OMIZ-P4 medium
A chemically defined, serum-containing medium based on OMIZ-W1 (Wyss, 1992) has
been used by several investigators. This medium, OMIZ-P4 (see Reagents and Solutions),
supports the growth of all cultivable oral treponemes described to date. An earlier version
of this medium has been particularly useful in isolating novel Treponema species from
periodontal lesions (Wyss et al., 1999) and bovine digital dermatitis lesions (Schrank
et al., 1999). OMIZ-P4 is listed by ATCC as Medium 2131 (http://www.atcc.org) and
is available commercially by special order (Anaerobe Systems) at a price reflective of
the complexity of its formulation. A chemically defined serum-free minimal medium for
growth of T. denticola is under development and is likely to become available in the near
future (C. Wyss, pers. comm.).
Laboratory
Maintenance of
Treponema
denticola
12B.1.2
Current Protocols in Microbiology
Several complex media formulations have been used for oral spirochete cultivation as
either surface or subsurface colonies. Generally, media formulations and considerations
are the same as for broth media: provision of required nutrients, near-neutral pH, and low
Eh. Earlier investigators often used various formulations of blood agar. More recently,
blood in the medium has been replaced by serum, usually from rabbit. The recipe given
in this unit for MTYGVS plate medium (see Reagents and Solutions) describes a solid
medium for plating spirochetes (or clinical samples) in an anaerobe chamber. For use
in isolation of spirochetes from clinical samples, this medium is supplemented with
rifampicin (Leschine and Canale-Parola, 1980; Salvador et al., 1987). The other two
solid media, NOS semisolid medium with low-melting-point (LMP) agarose and NOSGN (see Reagents and Solutions), were developed for growth of subsurface colonies
without overheating the spirochetes. These last two media, which utilize a pour-plate
method, are routinely used for selecting T. denticola genetic transformants (UNIT 12B.2).
Microscopy
Contamination of T. denticola cultures with other more rapidly growing organisms is
always a concern, especially when working with clinical isolates. Visualization of cultures by either dark-field or phase-contrast microscopy (see UNIT 2A.1) is a rapid and
reliable method for routine validation of culture purity. The unique morphology and
active motility of T. denticola enables the investigator to easily detect the presence of
nonspirochete contaminants. If species specificity is required, this can be determined by
PCR amplification and DNA sequencing of appropriate 16S rRNA sequences. Sequences
of genes encoding 16S rRNA are available in Genbank. Universal, spirochete-specific
and Treponema-specific oligonucleotide primer sets and appropriate PCR conditions
have been reported by several authors (Paster et al., 1991; Choi et al., 1994; Paster et al.,
2001).
Spirochetes
12B.1.3
Current Protocols in Microbiology
This particular system was developed specifically for isolation and maintenance of strict
anaerobes (Aranki et al., 1969; Rosebury and Reynolds, 1964).
AnaeroPack
The AnaeroPack system (Mitsubishi Gas Chemical America, distributed by REMEL)
requires no catalyst or water, produces no hydrogen, and is oxygen absorbing and carbon
dioxide generating (20% CO2 /0.1% O2 within 1 hr, according to the manufacturers
information). The system consists of a sachet packet that is opened just before sealing
the bacterial culture in the included container (sealable polystyrene box or disposable
bag). Containers are available in several sizes to accommodate 2 to 36 standard agar
plates. The disposable bag format easily handles several 15-ml culture tubes. Depending
on container volume, one to three sachets are recommended. Performance of this system
is similar to the GasPak system (Delaney and Onderdonk, 1997).
2. Conversely, never open the inner door before checking to see whether the outer door
is securely closed. If the entry lock has an anaerobic/aerobic warning light, always
check that the anaerobic light is on before opening the inner door.
12B.1.4
Current Protocols in Microbiology
3. Remove rings and watches before working in the chamber to avoid puncturing gloves
and chamber sleeves.
4. Avoid using sharp items in the chamber except as necessary.
5. Use absorbent bench paper in the chamber to contain spills and debris. Change
regularly.
6. Minimize the number of chamber entries by careful scheduling and planning of inchamber procedures, including having the necessary supplies and equipment in the
chamber as needed.
7. Maintain a regular schedule for changing desiccant and catalyst as appropriate to the
level of use of the anaerobic chamber.
8. Use white cotton gloves (available from photographic suppliers) to absorb perspiration while working in the chamber. This is for your comfort as well as for the comfort
of subsequent users.
9. Keep a roll of vinyl tape in the chamber, both for emergency repairs and for periodic
routine glove replacement.
10. Use standard laboratory safety practices for changing and handling gas tanks, with
special attention to ensuring that tanks are secured at all times.
BASIC
PROTOCOL 1
T. denticola can be revived from stored stock cultures by growth in one of the broth media
listed below. All of these media formulations provide nutrients required for growth of T.
denticola. Of the three, OMIZ-P4 is the closest to a defined medium. Even so, OMIZP4 contains neopeptone and serum. OMIZ-P4 will support growth of most cultivable oral
spirochetes. Although this medium is extremely useful for metabolic and biochemical
studies, it is very expensive for routine growth of T. denticola laboratory strains. NOS and
TYGVS are complex media with significant amounts of brain heart infusion and yeast
extract. TYGVS supplies higher concentrations of several nutrients, including nutrients
(such as gelatin) that are absent in NOS. Though NOS is practical for routine growth
of laboratory strains, growth is marginally faster and final densities typically higher in
TYGVS. Some recent clinical isolates will grow in TYGVS but will not grow in NOS.
Materials
NOS, TYGVS, or OMIZ-P4 broth medium (see recipes)
T. denticola stock culture (e.g., ATCC #35405)
100% glycerol, sterile, at 37 C, prereduced
Dry ice/ethanol bath or liquid nitrogen
Preservation medium (see recipe)
Spirochetes
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Current Protocols in Microbiology
3. Add contents of the vial to not more than 20 vol prereduced broth medium.
For typical strain growth, 10-ml cultures are grown in 15-ml glass or plastic (polystyrene
or polypropylene) tubes with caps loosened. Depending on the volume required, culture
volumes may be stepped up to 1 liter or more in large flasks or bottles.
The high level of inoculum helps to ensure recovery of the culture. Maintenance of
excellent aseptic technique is crucial at this step (and in step 6 below). T. denticola grows
more slowly than most environmental contaminants in rich medium.
5. If determining growth kinetics and using a low-viscosity broth medium, briefly mix
or vortex the culture gently to suspend the bacteria, then measure OD600 .
T. denticola cells accumulate at the bottom of the tube by late log phase even though they
retain motility as visualized by microscopy.
7a. For long-term storage by freezing: Add prereduced, prewarmed glycerol to actively
growing cultures to a final glycerol concentration of 15% (v/v). Aliquot to 2-ml
cryovials. If working in an anaerobic chamber, cap the vials at this point, remove
from chamber, and freeze quickly in a dry ice/ethanol bath or by immersion in liquid
nitrogen. Store vials at 70 C.
As with all procedures with T. denticola, viability is optimal when manipulations are
conducted under strict anaerobic conditions as much as is practicable, regardless of the
storage method used.
7b. For long-term storage by lyophilization: Lyophilize by the method of Cheng and
Chan, (1983).
Laboratory
Maintenance of
Treponema
denticola
7c. For long-term storage in liquid nitrogen: Store in liquid nitrogen in preservation
medium (Syed et al., 1993).
12B.1.6
Current Protocols in Microbiology
BASIC
PROTOCOL 2
Either 25-cm2 tissue culture flasks (Corning) or deep petri dishes may be used for plating
T. denticola.
Materials
NOS-GN 1% agar semisolid medium (see recipe) or NOS 1.5% LMP agarose
semi-solid medium (see recipe)
T. denticola cell suspension (see Basic Protocol 1)
37 to 39 C water bath
25-cm2 tissue culture flasks (Corning or equivalent)
6-well tissue culture plates (Corning or equivalent)
100 25mm extra-deep petri dishes (Nalgene Nunc or equivalent)
Ice packs
Anaerobic chamber (see Strategic Planning)
For tissue culture flasks
1a. Prepare semisolid medium, aliquot to tissue culture flask(s), and cool to 37 to 39 C
in a water bath. Add T. denticola cell suspension (up to 3 ml) to 25 ml medium in a
25-cm2 tissue culture flask.
2a. Mix by gentle inversion and incubate at room temperature until the medium solidifies.
The medium must be brought to 25 to 30 C to solidify.
3a. When the medium has solidified, overlay with a few milliliters of noninoculated
semisolid medium if desired.
Chan et al. (1993b) suggest this overlay as a means of sealing the medium from contact
with atmospheric gases. Refer to Chan et al., 1993b, for more information concerning
this technique.
5b. Return inoculated petri dishes or plates to the chamber for incubation until growth
is observed.
T. denticola growth is typically visible as diffuse subsurface colonies after one week,
though genetic transformants sometimes take two weeks or longer to form colonies (also
see UNIT 12B.2). Colonies will continue to grow larger as the organisms move through the
medium. The diffuse fuzzy appearance of T. denticola colonies in agar media is due
to spirochete motility through the semisolid medium. Subsurface colonies will be smaller
and less diffuse in media with higher concentrations of solidifying agent.
Spirochetes
12B.1.7
Current Protocols in Microbiology
Laboratory
Maintenance of
Treponema
denticola
Rifampicin inhibits growth of the vast majority of nonspirochete members of the oral flora.
Rifampicin resistance in most bacteria is the result of a single point mutation in the rpoB
gene (Carter et al., 1994). Thus, simple selection with rifampicin can result in isolation
of resistant organisms.
12B.1.8
Current Protocols in Microbiology
4. Expel the agar from the pipet into a culture tube containing 2 to 5 ml liquid TYGVS
medium containing 5 g/ml rifampicin.
It is important that each colony selected be grown in broth and monitored by darkfield microscopy (UNIT 2A.1) for culture purity and typical spirochete morphology (motile
helical organisms).
BASIC
PROTOCOL 4
Materials
Subgingival plaque sample
Reduced transfer fluid (RTF; see recipe)
OMIZ-P4 medium with 5 g/ml rifampicin and 100 g/ml phosphomycin (see
recipe; also see Strategic Planning)
OMIZ-P4 1.5% LMP agarose semisolid medium: prepare as described for NOS
1.5% LMP agarose semisolid medium (see recipe), substituting OMIZ-P4 basal
medium (see recipe) for NOS basal medium (see recipe)
96-well microtiter plates
Anaerobic jar (Becton Dickinson GasPak or equivalent; see Strategic Planning)
37 C incubator
Disposable cell spreaders, sterile (Fisher or equivalent)
Inoculating turntable
1. Disperse subgingival plaque sample in 0.5 ml RTF by vortexing 10 sec. Dilute
serially in 96-well microtiter plate with 0.2 ml per well OMIZ-P4 medium containing
rifampicin and phosphomycin.
Typically, dilutions of 1 105 or 1 106 result in growth in 50% of wells (Wyss et al.,
1996).
Conduct these manipulations on the benchtop with deliberate speed.
12B.1.9
Current Protocols in Microbiology
Materials
T. denticola broth culture (see Basic Protocol 1)
3
H-, 14 C-, or 32 P-labeled substrate
Phosphate-buffered saline (PBS; see APPENDIX 2A)
15-ml polypropylene centrifuge tubes with caps (Corning or equivalent)
Anaerobic jar (Becton Dickinson GasPak or equivalent; see Strategic Planning)
1. Grow T. denticola broth cultures to early logarithmic phase in 15-ml polypropylene
tubes in an anaerobic chamber.
This type of tube is used for growth of cultures in these experiments in order to minimize
transfer and handling of radioactive materials.
2. Remove the cultures from the chamber, uncap the tubes, and add the radioactive
label using conditions appropriate to the particular radioisotope.
The choice of radiolabeled substrate will depend on the cell component being studied.
3. Recap the tubes, place them in an anaerobic jar or pouch system and activate the
gas/catalyst system.
4. Incubate at 37 C in a standard incubator for 12 to 24 hr as required for efficient
labeling.
Time courses for more complex experiments such as pulse-chase studies must be determined empirically.
12B.1.10
Current Protocols in Microbiology
Spirochetes
12B.1.11
Current Protocols in Microbiology
Laboratory
Maintenance of
Treponema
denticola
12B.1.12
Current Protocols in Microbiology
12B.1.13
Current Protocols in Microbiology
10.0 ml neopeptone solution (see recipe for OMIZ-P4 supplement stock solutions)
Adjust pH to 6.9 with NaOH
Filter sterilize using a 0.22-m pore-size filter
Complete OMIZ-P4 medium can be stored for at least 4 weeks at 4 C before use
Human serum is more physiologically relevant for human-associated organisms. Other
media recipes use rabbit serum for convenience and economy with, in most reports, little
apparent difference in T. denticola growth behavior.
Equivalent amounts of yeast extract powder may be substituted.
CaCl2 solution
14.7 g CaCl2 H2 O
100.0 ml 10 mM HCl
Store at 20 C
Cholesterol solution
20.0 mg cholesterol
20.0 ml ethanol
Store at 20 C
Cu-Mn-Zn solution
0.798 mg CuSO4
0.169 mg MnSO4 H2 O
0.287 g ZnSO4 7H2 O
1.0 liter 10 mM HCl
Store at 20 C
DHNA solution
40.8 mg 1,4-dihydroxy-2-naphthoic acid
100 ml ethanol
Store at 20 C
Ferrous sulfate solution
278.0 mg FeSO4 7 H2 O
100 ml 10 mM HCl
Store at 4 C
Glutathione solution
30.73 g glutathione, reduced (Sigma)
150.0 ml distilled water
Adjust pH to 7.0 with NaOH
Adjust volume to 200.0 ml with distilled water
Store at 20 C
Hemin solution
12.5 mg hemin
100.0 ml 10 mM NaOH
Store at 20 C
Laboratory
Maintenance of
Treponema
denticola
continued
12B.1.14
Current Protocols in Microbiology
Spirochetes
12B.1.15
Current Protocols in Microbiology
Laboratory
Maintenance of
Treponema
denticola
12B.1.16
Current Protocols in Microbiology
TYGVS supplements
To 1 liter TYGVS basal medium add:
0.0125 g thiamine pyrophosphate (TPP)
1.0 g glucose
1.0 g L-cysteineHCl
0.25 g sodium pyruvate
0.27 ml acetic acid (glacial)
0.1 ml propionic acid
0.064 ml n-butyric acid
0.016 ml n-valeric acid
0.016 ml isobutryic acid
0.016 ml isovaleric acid
0.016 ml D,L-methylbutyric acid
100 ml heat-inactivated rabbit serum (see recipe)
Mix well and adjust pH to 7.0 with 1 N KOH
Filter sterilize through a 0.2-m filter
The original reference (Ohta et al., 1986) calls for 10% rabbit serum. Horse or bovine
serum can also be used with little difference in growth.
COMMENTARY
Background Information
Oral spirochetes, most notably Treponema
denticola, are associated with aggressive
forms of periodontal diseases (Loesche et al.,
1982; Loesche et al., 1985; Socransky and
Haffajee, 1991). Periodontal lesions contain
a highly complex microflora in which proteolytic Gram-negative anaerobes predominate.
Spirochetes alone often comprise up to 50%
of total bacteria in periodontal lesions, particularly in subgingival plaques directly adjacent to gingival tissue (Listgarten, 1976). Inflammatory responses to this microbial challenge, modulated by several bacterial factors,
leads to tissue destruction, loss of attachment
at the junctional epithelium between the gingiva and the tooth, alveolar bone resorption,
and tooth loss. The formation of a deep periodontal pocket between the tooth and the gingiva, coupled with the inflammatory response,
further enhances the environment for growth
of proteolytic Gram-negative anaerobes, and
the process continues. The overall picture of
periodontal disease is complex, involving numerous bacterial factors and host inflammatory responses, but it is an eventual result of
bacterial challenge (Socransky and Haffajee,
1992).
A consortium of three proteolytic Gramnegative organisms, Porphyromonas gingivalis (see UNIT 13C.1), Tannerella forsythia
(formerly Bacteroides forsythus), and Treponema denticola, has the highest association
with periodontal disease severity (Socransky
et al., 1998), especially in refractory cases.
Spirochetes
12B.1.17
Current Protocols in Microbiology
painful ulcerations of infected sites, the disease results in considerable economic loss due
to decreases in body weight and milk production (Blowey and Sharp, 1988). Spirochetes
of the genus Treponema are among the most
predominant organisms isolated from these lesions (Walker et al., 1995). Of five distinct
Treponema phylotypes in digital dermatitis
lesions identified by 16S rRNA analysis, T.
denticola-like spirochetes predominated (Choi
et al., 1997). The strong association of these
organisms with chronic ulcerative diseases of
both humans and cattle is reflected in increased
interest in characterization of their involvement in disease progression.
Laboratory
Maintenance of
Treponema
denticola
medium contains a higher serum concentration and a greater variety of volatile fatty acids
than are found in NOS medium (see Reagents
and Solutions). Not surprisingly, T. denticola
growth is somewhat more rapid in TYGVS.
Some strains (especially recent clinical isolates) that do not grow well in NOS medium
grow very well in TYGVS or OMIZ-P4. On
the other hand, some laboratories have been
able to maintain T. denticola strains with fewer
volatile fatty acids than in the standard NOS
recipe, or with serum replaced by alternate carriers of long-chain fatty acids (Socransky and
Hubersak, 1967; Suzuki and Loesche, 1989);
however, omitting or reducing concentrations
of these or other components (such as serum)
can be expected to result in slower growth rates
and lower overall cell yields.
As noted above (see Strategic Planning),
standard Bacto agar is inhibitory to growth
of T. denticola, and probably to that of other
oral treponemes. The higher cost and inconvenience of working with the alternative Noble agar, gelatin, or agarose formulations described here is well rewarded by increased viability of T. denticola plated on or in these solid
media.
Dispersion by sonication, though the most
effective method in terms of total numbers
of bacteria recovered from highly aggregated
plaque samples, results in extremely low recovery of oral spirochetes from clinical samples. Transport of freshly collected plaque
samples in RTF and gentle dispersion by vortexing can result in greatly increased recovery
of cultivable spirochetes (Salvador et al., 1987)
when combined with selection with rifampicin
(or rifampicin plus phosphomycin) and plating
on appropriate rich media.
Unless otherwise specified, all incubations
and (where feasible) all manipulations described here are performed under strictly
anaerobic conditions in an anaerobic glove
box chamber (37 C, in a nonhumidified, 85%
N2 /10% H2 /5% CO2 atmosphere; see Strategic Planning). While T. denticola can survive
some exposure to oxygen, and media formulations described here provide considerable
protection from oxidative processes, prudence
suggests minimizing this exposure. The alternative of using anaerobic jars is certainly feasible and is preferable for experiments involving radioactive labeling (see Basic Protocol
5). This is due to the relative difficulty of handling and monitoring radioactivity in an anaerobic glove box chamber versus an anaerobic
12B.1.18
Current Protocols in Microbiology
Anticipated Results
Isolation of T. denticola and other oral treponemes from clinical samples is typically
very low-yield. In one well designed study in
which spirochetes were 30% of microscopic
counts in subgingival dental plaque samples, spirochetes (primarily T. denticola) comprised <0.05% of total cultivable organisms
(Salvador et al., 1987). Even with rifampicin
selection against the hundreds of other species
present in plaque samples, contaminants are
very common, and new spirochete isolates
must be cloned and characterized extensively
for culture purity.
T. denticola cultures may be maintained
by weekly passage in fresh, prereduced broth
medium at dilutions of between 1:20 and
1:100. Although there have been no studies
documenting changes in T. denticola physiology or behavior attributable to long-term
passaging, prudent microbiological practice
would encourage periodic replacement of
stock cultures. This is likely to be more relevant when working with recent clinical isolates
as opposed to common laboratory strains.
Growth rates vary between strains and in
different media. Of the two most commonly
used lab strains, ATCC #35405 tends to grow
at a slightly more rapid rate than does ATCC
#33520. This difference is more notable when
the strains are grown in NOS medium, and
less apparent in the more nutrient-rich TYGVS
medium. Unlike other oral anaerobes such as
Porphyromonas gingivalis, viability of T. denticola does not decrease rapidly after logarithmic growth in liquid media, and it is possible
(though not recommended) to recover strains
that have been incubated in broth for over a
week. Growth of T. denticola on solid media
is comparably slow, with subsurface colonies
appearing in 1 week, depending on the strain
and the medium. Colonies are seldom observed growing on top of the medium. Plates
inoculated by spreading on solid agar medium
tend to show spirochete colonies growing
down into the medium. The diffuse appearance of spirochete colonies is due to motility through the agar. As might be expected,
colonies are more diffuse and spreading in
lower-concentration agar formulations.
In addition, it is important to note that environmental conditions are rather more difficult to control for T. denticola than for
Time Considerations
As noted in Basic Protocol 1, the generation time of T. denticola is 12 hr, yielding 1 109 cells/ml after 4 days growth
(late log phase) in NOS medium. Media formulation and serum concentration influence
growth rate, and there is considerable straindependent variability in growth rates. If possible, pilot experiments should be conducted
to validate growth conditions and kinetics for
individual strains and individual laboratories.
Literature Cited
Aranki, A., Syed, S.A., Kenney, E.B., and Freter,
R. 1969. Isolation of anaerobic bacteria from
human gingiva and mouse cecum by means
of a simplified glove box procedure. Appl.
Microbiol. 17:568-576.
Blakemore, R.P. and Canale-Parola, E. 1976. Arginine catabolism by Treponema denticola. J.
Bacteriol. 128:616-622.
Blowey, R.W. and Sharp, M.W. 1988. Digital dermatitis in dairy cattle. Vet. Rec. 122:505-508.
Brewer, J.H. and Allgeier, D.L. 1966. Safe selfcontained carbon dioxide-hydrogen anaerobic
system. Appl. Microbiol. 14:985-988.
Carter, P.E., Abadi, F.J., Yakubu, D.E., and Pennington, T.H. 1994. Molecular characterization
of rifampin-resistant Neisseria meningitidis.
Antimicrob. Agents Ch. 38:1256-1261.
Chan, E.C., Siboo, R., Keng, T., Psarra, N.,
Hurley, R., Cheng, S.L., and Iugovaz, I. 1993a.
Treponema denticola (ex Brumpt 1925) sp. nov.,
nom. rev., and identification of new spirochete
isolates from periodontal pockets. Int. J. Syst.
Bacteriol. 43:196-203.
Chan, E.C., Siboo, R., Touyz, L.Z., Qui, Y.S., and
Klitorinos, A. 1993b. A successful method for
quantifying viable oral anaerobic spirochetes.
Oral Microbiol. Immun. 8:80-83.
Chan, E.C.S., DeCiccio, A., McLaughlin, R.,
Klitorinos, A., and Siboo, R. 1997. An inexpensive solid medium for obtaining colony-forming
units of oral spirochetes. Oral Microbiol. Immun. 12:372-376.
Cheng, S.L. and Chan, E.C. 1983. The routine isolation, growth, and maintenance of
the intermediate-size anaerobic oral spirochetes
from periodontal pockets. J. Periodontal Res.
18:362-368.
Spirochetes
12B.1.19
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Laboratory
Maintenance of
Treponema
denticola
12B.1.20
Current Protocols in Microbiology
Key References
Chan et al., 1993b. See above.
Chan et al., 1997. See above.
These two articles describe efficient and repeatable methods of growing T. denticola as subsurface
colonies.
Cheng and Chan, 1983. See above.
Loesche, 1969. See above.
This article documents oxygen sensitivity and other
growth requirements of T. denticola.
Salvador et al., 1987. See above.
These two articles describe detailed methods for
isolating Treponema species from clinical samples.
Smibert, 1973. See above.
Smibert, 1976. See above.
These two classic reviews by an early investigator of
anaerobic bacterial metabolism provide an excellent introduction into microbial physiology and the
biochemistry of spirochetes. It is important to note
that nomenclature of oral spirochetes was rather
fluid before the advent of ribotyping. Most strains
reported as T. microdentium, T. dentium, or T. oralis
are currently classified as T. denticola.
Wyss, 1992. See above.
This article describes in great detail a defined
medium capable of supporting growth T. denticola.
Spirochetes
12B.1.21
Current Protocols in Microbiology
UNIT 12B.2
BASIC
PROTOCOL 1
Spirochetes
Contributed by Howard K. Kuramitsu, Bo Chi, and Akihiko Ikegami
Current Protocols in Microbiology (2005) 12B.2.1-12B.2.7
C 2005 by John Wiley & Sons, Inc.
Copyright
12B.2.1
The development of shuttle plasmids for use in T. denticola now allows for the potential expression of heterologous spirochete genes in the oral spirochete. This is now possible even
for organisms that cannot be cultivated in the laboratory, e.g., T. pallidum (Chi et al., 1999;
Sliviensky-Gebhardt et al., 2004). Even though such genes can be expressed in E. coli,
their expression in treponemes should be advantageous in terms of functional analyses.
Materials
Electrocompetent T. denticola, ATCC #35405 or ATCC #33520 (see Support
Protocol 1)
Appropriate shuttle plasmid: pKMR4PE (Chi et al., 1999), pKMCou (Chi et al.,
2002), or pBFC (Sliviesnki-Gebhardt et al., 2004)
TYGVS medium (UNIT 12B.1), with and without appropriate antibiotic (Sigma) for
growth: erythromycin at 40 g/ml, chloramphenicol at 10 g/ml, or
coumermycin A1 at 10 g/ml
TYGVS medium (UNIT 12B.1) containing 0.8% (w/v) SeaPlaque agarose (VWR) and
appropriate antibiotic (Sigma): erythromycin at 40 g/ml (for pKMR4PE),
chloramphenicol at 10 g/ml (for pBFC), or coumermycin A1 at 10 g/ml (for
pKMCou)
Gene Pulser (Bio-Rad)
0.1-cm-gap electroporation cuvettes (Bio-Rad)
15-ml plastic screw cap test tubes (VWR)
Anaerobic chamber (UNIT 12B.1)
100 15mm petri dishes
Sterile inoculation loops or sterile Pasteur pipets
Additional reagents and equipment for cultivation of T. denticola (UNIT 12B.1)
1. Chill 80 l competent T. denticola on ice. Add 1 to 10 g of the appropriate shuttle plasmid (pKMR4PE, pKMCou, or pBFC). Mix the suspension well by gentle
pipetting and incubate 30 min on ice.
2. Transfer the mixture to an ice-cold electroporation cuvette, with a 0.1-cm gap, placed
in a Gene Pulser. With the pulse controller set at 1.8 kV, capacitance at 25 F, and
resistance at 200 , administer a single pulse to the cells with a time constant of
4.1 msec.
3. Immediately add 1.0 ml TYGVS medium without antibiotics. Mix gently by pipetting
up and down a few times, then transfer to a 15-ml plastic screw-cap test tube. Add an
additional 1.0-ml aliquot of medium. Incubate 18 hr at 37 C in an anaerobic chamber
with the cap only loosely applied to allow for gas exchange (also see UNIT 12B.1).
4. Place 1.0 ml of the suspension from step 3 into a 100 15mm petri dish, then
add 35 ml precooled (37 C) 0.8% SeaPlaque agarose in TYGVS medium containing the appropriate antibiotic (40 g/ml erythromycin for pKMR4PE, 10 g/ml
coumermycin A1 for pKMCou, or 10 g/ml chloramphenicol for pBFC). Mix well
by gentle rotation of the dish and allow to solidify 1 hr at room temperature.
5. Incubate the plates at 37 C face-up for 18 hr in an anaerobic chamber, then turn
face-down and incubate an additional 5 to 7 days.
6. Remove individual colonies along with plug of agarose using a sterile loop or sterile
Pasteur pipet. Transfer to 10 ml TYGVS medium containing the appropriate antibiotic. Incubate at 37 C to expand, then characterize for subsequent experiments.
Genetic
Manipulation of
T. denticola
For example, examine under phase contrast or dark-eld microscopy to check for purity,
or extract DNA for agarose gel analysis to conrm plasmid transfer.
12B.2.2
Current Protocols in Microbiology
SUPPORT
PROTOCOL 1
The electroporation protocol for T. denticola is based upon general procedures developed
previously for other bacteria such as E. coli, with some modications. In addition, a
heat-treatment step was introduced in order to minimize the effects of host restrictionmodication systems on heterologous transforming plasmids.
Materials
T. denticola, ATCC #35405 or ATCC #33520
TYGVS medium (UNIT 12B.1)
10% (v/v) glycerol, ice-cold
Anaerobic chamber
50 C water bath
Sorvall RC5 or equivalent refrigerated centrifuge
Additional reagents and equipment for cultivation of T. denticola (UNIT 12B.1)
1. Grow T. denticola (also see
late-log phase (48 to 72 hr).
UNIT 12B.1)
2. Take a 100-ml aliquot of the cell suspension. Heat to 50 C for 30 min, then chill on
ice for 30 min.
Heating increases the rate of plasmid transformation, probably by inactivating host
restriction-modication enzymes.
3. Wash cells three times, each time by centrifuging 10 min at 4000 g, 4 C, resuspending cells in 10 ml of 10% ice-cold glycerol, centrifuging again as before, then
removing the supernant. Resuspend pellet in 700 l 10% glycerol.
4. If desired, divide into 80-l aliquots and store indenitely at 70 C.
BASIC
PROTOCOL 2
Materials
Electrocompetent T. denticola, ATCC #35405 or ATCC #33520 (see Support
Protocol 2)
Plasmid pVA2198 (Fletcher et al., 1995; contact H. Fletcher, Loma Linda
University, Loma Linda, Calif.)
T. denticola (ATCC #35405 or ATCC #33520)
TYGVS medium (UNIT 12B.1) with and without 40 g/ml erythromycin (Sigma)
TYGVS medium (UNIT 12B.1) containing 0.8% (w/v) SeaPlaque agarose (VWR) and
40 g/ml erythromycin (Sigma)
Spirochetes
12B.2.3
Current Protocols in Microbiology
Materials
T. denticola, ATCC #35405 or ATCC #33520
TYGVS medium (UNIT 12B.1)
10% (v/v) glycerol, ice-cold
Anaerobic chamber
50 C water bath
Sorvall RC5 or equivalent refrigerated centrifuge
Additional reagents and equipment for cultivation of T. denticola (UNIT 12B.1)
Genetic
Manipulation of
T. denticola
12B.2.4
Current Protocols in Microbiology
2. Place the cells on ice for 15 min. Centrifuge 10 min at 4000 g, 4 C. Wash by
resuspending cells in 500 ml ice-cold distilled water, centrifuging again as before,
and removing the supernatant.
3. Resuspend cells in 250 ml ice-cold distilled water. Centrifuge as in step 2, then
resuspend cells in 10 ml ice-cold 10% glycerol.
4. Centrifuge cells and wash twice more, each time with 10 ml of 10% glycerol using
the technique described in step 2, to ensure complete removal of the medium salts.
After the last centrifugation, resuspend the cells in 1.0 ml of 10% glycerol.
5. Divide into 80-l aliquots and store indenitely at 70 C.
SUPPORT
PROTOCOL 3
The Erm cassette from plasmid pVA2198 (Fletcher et al., 1995), containing the ermF
and ermAM genes, is isolated from the plasmid following PvuII digestion and inserted
into the target gene fragment following cleavage (and blunt-ending with Klenow DNA
polymerase if necessary). The resulting plasmid is then linearized with a restriction
enzyme that cleaves a unique restriction site outside of the mutagenized target gene for
transformation.
It should also be possible to generate monospecic mutants using either the chloramphenicol or coumermycin resistance cassettes (Chi et al., 2002; Slivienski-Gebhart et al.,
2004) via the same approach as that utilized for the generation of erythromycin-resistant
mutants. In addition, these cassettes will allow for the isolation of mutants defective in
multiple genes of T. denticola.
COMMENTARY
Background Information
T. denticola strains are among the most
commonly occurring oral bacteria associated
with adult periodontitis (Socransky et al.,
1998). More recently, these organisms have
also been identied in cardiac tissue, and
it has been suggested that they are associated with atherosclerosis (Okuda et al., 2004).
Therefore, the identication of potential virulence factors in these organisms is of interest.
The development of techniques to construct
monospecic mutants of these organisms is
crucial for identifying some of these factors.
In addition, the ability to express genes from
shuttle plasmids in these organisms would allow for complementation of these mutants for
further verication of their respective roles in
pathogenicity. Moreover, these shuttle plasmids are also useful in expressing genes from
heterologous treponemes, including those that
cannot be readily cultivated in the laboratory
(Chi et al., 1999; Slivienski-Gebhardt et al.,
2004). Finally, these techniques might also be
Spirochetes
12B.2.5
Current Protocols in Microbiology
Genetic
Manipulation of
T. denticola
Anticipated Results
The frequency of transformation of T. denticola 33520 or 35405 was found to be approximately one colony per microgram of
transforming DNA for gene inactivation (Li
and Kuramitsu, 1996; Chi et al., 1999), but
almost one-hundred fold higher following
plasmid transformation (Chi et al., 2002).
Depending upon the antibiotic marker selected, some spontaneous mutant colonies
might also be detected. This is especially a
problem using plasmid pKMCou and Cou as a
selective marker (Chi et al., 2002; SlivienskiGebhardt et al., 2004). The utilization of Ermor Chl-resistance-expressing plasmids should
minimize the isolation of spontaneous antibiotic resistant colonies. However, if another
readily detectable phenotypic property can be
detected for the transformants on the agarose
plates (i.e., swarming), it is still possible to utilize plasmid pKMCou and the coumermycin
selection strategy (Chi et al., 2002).
Time Considerations
Because of the relatively slow growth rate
of T. denticola, 2 weeks are necessary from
initiation of the growth of the cells until transformants can be detected following electroporation. In addition, because of the relatively
low frequency of transformation of T. denticola, it is recommended that multiple electroporation experiments be carried out over the
span of several days prior to examining the results of the initial transformation.
Literature Cited
Cheng, S.-L., Siboo, R., Quee, T.C., Johnson, J.L.,
Mayberry, W.R., and Chan, E.C.S. 1985. Comparative study of six random oral spirochete isolates. J. Periodontal Res. 20:602-612.
12B.2.6
Current Protocols in Microbiology
Okuda, K., Kato, T., and Ishihara, K. 2004. Involvement of periodontopathic biolms in vascular
disease. Oral Dis. 10:5-12.
Scshadri, R., Meyers, G.S., Tettilin, H., Eisen,
J.A., Heidelberger, J.F., Dodson, R.J., Davidsen,
T.M., DeBoy, R.T., Fouts, D.E., Haft, D.H.,
Selengut, J. Ren, Q., Brinkac, L.M., Madupu,
R., Kolony, J., Durkin, S.A., Daughterty, S.C.,
Shetty, J., Shvartsbeyn, A., Gebregeorgis, E.,
Geer, K., Tsegaye, G., Malek, J., Ayodeji,
B., Shatsman, S., McLeod, M.P., Smajs, D.,
Howell, J.K., Pal, S., Amin, A., Vashisth, P.,
McNeill, T.Z., Xiang, Q., Sodergren, F., Baca,
E., Weinstock, G.M., Norris, S.J., Fraser, C.M.,
and Paulsen, I.T. 2004. Comparisons of the
genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc. Natl.
Acad. Sci. U.S.A. 101:5646-5651.
Slivienski-Gebhardt, L.L., Izard, J., Samsonoff,
W.A., and Limberger, R.J. 2004. Development
of a novel chloramphenicol resistance expression plasmid used for genetic complementation
of a iG deletion mutant in Treponema denticola.
Infect. Immun. 72:5493-5497.
Socransky, S.S., Haffajee, A.D., Cugini, M.A.,
Smith, C., and Kent, R.L.J. 1998. Microbial
complexes in subgingival plaque. J. Clin. Periodontol. 25:134-144.
Bo Chi
U.S. Food and Drug Administration
Bethesda, Maryland
Akihiko Ikegami
Case Western Reserve University
Cleveland, Ohio
Spirochetes
12B.2.7
Current Protocols in Microbiology
UNIT 12C.1
This unit describes the methods for growth and storage of Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. It provides the recipe of the standard culture medium,
Barbour-Stoenner-Kelly-II (BSK-II) and details its use in broth and solid culture. Methods for short and long-term storage of Borrelia cells are also provided.
CAUTION: B. burgdorferi is considered a Biosafety Level 2 (BSL-2) category organism.
Work should be performed in a BSL-2 biosafety hood. See UNIT 1A.1 and other pertinent
resources (APPENDIX 1B) for more information. The use of a biosafety hood also further
protects cultures from potential contaminants, which will readily outgrow the spirochetes
in the rich culture medium (see Commentary).
STRATEGIC PLANNING
Borrelia burgdorferi characteristics
Borrelia burgdorferi are slender, highly motile spirochetes 0.2- to 0.5-m in diameter and
up to 30 m in length. They can be visualized by phase-contrast or dark-field microscopy
(UNIT 2A.1). B. burgdorferi stain weakly Gram negative; other stains such as Giemsa or
Wright are more appropriate. B. burgdorferi are motile in both low- and high-viscosity
media due to periplasmic bundles of seven to eleven flagella attached at each end of the
cell cylinder. Intriguingly, these flagella also determine the spirochetes flat-wave cell
shape (Motaleb et al., 2000). Opaque white colonies form within or on the surface of
solid medium (see Basic Protocol 3). In general, colonies are regularly shaped, up to
3 mm in diameter, with a dense center and a diffuse border. However, colony size and
morphology can vary from strain to strain or even within clones of the same isolate (Elias
et al., 2002).
Borrelia sensitivity to detergents
The use of disposable labware is recommended since Borrelia cells are sensitive to
detergents, residues of which might be present on reusable plastic and glass labware.
All reusable labware, such as beakers for medium preparation, should be rinsed with
double-distilled water before use.
Stock solutions
The formulation for the standard Borrelia growth medium BSK-II, provided in Reagents
and Solutions, follows the recipe described by Barbour in 1984. The medium is made
in two steps: an incomplete or basal medium, which can be stored indefinitely at 4 C or
lower, and a complete medium, which is stored up to 2 to 3 weeks at 4 C. In addition to
the basal BSK-II medium, gelatin or agarose stock solutions for liquid or solid BSK-II,
respectively, are prepared beforehand and stored indefinitely at room temperature. Rabbit
serum is aliquoted in ready-to-use volumes and stored indefinitely at 20 C or lower.
CMRL-1066
CMRL-1066 without L-glutamine is available as liquid or powder from a limited number
of companies, e.g., Invitrogen (1 solution), or U.S. Biological (powder). The 10
solutions previously sold by different suppliers (e.g., Invitrogen and Sigma) have been
discontinued. In powder form, CMRL-1066 should be stored protected from light and
humidity (e.g., in a dessicator) at room temperature. Solutions should be stored protected
from light at 4 C.
Spirochetes
12C.1.1
Supplement 4
Materials
Complete BSK-II medium (see recipe)
Late-exponential-phase B. burgdorferi culture or frozen stock (see Basic
Protocol 2)
8-ml polystyrene round-bottom screw cap tubes (e.g., Falcon 352027)
34 C incubator
Petroff-Hausser counting chamber (optional; APPENDIX 4A)
1. Place 6 ml fresh complete BSK-II medium into a sterile 8-ml screw cap tube.
2. Inoculate with 50 to 500 l of a late-exponential-phase B. burgdorferi culture (about
5 107 cells/ml) or with entire 1 ml of stock (Basic Protocol 2) immediately after
thawing it at room temperature or in 34 C incubator.
Microscopic assessment of thawed stock culture viability (i.e., motility) is suggested prior
to inoculation. Thawed stocks should not be refrozen to preserve optimal cell viability.
Cultures can also be inoculated with scrapings from a frozen stock surface using a sterile
toothpick or platinum loop. Yet, inoculation with the entire stock culture is suggested to
prevent selection of a subpopulation of cells (see Commentary).
12C.1.2
Supplement 4
BASIC
PROTOCOL 2
Materials
Late-exponential-phase B. burgdorferi BSK-II culture (Basic Protocol 1)
Sterile 100% DMSO or 80% (v/v) glycerol solution
2-ml polypropylene cryogenic vial (Corning cat no. 430659), sterile
1a. To cryopreserve with DMSO: Add 900 l of a late-exponential-phase B. burgdorferi
BSK-II culture to 100 l of sterile DMSO into a sterile 2-ml polypropylene cryogenic
vial.
1b. To cryopreserve with glycerol: Add 750 l of a late-exponential-phase B. burgdorferi
BSK-II culture to 250 l of a sterile (i.e., autoclaved) solution of 80% (v/v) glycerol
in distilled water into a sterile 2-ml polypropylene cryogenic vial.
2. Mix thoroughly by pipetting or vortexing.
3. Transfer tubes to a 80 C freezer for indefinite storage.
Standard protocols for freezing bacterial cultures on dry ice or in liquid nitrogen prior to
storage can be followed, but no empirical benefit for cell survival has been observed.
PLATING OF B. BURGDORFERI
The following protocol details the modification of BSK-II for subsurface plating of
Borrelia burgdorferi isolates as described by Hinnebusch and Barbour (1992). This
technique, in addition to limiting dilutions in liquid broth, allows for the cloning of
B. burgdorferi isolates and represents an essential step in molecular genetics experiments.
The plates consist of a bottom and top agar containing agarose and BSK-II medium, with
the bacteria embedded in the top agar. In contrast to the air-tight culture tubes, the
air-permeable plates need to be incubated in a CO2 atmosphere.
BASIC
PROTOCOL 3
Three stock solutions need to be made beforehand: a 2 BSK-II solution, and two
separate agarose solutions. In comparison to the liquid BSK-II medium, the 2 solution
lacks gelatin. All stock solutions need to be prewarmed prior to use and can be kept at
their appropriate temperatures for several hours.
Plates are poured in two steps: First, the bottom agar is poured and allowed to solidify.
Next, B. burgdorferi cells are added to the top agar and plated on top of the bottom agar.
Solidified culture plates are then incubated in a CO2 atmosphere.
Materials
2% (top) and 3% (bottom) agarose stock (see recipe)
2 BSK-II (see recipe), 37 and 45 C
B. burgdorferi cell suspension (in BSK-II or PBS; Basic Protocol 1)
Spirochetes
12C.1.3
Current Protocols in Microbiology
Supplement 4
6. Add 5 ml melted 2% top agarose and mix by inverting. Pour immediately on top of
bottom agar and allow to solidify.
The final 1% low melt top agar solidifies much slower than the bottom agar, i.e., within
about 2 min. This allows for a less frantic pace of plate pouring.
8. Pick agarose plugs containing the bacterial colonies using sterile 1000-l pipet tips
or Pasteur pipets.
9. Eject plugs into medium to inoculate liquid cultures or squash under coverslips on
glass slides for microscopic observation (UNIT 2A.1).
Alternatively, sterile toothpicks can be used for inoculation of liquid medium.
Laboratory
Maintenance of
B. burgdorferi
12C.1.4
Supplement 4
1000 ml H2 O
9.7 g CMRL-1066 (see Strategic Planning)
50.0 g BSA fraction V
5.0 g neopeptone
6.6 g HEPES, sodium salt
0.7 g sodium citrate
5.0 g glucose
2.0 g yeastolate
2.2 g sodium bicarbonate
0.8 g pyruvic acid
0.4 g N-acetyl-glucosamine
Spirochetes
12C.1.5
Supplement 7
Filter solution through a 0.45-m filter unit. Place a glass-fiber prefilter on top of
the 0.45-m cellulose acetate filter unit to reduce clogging. Apply vacuum and wet
the glass-fiber prefilter with 10 ml medium before pouring the remaining volume
into the filter unit. Change filter unit if it becomes clogged and flow rate declines
significantly. Store indefinitely in 500-ml filter-bottle units protected from light at
4 C or lower.
The use of low-protein-binding cellulose acetate 0.45-m filter-bottle systems in combination with glass fiber prefilters (e.g., Corning no. 430770 with no. 431353) is suggested to
prevent clogging during medium filtration. Prewetting attaches the prefilter to the filter and
prevents it from floating when the rest of the medium is poured into the filter unit.
The use of HEPES sodium salt instead of HEPES free acid leads to a final pH of the
medium that is close to the desired 7.6. Thus, adjusting the pH with 1 M NaOH is usually
not necessary when HEPES salt is used.
Although this recipe uses CMRL-1066 in powder form, the water and CMRL-1066 powder
can be replaced with 1 CMRL-1066 solution, available commercially. The addition of
solid medium components, mainly BSA, will increase the volume of prepared incomplete
BSK-II to about 1200 ml.
Also see Strategic Planning.
Gelatin solution, 7%
Dissolve 7 g gelatin in 100 ml double-deionized water in a 250-ml glass bottle
previously rinsed with distilled water (to remove potential detergent residues; see
Strategic Planning). Autoclave 20 min at 121 C on liquid cycle. For immediate
use, cool to 37 C in a water bath. Alternatively, 100-ml aliquots can be stored
indefinitely at room temperature. Warm to 37 C before use.
Heating in a microwave or on a heating plate can be used to dissolve the gelatin, but
autoclaving is the most effective way to get it fully into solution.
COMMENTARY
Background Information
Epidemiology
Borrelia spirochetes are the causative
agents of vector-borne Lyme borreliosis and
relapsing fever (Barbour, 2003). Tick-borne
Lyme borreliosis, caused by B. burgdorferi
sensu lato (Burgdorfer et al., 1982; Steere
et al., 1983), is the most common vector-borne
disease in the United States and is endemic
in moderate climates of the Northern hemisphere, including Europe and Asia. Tick-borne
relapsing fever, caused by a variety of Borrelia species, is a relatively rare infectious syndrome in the Western United States from Texas
to Washington State, but more common in subSaharan Africa, the Middle East, and Central
Asia. A louse-borne form of relapsing fever
has surfaced in areas affected by war, poverty,
and overcrowding (Barbour, 2003).
Laboratory
Maintenance of
B. burgdorferi
the most fragmented of any prokaryote analyzed to date. A 1-megabase linear chromosome is accompanied by multiple linear and
circular plasmids ranging in size from about 5
to 50 kb. The recent genome analysis of other
Borrelia, including another B. burgdorferi
sensu lato strain (B. garinii Pbi; Glockner
et al., 2004), as well as two relapsing fever
B. hermsii and B. turicatae strains (Guyard
et al., 2005), confirmed earlier studies (Casjens
et al., 1995; Palmer et al., 2000; Stevenson
et al., 2000) that the overall genome structure
of Borrelia spirochetes is conserved.
Most of the B. burgdorferi plasmids provide genes essential for survival in the infectious tick-mammal cycle and are readily lost
upon in vitro propagation (Schwan et al., 1988;
Sadziene et al., 1993a; see Critical Parameters and Troubleshooting, Borrelia Stocks:
Plasmid Instability). Yet, at least one of these
extrachromosomal elements, a 26-kb circular plasmid, carries genes essential for in
vitro growth (Byram et al., 2004), such as a
telomere resolvase essential for the replication
of the bacteriums linear replicons (Kobryn
12C.1.6
Supplement 7
and Chaconas, 2002). This extrachromosomal presence of essential genes has led to the
proposed term minichromosome (Barbour,
1993) for some of these plasmids.
Although the genetic tools to manipulate
B. burgdorferi are not as numerous and refined as for other pathogens, they are sufficiently advanced to allow for detailed studies
of bacterial physiology and virulence (comprehensively reviewed in Rosa et al., 2005).
B. burgdorferi can therefore serve as a model
organism for this bacterial phylum and as a
transgenic expression host to study other
spirochetal virulence factors (Schwan et al.,
2003; Zuckert et al., 2004).
Cultivation of Borrelia and other spirochetes
The cultivation of spirochetes has been a
significant challenge for researchers. As a particularly stark example, the syphilis spirochete
Treponema pallidum can only be recovered in
significant numbers from the testicles of experimentally infected rabbits (Stamm et al., 1987;
also see UNIT 12A.1). Borrelia lonestari, the
suspected causative agent of Southern Tickassociated rash illness (STARI; Armstrong
et al., 1996; Barbour et al., 1996), long considered uncultivable due to lack of growth in
BSK-II and other media, was recently recovered from infected ticks in a coculture with an
embryonic tick cell line (Varela et al., 2004), an
approach that was previously used to study
B. burgdorferi-tick cell interactions (Kurtti
et al., 1988, 1993). In vitro cultivation methods are available for many other spirochetes
(see other units in this chapter).
The current BSK-II medium is based on
a formulation by Kelly, which supported the
continual growth of B. hermsii (Kelly, 1971).
The recipe was subsequently enriched by
Stoenner to enable the outgrowth of a single B.
hermsii cell (Stoenner et al., 1982). Ultimately,
this fortified Kelly medium was modified by
Barbour (Barbour, 1984) to reliably achieve
high spirochete densities (1 to 4 108 /ml),
generation times of 11 to 12 hr and outgrowth
of low inocula, i.e., one to two infectious organisms of both Lyme disease and relapsing
fever spirochetes. Modifications of the BSKII recipe also showed that relapsing fever borreliae are more fastidious than B. burgdorferi
(Barbour, 1984).
Variations of the above described Borrelia
culture medium, e.g., modified Kelly medium
and BSK-H, have been used in different laboratories. Modified Kelly (MK) medium is
detailed by Preac-Mursic et al. (1986) and
Spirochetes
12C.1.7
Current Protocols in Microbiology
Supplement 4
cultures can be passaged through BSK-II containing these two antibiotics. Final concentrations are 50 g/ml rifampicin (from a stock of
50 mg/ml in DMSO) and 100 g/ml phosphomycin (from a stock of 100 mg/ml in distilled
water). Other compounds that might help in removing contaminants are nalidixic acid (100
g/ml) and 5-fluorouracil (200 g/ml; Barbour, 1984). If needed, 100 g/ml of the fungicide amphotericin B (e.g., Fungizone) can be
used to supress fungal growth. Although these
compounds should be superfluous during standard laboratory maintenance of B. burgdorferi,
they can also be added to BSK-II to prevent
contamination.
Borrelia stocks: plasmid instability
Researchers should be keenly aware of the
well-documented phenomenon of plasmid instability: the majority of Borrelia burgdorferi plasmids (see Commentary above) are
dispensable for in vitro growth and readily lost upon extended cultivation. Therefore,
passages should be kept to a minimum and
cell stocks should be generated as early as
possible.
Anticipated Results
Properly prepared BSK-II should support
the outgrowth of one to two Borrelia cells to
an approximate density of 108 cells. Assuming exponential growth conditions and cell divisions every 5 to 18 hr, this would require
<30 generations, i.e., 6 days to 3 weeks. Microscopic observation of a wet mount under phase-contrast or dark-field microscopy
(UNIT 2A.1) should show motile spirochetes.
Large aggregates of bacterial cells (clumps)
have been observed, especially in early passages of original isolates (Barbour, 1984).
These aggregates can be temporarily dissolved
by vortexing or pipetting and have no significant impact on further cultivation.
Borrelia grow in solid BSK-II in white,
opaque colonies. Depending on the strain, they
are small (<1 mm) and pointed, to larger (2
to 4 mm) and diffuse. Colonies should appear
within 1 to 4 weeks of plating.
Time Considerations
Laboratory
Maintenance of
B. burgdorferi
Acknowledgments
The author wishes to thank Dr. Alan G.
Barbour, University of California at Irvine, for
comments on the manuscript.
Literature Cited
Armstrong, P.M., Rich, S.M., Smith, R.D., Hartl,
D.L., Spielman, A., and Telford, S.R. III 1996.
A new Borrelia infecting Lone Star ticks. Lancet
69:67-68.
Babb, K., El-Hage, N., Miller, J.C., Carroll,
J.A., and Stevenson, B. 2001. Distinct regulatory pathways control expression of Borrelia
burgdorferi infection-associated OspC and Erp
surface proteins. Infect. Immun. 69:4146-4153.
Barbour, A.G. 1984. Isolation and cultivation of
Lyme disease spirochetes. Yale J. Biol. Med.
57:521-525.
Barbour, A.G. 1993. Linear DNA of Borrelia
species and antigenic variation. Trends Microbiol. 1:236-239.
Barbour, A.G. 2003. Antigenic variation in Borrelia: Relapsing fever and Lyme borreliosis. In
Antigenic Variation (A. Craig, and A. Scherf,
eds) pp. 319-356. Academic Press, London.
Barbour, A.G., Maupin, G.O., Teltow, G.J., Carter,
C.J., and Piesman, J. 1996. Identification of
an uncultivable Borrelia species in the hard
tick Amblyomma americanum: Possible agent
of a Lyme disease-like illness. J. Infect. Dis.
173:403-409.
Burgdorfer, W., Barbour, A.G., Hayes, S.F.,
Benach, J.L., Grunwaldt, E., and Davis, J.P.
1982. Lyme disease-a tick-borne spirochetosis?
Science 216:1317-1319.
Byram, R., Stewart, P.E., and Rosa, P. 2004. The
essential nature of the ubiquitous 26-kilobase
circular replicon of Borrelia burgdorferi. J. Bacteriol. 186:3561-3569.
Casjens, S., Delange, M., Ley, H.L.R., Rosa, P.,
and Huang, W.M. 1995. Linear chromosomes of
Lyme disease agent spirochetes: Genetic diversity and conservation of gene order. J. Bacteriol.
177:2769-2780.
Casjens, S., Palmer, N., van Vugt, R., Huang, W.M.,
Stevenson, B., Rosa, P., Lathigra, R., Sutton, G.,
Peterson, J., Dodson, R.J., Haft, D., Hickey, E.,
Gwinn, M., White, O., and Fraser, C.M. 2000.
A bacterial genome in flux: The twelve linear
and nine circular extrachromosomal DNAs in an
infectious isolate of the Lyme disease spirochete
Borrelia burgdorferi. Mol. Microbiol. 35:490516.
12C.1.8
Supplement 4
Spirochetes
12C.1.9
Current Protocols in Microbiology
Supplement 4
Stevenson, B., Porcella, S.F., Oie, K.L., Fitzpatrick, C.A., Raffel, S.J., Lubke, L., Schrumpf,
M.E., and Schwan, T.G. 2000. The relapsing
fever spirochete Borrelia hermsii contains multiple, antigen-encoding circular plasmids that
are homologous to the cp32 plasmids of Lyme
disease spirochetes. Infect. Immun. 68:39003908.
Stoenner, H.G., Dodd, T., and Larsen, C. 1982.
Antigenic variation of Borrelia hermsii. J. Exp.
Med. 156:1297-1311.
Varela, A.S., Luttrell, M.P., Howerth, E.W., Moore,
V.A., Davidson, W.R., Stallknecht, D.E., and
Little, S.E. 2004. First culture isolation of
Borrelia lonestari, putative agent of southern
tick-associated rash illness. J. Clin. Microbiol.
42:1163-1169.
Wang, G., Iyer, R., Bittker, S., Cooper, D., Small,
J., Wormser, G.P., and Schwartz, I. 2004.
Variations in Barbour-Stoenner-Kelly culture
medium modulate infectivity and pathogenicity
of Borrelia burgdorferi clinical isolates. Infect.
Immun. 72:6702-6706.
Key References
Barbour, 1984. See above
Describes the cultivation of Borrelia in BSK-II
medium, including its original recipe.
Hinnebusch & Barbour, 1992. See above
Describes the cloning of B. burgdorferi by subsurface plating in BSK-II agar.
Laboratory
Maintenance of
B. burgdorferi
12C.1.10
Supplement 4
UNIT 12C.3
The dialysis membrane chamber (DMC) model is an animal model for studying changes
in bacterial gene expression in response to growth within the mammalian host environment, a process referred to as host adaptation. Briey, diluted bacterial cultures are
placed within chambers constructed from sterile dialysis membrane tubing (DMCs) and
implanted into the peritoneal cavities of rats. Following a 9- to 12-day incubation, DMCs
are recovered by explantation and the resulting host-adapted bacteria are harvested for
subsequent analyses. Bacteria cultivated in this manner are suitable for use with a wide
range of molecular techniques including, but not limited to, quantitative PCR/RT-PCR
(UNIT 1D.3), microarray analyses, immunoblotting, and proteomics. This unit outlines a
method for preparing bacteria-lled dialysis membrane chambers, surgically implanting
them into rat peritoneal cavities, and then isolating the resulting host-adapted bacteria
(see Basic Protocol). A method for preparing sterile dialysis tubing is also described (see
Support Protocol).
BASIC
PROTOCOL
In this technique, a small length of sterile dialysis membrane tubing, lled with a diluted
culture aliquot of Borrelia burgdorferi, is tied off at both ends to form a chamber that
is then surgically implanted into the peritoneal cavity of a small mammal, typically
a rat. The spirochetes inside the DMC are then cultivated within the mammalian host
for a period of 9 to 12 days, following which the DMC is explanted and the dialysate
containing the host-adapted spirochetes is harvested. Although spirochetes have been
shown to survive for as long as 6 months post-implantation, the overall viable spirochetal
density does not appear to increase above that observed at the 9- to 12-day incubation
period. The spirochete material resulting from normal culture turnover will be retained
within DMCs and may reduce the quality of the explanted bacterial cultures obtained.
This protocol describes preparation of bacteria-lled DMCs and the subsequent
surgical implantation procedure. In addition to being used in numerous studies to
examine various aspects of Lyme disease pathogenesis, this procedure has been used
to cultivate the pathogenic spirochete Leptospira interrogans, as well as Pseudomonas
aeruginosa (Mashburn et al., 2004). It may also be modied for use with other bacterial
pathogens.
CAUTION: Borrelia burgdorferi is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: This experiment requires Animal Biosafety Level 2 (ABSL-2) conditions.
Follow all appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1
and other pertinent resources (APPENDIX 1B) for more information. Protocols using live
animals must rst be reviewed and approved by an Institutional Animal Care and Use
Committee (IACUC) or must conform to governmental regulations regarding the care
and use of laboratory animals.
IMPORTANT NOTE: The success of this protocol is dependant on the proper use of
aseptic techniques during all stages of both dialysis membrane chamber preparation and
animal surgery.
Spirochetes
Contributed by Melissa J. Caimano
Current Protocols in Microbiology (2005) 12C.3.1-12C.3.13
C 2005 by John Wiley & Sons, Inc.
Copyright
12C.3.1
Materials
Female Sprague-Dawley rats weighing 150 to 174 g upon arrival
70% ethanol in spray bottle
BSK-H (or BSK-II) medium (Sigma), prewarmed to room temperature
Borrelia burgdorferi growing in culture (UNIT 12C.1)
Ketamine/xylazine anesthetic cocktail (see recipe)
Ophthalmic ointment, sterile (e.g., Puralube from Pharmaderm Animal Health)
0.3 mg/ml buprenorphineHCl (e.g., Buprenex Injectable)
Betadine surgical scrub solution
10-ml individually wrapped, sterile disposable pipets
Surgical drape, cut into 18-in. (46-cm) squares, sterile
Battery-operated pipet ller (e.g., Pipet-Aid, Drummond Scientic)
Petroff-Hausser counting chamber (Hausser Scientic)
50-ml conical polypropylene centrifuge tubes
Surgical gloves, sterile, individually wrapped
Sterile blunt-end forceps
Sterile dialysis tubing (see Support Protocol)
Sterile petri dishes
0.5-ml syringes with 28-G, 1/2-in. (1.3-cm) needles
Sterile 4 4in. (10.2 10.2cm) gauze
Surgical instruments, sterilized and kept within sterile packaging inside surgical
eld:
Scalpel blade and holder, no. 10
4-in. (10.2-cm) iris scissors (optional)
Tissue forceps (1 2tooth dissecting or Adson-Brown, available from Henry
Schein Medical)
5 1/2-in. (14-cm) needle holder with built-in scissors (e.g., Olsen Hegar,
available from Henry Schein Medical)
Ethicon 4-0, SH-1, 27-in. (68.6-cm) coated Vicryl, violet-braided suture
(available from Henry Schein Medical)
Auto-clip 9-mm stainless steel wound closure clips and applicator (Fisher
Scientic)
10-ml syringe with 18-G, 1-in. (2.54-cm) needle, sterile
Additional reagents and equipment for injection (Donovan and Brown, 1995a) and
euthanasia (Donovan and Brown, 1995b) of rodents
1. Acclimate female Sprague-Dawley rats, weighing 150 to 174 g upon arrival, to the
animal facility environment for no less than 1 week prior to surgical procedure.
Animals should be acclimated under standard lighting and temperature conditions with
food and water ad libitum.
DMC Cultivation
of B. burgdorferi
12C.3.2
Current Protocols in Microbiology
Each dialysis membrane chamber will require 8 ml medium containing spirochetes, and
each bacterial isolate should be implanted in a minimum of two rats per experiment.
6. Place tubes containing the diluted culture(s) within the surgical drape area (see step 2).
Spray the outside of each tube with 70% ethanol and wipe with a Kimwipe. Gently
loosen the lid of each tube to allow for easy removal during subsequent steps
7. Place one of the disposable 10-ml pipets that were previously placed in the surgical
eld (step 1) into the nosepiece of the cleaned pipet ller (step 3), but do not remove
from the sterile wrapping until just prior to use.
9. Using sterile blunt-end forceps, remove a tubing strip from its storage container and
place between the index nger and thumb of the free hand. With the other hand,
remove the cap from the tube containing the diluted culture and transfer up to 8 ml
to the interior of the tubing.
Dialysis membrane tubing must be prepared as described in the Support Protocol. One
end of the tubing will already have been tied off at this point (Support Protocol, step 2).
10. While continuing to hold the open end, atten the unlled part of the tubing to
eliminate any air bubbles that may have been introduced during lling. Gently twist
the lled tubing until essentially all remaining void volume has been eliminated and
the tubing becomes taut.
Try to make the dialysis membrane chamber as taut as possible. This will help to maneuver
the chamber into the rat peritoneal cavity during surgery.
11. Begin tying off the top of the tubing strip by holding the twisted tubing tightly
between thumb and index nger, and wrapping the loose tubing end one time around
the index nger.
12. Bring the open tubing end through the loop that was made around the index nger
so as to create a knot, moving the index nger just enough to allow the open tubing
end to pass through the loop. Hold the twisted tubing closed by switching the grasp
to thumb and middle nger if necessary.
13. Once the open tubing end has been pulled through the twisted tubing loop, pull down
on the knot while holding the open end to tighten.
It may be easier to use blunt-end forceps to manipulate the open tubing end as it is passed
through the loop.
The resulting lled, tied dialysis membrane chamber should be no more than 1.5 in.
(3.8 cm). It is important that the maximum culture volume (i.e., 8 ml) not be exceeded
when lling the dialysis tubing. Larger DMCs may result in animal discomfort due to
interference with normal intestinal or bladder functions.
14. Once a dialysis membrane chamber has been prepared, place it into a sterile storage
container (e.g., petri dish) containing enough BSK-H (or BSK-II) medium to partially
cover each chamber.
It is not recommended that lled dialysis membrane chambers be stored in this manner
for extended periods of time, as this may increase the risk of contamination. Once lled,
proceed directly to preoperative preparation of animals.
Perform preoperative preparation of animals.
Spirochetes
12C.3.3
Current Protocols in Microbiology
16. Peform a toe pinch on the animal to ensure anesthesia. If the animal inches or
otherwise responds to the pinch, wait 5 min before checking again. If the animal
continues to be responsive, administer a second dose of anesthetic cocktail equivalent
to half of the original dose, wait 10 min, then retest by toe pinch.
If difculties are experienced even after a second dosage, contact institutional veterinary
staff for technical assistance.
Once animal becomes unresponsive to toe pinch, it is safe to begin.
17. Transfer the anaesthetized animal to the surgical eld, placing it supine on a fresh
sterile drape. Apply a small amount of ophthalmic ointment to each eye. Check that
the rats tongue has not fallen back into the throat, potentially obstructing its airway.
18. Administer preoperative analgesic medication (i.e., buprenorphineHCl, Buprenex)
by subcutaneous injection at 0.05 to 0.1 mg/kg body weight using a 0.5-ml syringe
with a 28-G, 1/2-in. (1.3 cm) needle.
19. Test the level of anesthesia by performing a second toe pinch and begin preparing
the implant site by cleaning with 4 4 sterile gauze soaked in Betadine surgical
scrub solution. Once nished cleaning the implant site, put on a fresh pair of surgical
gloves. From this point on, avoid contact with any nonsterile areas.
20. Open a fresh sterile surgical instrument pack within the surgical eld, leaving the
instruments in their sterile packaging until needed.
DMC Cultivation
of B. burgdorferi
Figure 12C.3.1
12C.3.4
Current Protocols in Microbiology
22. Using tissue forceps, hold up a section of abdominal muscle to avoid injuring any
internal organs, then make a small vertical midline incision using either the scalpel
blade tip or, alternatively, 4-in. (10.2-cm) iris scissors.
23. Open the abdominal wall to expose the peritoneal cavity by extending the length of
the incision to 2 in. (5.1 cm), or slightly smaller than the outer (rst) incision
(Figure 12C.3.1).
Use the white line along the abdomen (linea alba) as a guide to keep the incision straight
and clean.
24. Using tissue forceps, hold up one side of the abdominal incision and, with the other
hand, use blunt-end forceps to remove one dialysis membrane chamber from the
medium in which it was placed at step 14. Carefully situate the DMC within the rat
peritoneal cavity by pushing it to either side of the peritoneal cavity.
26. Use sterile 9-mm wound closure clips to close the outer incision.
It is helpful to grasp both sides of the skin together with tissue forceps held in the right
hand while applying the wound closures with the left, moving down the incision in one
contiguous line until the incision is completely closed.
27. Return the rat to a cage containing fresh bedding, overlaid with a sterile surgical
drape. To help maintain body temperature, apply gentle heating to the cage bottom
using a circulating water pad until animals are alert and responsive.
If a circulating water pad is not available, one may also use an incandescent lamp (50-to
75-W) placed 12 to 14 in. (30.5 to 35.6 cm) away from the animal to provide supplemental
heat.
Be sure to closely monitor the heat being applied to ensure that the animal does not
become overheated.
Spirochetes
12C.3.5
Current Protocols in Microbiology
33. Use tissue forceps to grasp the sutured inner incision and scissors to cut an opening
larger than the original implant opening, to give a better view of the peritoneal
cavity.
34. Locate and remove the dialysis membrane chamber using blunt-ended forceps. Transfer to a sterile, empty petri dish.
35. To ensure death, bisect the thoracic cavity using surgical scissors following DMC
explantation.
CAUTION: Dispose of the animal as infectious material.
36. Remove the chamber dialysate using an 18-G, 1-in. (2.54-cm) needle attached to a
sterile 10-ml syringe by holding one end of the explanted dialysis membrane chamber
with blunt-ended forceps. Gently transfer the uid from the syringe to a centrifuge
tube.
CAUTION: Be careful to avoid needle stick with infectious material.
Materials
1 mM EDTA, pH 8.0
Three 2-liter sterile Pyrex beakers
Regenerated dialysis membrane tubing (Spectra/Por 6, 6000 to 8000 MWCO,
32-mm width; Spectrum Labs)
Surgical gloves, sterile, individually wrapped
5 1/2 in. (14 cm) straight surgical scissors, sterile
11 13/16 in. (30 cm) extra-long blunt-end forceps, sterile
250-ml sterile Pyrex beakers or 150-mm sterile petri dishes with lids
DMC Cultivation
of B. burgdorferi
12C.3.6
Current Protocols in Microbiology
2. Wearing sterile surgical gloves, cut a strip of dialysis tubing 18- to 20-cm (6- to 7-in.)
in length using sterile surgical scissors. Using ngers, tie off one end of the tubing.
Remove any excess tubing at the tied end using sterile scissors.
When rst learning to handle the tubing, it may be helpful to cut longer strips. Any excess
tubing will be removed from the open end during subsequent steps.
3. Transfer the tied strip of dialysis membrane tubing to one of the beakers containing
sterile Milli-Q water (Beaker 1). Replace the aluminum foil cover to maintain sterility.
4. Cut and tie off additional tubing strips as required, preparing one strip of tubing for
each rat to be implanted. Transfer to Beaker 1, replacing the aluminum foil lid after
each addition.
It is recommended that at least one extra bag be added in case a bag becomes contaminated
during subsequent steps.
8. While strips are boiling, place Beaker 3 (containing water) on a hot plate/magnetic
stirrer and bring to a rolling boil with constant stirring.
9. Transfer tubing strips from Beaker 2 to Beaker 3 using sterile extra-long blunt-end
forceps. Replace sterile aluminum foil cover and continue to boil tubing strips for
20 min with constant stirring.
10. Turn off hot plate/magnetic stirrer and allow tubing to cool until the outside surface
of Beaker 3 is warm to the touch.
The tubing is now ready to use. Although it is preferable to use freshly prepared dialysis
membrane tubing, strips may also be prepared several days in advance and stored at 4 C
submerged in sterile Milli-Q water. If prepared in advance, however, dialysis membrane
tubing should be stored in small quantities of tubing per container (i.e., 5 to 9 strips per
container), then opened only once using strict aseptic technique to avoid contamination.
Spirochetes
12C.3.7
Current Protocols in Microbiology
11. If desired for convenience, transfer tubing to a smaller container (e.g., 250-ml sterile
Pyrex beaker or 150-mm sterile Petri dish with lid) containing freshly autoclaved
Milli-Q water.
COMMENTARY
Background and Information
DMC Cultivation
of B. burgdorferi
12C.3.8
Current Protocols in Microbiology
the use of more extensive genome-wide molecular approaches, such as microarrays and
proteomics.
In order to circumvent the technical limitations imposed by the paucity of spirochetes
within infected tissues, a number of alternate animal models have been developed to
cultivate and, more importantly, recover B.
burgdorferi in a host-adapted state. Several of
these models have explored the use of various growth chamber implantation techniques,
including the use of steel net (Jonsson et al.,
1995) and Teon chambers (Hurtenbach et al.,
1995) containing B. burgdorferi, implanted
subcutaneously into mice. Each of these strategies, however, is hampered by the relatively
small number of spirochetes that can be analyzed. Thus, their use has been primarily restricted to immune protection assays, antibody
proling studies, or both. In order to isolate
the quantities of host-adapted spirochetes required for global expression proling, Akins
et al. (1998) developed a model for cultivation
of B. burgdorferi within dialysis membrane
tubing implanted into the peritoneal cavities
of rats. This method was based, in part, on a
modication of an earlier murine model using implanted plastic ring chambers (de Silva
et al., 1998). This new animal model, described within this unit, has since been used
successfully to examine the expression proles and functions of a number of borrelial
loci, including ospA, ospC, dbpBA, vlsE, oppA,
and pncA (Akins et al., 1998; Elias et al.,
2002; Parveen et al., 2003; Purser et al., 2003).
The cellular material generated using this
methodology has proved to be sufcient for
both microarray analyses (Revel et al., 2002;
Brooks et al., 2003) and proteomics (Akins
et al., 1998).
One signicant additional benet of this
model lies in its ability to help distinguish
between the physiological and nonphysiological demands placed upon spirochetes during
growth within the mammalian host (Elias et al.,
2002; Purser et al., 2003; Caimano et al.,
2004). The pore size of the dialysis tubing used
to make the DMC chambers allows for the free
diffusion of small molecules and nutrients, but
does not permit the entry of immune effector
cells. The use of DMCs, therefore, allows for
an analysis of growth in vivo, independently
of the ability to disseminate or evade host immune defenses. For example, isolates containing mutations within loci whose gene products are involved in generalized metabolism
(e.g., Opps and PncA) exhibit partial or complete growth defects within DMCs (Elias et al.,
2002; Purser et al., 2003), while isolates containing mutations within loci involved in more
traditional virulence factors, e.g., antigenic
variations such as VlsE (Elias et al., 2002;
Purser et al., 2003; Caimano et al., 2004) grow
in a manner that is indistinguishable from that
of their wild-type parent. Use of the DMC
model, or any animal model for that matter,
to dene a phenotype associated with a genetic mutation requires a note of caution. Given
that the ability to survive within DMCs, and
subsequently host-adapt, is almost certainly a
complex process involving multiple loci, it is
critical that any mutant (or complemented mutant) isolate be examined in the context of its
wild-type isogenic parent to ensure that any
phenotypes observed are due to the mutation
of interest and not a spontaneous secondary
mutation.
Critical Parameters
Pilot experiments
The DMC model should be suitable for use
with any low-passage, virulent, B. burgdorferi
isolate. Prior to beginning use of this technique, however, a number of variables should
be considered.
Although little strain-to-strain variation has
been observed when the DMC procedure
has been employed to examine virulent lowpassage isolates of strains 297, B31, and N40,
these isolates represent B. burgdorferi whose
in vitro growth characteristics, antigenic composition, and virulence properties have all been
well established in numerous published studies. When using a strain other than these, a
small pilot experiment should be considered.
This is particularly important when contemplating the use of uncharacterized isolates,
such as those obtained from clinical specimans. To facilitate the design of such a pilot
experiment, it is highly advisable that one rst
determine the growth kinetics of each isolate
in BSK medium following temperature shift
from 23 C to 37 C (Fig. 12C.3.2). This preliminary in vitro study will help take into account
any signicant variations in growth kinetics
between isolates when estimating the implantation time course. Growth kinetics within
DMCs will likely be similar to that of cultures
grown in vitro at 37 C, although the DMCcultivated cultures do not appear to be able
to attain the same maximal density as their in
vitrogrown counterparts (Fig. 12C.3.2).
A second variable to consider is that at least
one borrelial locus, pncA (bbe22), encoding a
nicotinamidase, has been shown to be required
Spirochetes
12C.3.9
Current Protocols in Microbiology
Figure 12C.3.2 Growth kinetics of B. burgdorferi strain 297 cultivated in vitro or within DMCs.
Spirochetes cultivated in BSK-II medium at 23 C (open squares), 34 C (triangles), or 37 C after
temperature-shift (circles), or within DMCs (diamonds), were enumerated by dark-eld microscopy
over a 15-day period after inoculation. In all four cultures, 5-ml volumes were inoculated with 103
mid-logarithmic-phase organisms. Data points represent the means of duplicate samples. Adapted
with permission of the publisher from Akins et al. (1998).
DMC Cultivation
of B. burgdorferi
perature shift. Lysates prepared from bacteria cultivated in vitro and within DMCs may
be compared visually for well expressed proteins such as OspA and OspC using SDSPAGE gel electrophoresis. Alternatively, immunoblotting may be used to examine borrelial
antigens whose expression is not readily apparent by gel electrophoresis (e.g., DbpA, Lp6.6).
When testing lysates by immunoblot, however,
it is important to keep in mind that strain-tostrain sequence variability may result in limited to no antigenic cross-reactivity. Thus, one
should be sure to use antisera that are known to
react against the corresponding antigens within
the isolate of interest.
Troubleshooting
The most common problem associated with
the procedure is contamination of the DMC
during the implant surgery. It is critical that
aseptic technique be maintained at all times
during both the preparation of the dialysis membrane tubing strips and the implant
surgery. To reduce contamination, be sure that
the surgical area is thoroughly cleaned with a
surface sterilant immediately prior to the procedure. Ideally, instruments and drapes should
12C.3.10
Current Protocols in Microbiology
Figure 12C.3.3 Wild-type B. burgdorferi strain 297 (107 per lane) was cultivated in vitro in BSKH medium at 23 C, at 37 C following temperature shift, at 34 C, and within dialysis membrane
chambers (DMC). The whole-cell lysates were separated by SDS-PAGE, then stained with silver.
Molecular mass markers (kDa) are indicated. Asterisks indicate polypeptides that were absent
or dramatically down-regulated in chamber-grown spirochetes. Arrows designate proteins either
dramatically up-regulated or observed only in chamber-cultivated organisms. Well characterized
borrelial proteins are indicated on the right and molecular mass markers in kilodaltons are shown
on the left. Adapted with permission of the publisher from Akins et al. (1998).
(see above), it is recommended that the isolate be tested for survival alone by implanting spirochetes at higher density (1 107
spirochetes per ml) for a short incubation period (5 days), and then evaluating for spirochete viability, following explantation, by either dark-eld microscopy to assess motility
or by limiting-dilution plating.
Anticipated Results
The average recovery volume per DMC
chamber following explantation will vary
between 6.5 to 8 ml dialysate-containing spirochetes. The theoretical yield of spirochetes
contained within the DMCs should be between
2 106 and 2 107 spirochetes per ml, or an
average 700- to 3000-fold increase in bacterial number (Fig. 12C.3.2). Following explantation, spirochetes should be examined using
dark-eld microscopy to assess spirochetal viability. Spirochete density should be calculated
Spirochetes
12C.3.11
Current Protocols in Microbiology
Time Considerations
DMC Cultivation
of B. burgdorferi
Literature Cited
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model for studying Lyme disease spirochetes in
a mammalian host-adapted state. J. Clin. Invest.
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Anguita, J., Hedrick, M.N., and Fikrig, E. 2003.
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and the mammalian host. FEMS Microbiol. Rev.
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Brooks, C.S., Hefty, P.S., Jolliff, S.E., and Akins,
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Caimano, M.J., Eggers, C.H., Hazlett, K.R.O., and
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de Silva, A.M., Fikrig, E., Hodzic, E., Kantor, F.S.,
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S.K., Radolf, J.D., and Akins, D.R. 2001. Regulation of OspE-related, OspF-related, and Elp
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Hodzic, E., Feng, S., Freet, K.J., and Barthold, S.W.
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mice. Infect. Immun. 71:5042-5055.
Hurtenbach, U., Museteanu, C., Gasser, J., Schaible,
U.E., and Simon, M.M. 1995. Studies on
early events of Borrelia burgdorferiinduced
cytokine production in immunodecient SCID
mice by using a tissue chamber model for
acute inammation. Int. J. Exp. Pathol. 76:
111-123.
12C.3.12
Current Protocols in Microbiology
Key References
Akins et al., 1998. See above.
Original report describing use of dialysis membrane chamber technique with B. burgdorferi.
Fox et al., 2002. See above.
Contains practical advice administering anesthetics and analgesics in common laboratory animal
species.
Spirochetes
12C.3.13
Current Protocols in Microbiology
UNIT 12E.1
Leptospira are long, thin, helically shaped bacteria. Pathogenic Leptospira utilize fatty
acids and fatty alcohols as primary carbon and energy sources (Baseman and Cox, 1972).
These bacteria consume, preferentially, saturated long-chain fatty acids by -oxidation.
This requirement for fatty acids as a critical component of Leptospira media poses
difficulties for reliable cultivation of cells in vitro, because, at elevated levels, these
compounds are toxic to bacterial growth. This toxicity is often circumvented by the addition of albumin. Both defined and complex media have been formulated for Leptospira
growth. The purpose of this unit is to describe the preparation of various Leptospira
growth media and methods for bacterial propagation (see Growth of Pathogenic Leptospira; Basic Protocols 1 through 3). Methods for the long-term storage (see Storage
and Recovery of Pathogenic Leptospira; Basic Protocols 4 and 5), isolation (see Isolation of Pathogenic Leptospira; Basic Protocols 6 and 7), and detection of pathogenic
Leptospira by immunofluorescence (see Basic Protocol 8) are also described.
CAUTION: Leptospira interrogans sensu lato is a Biosafety Level 2 (BSL-2) pathogen
that can cause acute, fatal infections in humans. Follow all appropriate guidelines and
regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and
other pertinent resources (APPENDIX 1B) for more information.
IMPORTANT NOTE: Water for all media solutions must be sterilized by autoclaving prior
to use to remove saprophytic Leptospira that can pass through microbiological filters.
Saprophytic Leptospira are ubiquitous in water supplies and are a common contaminant
of improperly prepared media. When preparing the media supplements, it is important
to follow the order of constituent additions.
STRATEGIC PLANNING
Recipes for Media
Recipes are provided for standard media commonly used to propagate pathogenic Leptospira (see Reagents and Solutions). The order in which media components are added
is critical for successful preparation.
Additional media recipes are described elsewhere (Faine, 1994). Commercially prepared
media are available and are often suitable for routine maintenance of some strains,
but some strains either do not grow well in commercial media or have altered growth
characteristics.
Stock Solutions
Several stock solutions must be prepared before assembling media components. All
solutions are made with glass-distilled water that must be sterilized by autoclaving 20
min at 121 C and allowed to cool before used. The complete medium is prepared by
mixing an albumin supplement with a phosphate-buffered basal salt solution, which is
filter-sterilized before use.
EMJH Medium
Reagents and Solutions describes the preparation of the common, defined base media
used for isolation and growth of Leptospira. Many laboratories utilize the Johnson and
Harris (1967) modification of Ellinghausen McCullough (1965) medium (EMJH) to
cultivate Leptospira. The EMJH base medium can be used with or without serum for the
Contributed by Richard L. Zuerner
Current Protocols in Microbiology (2005) 12E.1.1-12E.1.13
C 2005 by John Wiley & Sons, Inc.
Copyright
Spirochetes
12E.1.1
Figure 12E.1.1 Growth of Leptospira in a semisolid agar medium. The formation of a Dingers
disk is typical of Leptospira grown in semisolid media. After prolonged incubation, growth extends
deeper into the tube and may form several disks farther from the air/medium interface. Photograph
courtesy of Richard Hornsby.
propagation of leptospires, and has been subjected to minor modifications to support the
isolation of various strains from clinical samples. Notes for common alterations of the
base media are included. Modified Stuart medium (see recipe in Reagents and Solutions)
is rich in rabbit serum and is a convenient alternative to EMJH for routine propagation
of laboratory-adapted strains of Leptospira.
12E.1.2
Current Protocols in Microbiology
tube. With some cultures, little growth is seen near the medium/air interface, and limited
growth is seen near the bottom of the tube.
For growth in a semisolid medium, transfer a small amount of culture, 100 to 250 l,
using a pipettor fitted with a sterile filtered tip or a 1-ml sterile pipet to a fresh tube
containing 6 to 8 ml of semisolid medium. To keep cells active, transfer most strains to
fresh medium weekly. Some strains grow very slowly, requiring transfers less frequently,
occasionally as infrequently as once every 6 months. Typically, transfers are made when
a Dingers disk forms with visible density. The time between transfers is determined
empirically because of differences in a variety of factors, including strain differences
and type of media being employed to cultivate these bacteria. The critical factor in
determining when to transfer cultures is density. Some strains achieve sufficient density
within 1 week, while comparable culture density with other strains may take months.
Continuous in vitro passage of pathogenic Leptospira often results in reduced virulence.
However, carefully controlled studies have not been performed that demonstrate how
many passages lead to a reduction of virulence, and the mechanisms causing these
phenotypic changes are uncharacterized.
BASIC
PROTOCOL 2
To isolate individual colonies, remove the colony using either a sterile Pasteur pipet
or a micropipet (e.g., Rainin Pipetman 200) fixed with a sterile, filtered tip and gently
aspirate the colony into the tip. Place the colony in a semisolid medium and incubate
until a Dingers disk is obtained (7 to 10 days). Direct passage of pathogenic Leptospira
on a solid medium often fails and recovery of isolated colonies in a semisolid medium is
recommended.
BASIC
PROTOCOL 3
Spirochetes
12E.1.3
Current Protocols in Microbiology
Materials
Starter cultures of Leptospira
Semisolid Leptospira medium (see recipe)
Liquid medium (see recipes for EMJH medium, modified Stuart medium, or
T80/40/LH medium)
29 to 30 C incubator
50-ml culture tubes with loosely fitting caps
1. Grow starter cultures of Leptospira in semisolid medium until a Dingers disk forms.
2. Gently suspend the Dingers disk in 2 to 3 ml of fresh liquid medium, and incubate
for several days at 29 to 30 C.
The liquid portion of the culture should appear slightly turbid.
3. Transfer liquid culture and some semisolid medium (3 ml) to a 50-ml culture tube
containing 8 ml fresh liquid medium.
Culture tubes should have either Morton closures or loosely fitting caps.
At this point, the culture may be gently shaken during growth (at 50 rpm).
Laboratory
Maintenance of
Pathogenic
Leptospira
12E.1.4
Current Protocols in Microbiology
BASIC
PROTOCOL 5
BASIC
PROTOCOL 6
Materials
Urine sample
Leptospira storage medium (see recipe)
Semisolid Leptospira culture medium (see recipe)
16 125mm capped tubes
29 C incubator
Dark-field microscope
1. Place 1 ml urine in 9 ml Leptospira storage medium to make a 101 dilution of the
sample.
2. Cap tube and vortex for 10 sec.
3. Transfer 1 ml of the diluted sample to a second tube containing 9 ml of storage
medium to make a 102 dilution.
4. Cap tube and vortex for 10 sec.
5. Transfer 0.3 ml of each dilution to a tube containing 6 ml semisolid Leptospira
culture medium. Incubate at 29 C.
6. Check for growth after 7, 14, 21, and 28 days of incubation and thereafter monthly
for 6 months. Examine for growth using dark-field microscopy (UNIT 2A.1).
Spirochetes
12E.1.5
Current Protocols in Microbiology
BASIC
PROTOCOL 7
Materials
Tissue sample
Leptospira storage medium (see recipe)
Culture medium
Whirlpak bag
Tissue homogenizer (e.g., Stomacher 400, Seward Medical)
29 C incubator
Dark-field microscope
1. Place 1 g tissue sample in 9 ml Leptospira storage medium in a Whirlpak bag and
homogenize for 5 min with a Stomacher tissue homogenizer.
2. Transfer 1 ml tissue homogenate to 9 ml storage medium to make a 102 dilution.
3. Cap tube and vortex for 10 sec.
4. Transfer 1 ml tissue homogenate (from step 3) to 9 ml storage medium to make a
103 dilution.
5. Transfer 0.1 ml of each dilution to 6 ml culture medium. Incubate at 29 C.
6. Check for growth after 7, 14, 21, and 28 days of incubation and thereafter monthly
for 6 months. Examine for growth using dark-field microscopy (UNIT 2A.1).
BASIC
PROTOCOL 8
Materials
Laboratory
Maintenance of
Pathogenic
Leptospira
Urine sample (see Basic Protocol 6) or tissue homogenate (see Basic Protocol 7)
PBS (APPENDIX 2A)
Acetone
Rabbit anti-Leptospira antisera conjugated with fluorescent tag (available from
Mark Wilson, NVSL, Ames, IA 50010, 515-663-7595)
Flazo Orange counter stain (diluted 1:10 to 1:20 with PBS)
Buffered glycerol
2-ml microcentrifuge tubes
Microscope slides
12E.1.6
Current Protocols in Microbiology
Figure 12E.1.2 Fluorescent antibody detection of L. interrogans serovar Pomona. Bacteria obtained directly from urine of an infected steer were xed and stained with anti-Leptospira antisera
conjugated with a uorescent dye. The resulting micrograph shows characteristic green uorescent
staining of the Leptospira (arrows) with an orange and black background. Photograph courtesy of
Richard Hornsby. For the color version of this gure go to http://www.currentprotocols.com.
Moist chamber: petri dish with lid, containing a circle of filter paper moistened
with water
37 C incubator
Coverslips
Fluorescent microscope
To prepare urine samples
1a. Concentrate 1.5 ml urine sample in a 2-ml microcentrifuge tube by microcentrifuging 10 min at 13,000 g, room temperature. Remove all but the final 100 l of
supernatant and discard.
2a. Add 500 l distilled water and resuspend the pellet. Centrifuge 10 min at 13,000
g, room temperature, and remove all but the final 25 to 50 l of supernatant.
3a. Resuspend pellet by adding 150 l distilled water. Spot 10 to 15 l of sample on a
clean glass slide. Proceed to step 4.
Process slides
4. Air dry samples on microscope slides at room temperature, typically overnight.
5. Place slides in acetone for 10 min to fix. Air dry samples.
6. Place each slide into a moist chamber (supported by two parallel toothpicks laid
flat upon the moist filter paper) and add 15 l of rabbit anti-Leptospira antisera
conjugated with fluorescent tag to each sample. Incubate for 1 to 1.5 hr at 37 C.
Current Protocols in Microbiology
Spirochetes
12E.1.7
7. Rinse slides briefly with PBS, then wash well with PBS for 10 min with stirring
using a magnetic stir bar and plate at room temperature.
8. Blot away excess moisture, then spot 15 l of Flazo Orange counter stain (diluted
1:10 to 1:20 with PBS) for 20 sec.
9. Rinse slide briefly with PBS, and blot away excess moisture.
10. Apply a small amount of buffered glycerol as a mounting medium and attach coverslip.
11. Observe samples using a fluorescent microscope (Figure 12E.1.2).
Laboratory
Maintenance of
Pathogenic
Leptospira
12E.1.8
Current Protocols in Microbiology
Phosphate buffer
0.087 g KH2 PO4
0.664 g Na2 HPO4
Bring volume to 1 liter with glass-distilled, sterile water
Mix by stirring
Prepare fresh
Historically, a combination of sodium and potassium salts has been used to prepare this
buffer, presumably to limit exposure of the bacteria to higher concentrations of either
cation.
Spirochetes
12E.1.9
before sterilization. Cool basal salt solution to 50 C before adding 100 ml complete BSA supplement (see recipe) per liter. The semisolid medium has properties
similar to a suspended slurry, and can be stored in 1-liter bottles and dispensed as
needed. Store up to 1 year at room temperature.
1 g lactalbumin hydrolysate
0.1 g superoxide dismutase
0.04 g sodium pyruvate
During preparation of the complete BSA supplement (see recipe), add:
0.1 ml manganese sulfate stock solution (0.3 g/100 ml) after the addition of zinc
sulfate
9 ml Tween 80 per 100 ml of supplement
3.5 ml Tween 40 per 100 ml of supplement
Upon final assembly of the medium, add 4 to 10 ml fresh rabbit serum (heat
inactivated 30 min at 56 C) per liter of complete BSA supplement.
For primary isolation of pathogenic Leptospira, suppression of contaminants is aided by
the addition of 10 ml of 5-FU stock solution (5-FU; 1 g/100 ml) (optional) per liter.
COMMENTARY
Background Information
Laboratory
Maintenance of
Pathogenic
Leptospira
muscle aches (Levett, 2001). In the more serious cases, death occurs from cardiac, renal, or
hepatic failure (Faine et al., 1999). Antibiotic
treatment can be successful, and in potential
laboratory exposures, prophylactic antibiotic
treatment is usually recommended.
Relatively few laboratories have successfully isolated Leptospira from clinical samples, yet successful culture of isolates is
critical to determining the prevalence of different antigenic types (serovars) of pathogenic
Leptospira. There are >200 recognized
serovars of pathogenic Leptospira (Brenner
et al., 1999), and each appears to form a
close association with one mammalian host
species (Faine et al., 1999). While a variety of
techniques are available to detect leptospiral DNA or anti-leptospiral antibodies, these
12E.1.10
Current Protocols in Microbiology
Anticipated Results
Pathogenic Leptospira do not grow to the
same cell densities as many other bacteria.
Under optimal conditions using laboratory
adapted cultures, cell densities of 1 109 /ml
may be obtained. Clinical isolates typically
reach saturation approximately one to two logs
lower cell densities. Therefore, it is often necessary to use considerably more volume of
Leptospira culture to obtain equivalent quantities of cells as compared to other bacteria.
Routine passage of Leptospira cultures in
vitro in semisolid media (see Basic Protocol 1)
will result in reliable growth rates and zones
of growth (Dingers disks) on average in 7 to
14 days with most strains.
Cultures of pathogenic Leptospira grown
on solid media (see Basic Protocol 2) should
result in the formation of small white colonies
that appear partially embedded in the agar.
Growth of pathogenic Leptospira in liquid
media (see Basic Protocol 3) should result in
Spirochetes
12E.1.11
Current Protocols in Microbiology
Time Considerations
Laboratory
Maintenance of
Pathogenic
Leptospira
Acknowledgement
This unit was prepared with the assistance
of Richard Hornsby (National Animal Disease Center, U.S. Department of Agriculture,
Ames, IA).
12E.1.12
Current Protocols in Microbiology
Literature Cited
Baseman, J.B. and Cox, C.D. 1972. Intermediate
energy metabolism of Leptospira. J. Bacteriol.
97:992-1000.
Brenner, D.J., Kaufmann, A.F., Sulzer, K.R.,
Steigerwalt, A.G., Rogers, F.C., and Weyant,
R.S. 1999. Further determination of DNA relatedness between serogroups and serovars in the
family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int. J. Sys. Bacteriol.
49:839-858.
Ellinghausen, H.C. and McCullough, W.G. 1965.
Nutrition of Leptospira pomona and growth of
13 other serotypes: Fractionation of oleic albumin complex and medium bovine albumin and
polysorbate 80. Am. J. Vet. Res. 26:45-51.
Ellis, W.A. and Thiermann, A.B. 1986. Isolation of
Leptospira interrogans serovar bratislava from
sows in Iowa. Am. J. Vet. Res. 47:1458-1460.
Faine, S. 1994. Leptospira and Leptospirosis. CRC
Press, Boca Raton, Florida.
Faine, S., Adler, B., Bolin, C., and Perolat, P. 1999.
Leptospira and Leptospirosis, 2nd ed. MediSci,
Melbourne, Australia.
Johnson, R.C. and Harris, V.G. 1967. Differentiation of pathogenic and saprophytic leptospires. I.
Growth at low temperatures. J. Bacteriol. 94:2731.
Key References
Faine, S. 1994. See above.
Faine et al. 1999. See above.
These two books provide comprehensive coverage
of the organism, metabolism, and the disease.
Spirochetes
12E.1.13
Current Protocols in Microbiology
UNIT 12E.2
Animal inoculation remains essential for many aspects of leptospirosis research. Although pathogenic leptospiral strains may be able to infect a wide variety of animals,
the Golden Syrian hamster is the preferred model because its susceptibility to infection
and the reproducibility of the results. Common applications include determination of
strain infectivity, restoring virulence to culture-attenuated strains, assessing usefulness
of potential vaccines or diagnostic antigens, and examining pathology of mammalian
infection. In many respects, acute leptospirosis in the hamster reproduces the severe
form of human leptospirosis (see Commentary).
This unit provides a detailed description of the procedures involved in the hamster
model of leptospirosis, including housing and handling of hamsters and intraperitoneal
challenge (Basic Protocol 1), and monitoring response to challenge (Basic Protocols
2 and 3, and Alternate Protocol). Methods are provided for demonstrating infection,
including culture isolation of leptospires from blood and tissues (Basic Protocol 2) and
serologic studies and histopathology (Basic Protocol 3). Quantitative PCR is provided as
an alternative detection method (Alternate Protocol). Although leptospires disseminate
to many organs, the tissues of the liver and kidney are the primary targets of infection.
The liver is heavily infected during the initial stage of hematogenous dissemination.
Persistent infection occurs primarily in the kidneys of animals that survive acute disease.
Infection of the renal tubules leads to shedding of infectious organisms in the urine,
which is the primary mode of transmission in nature. A safety policy memorandum is
provided (see Strategic Planning) that should be read and signed by all staff.
In the Commentary section, a detailed rationale is presented for use of hamsters as an
animal model of leptospirosis. The effects of challenge dose on the hamster model,
LD50 , in vitro passage of leptospiral strains, and immunological maturity are discussed.
Alternative animal models are evaluated with reference to the concept of accidental
versus reservoir hosts. The advantages and disadvantages of the hamster model are
considered, with reference to use of alternative host animals to address specific research
or vaccine-development issues.
CAUTION: Pathogenic Leptospira species are Biosafety Level 2 (BSL-2) pathogens.
L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii
are known to be pathogenic for humans. Certain strains of L. inadai, L. meyeri, L. fainei,
and L. alexanderi may also be pathogenic. L. biflexa, L. wolbachii, and L. parva are
thought to be nonpathogens. Follow all appropriate guidelines and regulations for the use
and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
CAUTION: Protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals. This experiment requires
Animal Biosafety Level 2 (ABSL-2) conditions. Follow all appropriate guidelines for the
use and handling of infected animals. See UNIT 1A.1 and other pertinent resources (APPENDIX
1B) for more information. Fluids and tissues of infected animals are highly infectious.
Numerous laboratory-associated infections, including deaths, have been reported (Miller
et al., 1987; Pike, 1976). A safety policy memorandum is provided that should be signed
by all staff (see Strategic Planning).
Spirochetes
Contributed by David A. Haake
Current Protocols in Microbiology (2006) 12E.2.1-12E.2.13
C 2006 by John Wiley & Sons, Inc.
Copyright
12E.2.1
Supplement 2
STRATEGIC PLANNING
Successful challenge studies require the simultaneous availability of Golden Syrian
hamsters and virulent leptospiral cultures. Hamsters typically require a 1-week quarantine
after shipment and should be no more than 4 weeks old on the day of challenge. Virulent
leptospires typically require at least 1 week of growth after inoculation of culture medium
from frozen stocks. However, leptospiral growth varies depending on the strain and the
quality of the medium (UNIT 12E.1).
Prior to initiating animal challenge studies, the principal investigator should ask all
personnel to read and sign a safety policy memorandum stating that they understand and
agree to the following:
a. Leptospirosis is a well-documented laboratory hazard. Virulent Leptospira may
be present in urine, blood, and tissues of infected animals and humans. Ingestion,
accidental parenteral inoculation, and direct and indirect contact of skin or mucous
membranes with cultures or infected tissues or body fluidsespecially urineare
the primary laboratory hazards.
b. All personnel must submit a baseline serum sample to employee health to test for
leptospiral antibodies.
c. All personnel must wear a gown, eyeware, gloves, mask, hat, and shoe coverings
while in any room containing infected animals.
d. In addition to standard microbiological safety practices, cultures and infected
materials will be handled in a laminar flow biological safety cabinet that has been
designated for use exclusively by the principal investigator and personnel in his
or her laboratory. Infected materials will be disinfected with bleach or autoclaved
before disposal.
e. In the event of an accidental percutaneous or mucous membrane exposure to
an infected animal or its tissues, the principal investigator and the employee
health department should be contacted immediately for consideration of antibiotic
prophylaxis with amoxicillin (500 mg, orally, three times daily) or doxycycline
(100 mg, orally, twice daily) for 7 days.
BASIC
PROTOCOL 1
Hamster Model of
Leptospirosis
12E.2.2
Supplement 2
Materials
3- to 4-week-old Golden Syrian hamsters (Harlan Bioproducts for Science)
Log-phase Leptospira culture for challenge (UNIT 12E.1): low-passage, virulent strain
(e.g., L. interrogans serovar Copenhageni strain L1-130 or L. kirschneri serovar
Grippotyphosa strain RM52)
Current Protocols in Microbiology
12E.2.3
Current Protocols in Microbiology
Supplement 2
BASIC
PROTOCOL 2
Materials
Hamsters infected with Leptospira (see Basic Protocol 1)
Isoflurane
Small, sealable container (e.g., Tupperware) to accommodate hamster for
anesthesia
Gauze or cotton sponges
50-ml conical polypropylene centrifuge tubes
Dissecting equipment: two sets of dissecting scissors, tweezers, and scalpel
Additional reagents and equipment for blood collection by cardiac puncture
(Donovan and Brown, 2006c)
NOTE: All reagents and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.
1. Place animal in a small container (Tupperware works well) containing gauze or
cotton sponges soaked with isoflurane to induce anesthesia.
2. When the animals breathing rate has slowed to less than one breath per 3 sec,
remove the animal from the container and place the open end of a 50-ml centrifuge
tube containing gauze or cotton sponges soaked with isoflurane over the nose of
the animal to maintain anesthesia throughout the procedure. Ensure a snug fit by
wrapping gauze around the animals nose.
CAUTION: Surgery on infected animals should be performed inside a biological safety
cabinet to prevent exposure to infectious material.
The anesthesia should be deep enough that the animal does not respond to toe pinch but
not so deep that the heart stops. Optimal blood collection is performed when the heart is
still beating.
3. With animal placed on its back, open the skin via a vertical midline incision with
one set of dissection scissors and tweezers.
4. To minimize risk of contaminating specimens with the animals skin flora, retract
the skin and use a second set of dissection scissors, tweezers, and scalpel to open the
peritoneal and chest cavities.
Opening of the chest cavity exposes the heart and lungs, preventing the animal from
breathing and regaining consciousness.
5. Obtain blood for culture and serology by cardiac puncture (Donovan and Brown,
2006c).
6. Obtain liver and kidney tissue for culture and histopathology.
These tissues contain the highest densities of organisms.
Hamster Model of
Leptospirosis
The liver is the dark red multilobed organ in the right upper quadrant of the peritoneal
cavity. Note any areas of hemorrhage. Using scissors or scalpels, obtain 1-cm 1-cm
segments of liver for culture and histopathology. To avoid crush artifacts, grasp only the
margin of the organ segment with tweezers rather than the body of the organ.
12E.2.4
Supplement 2
Hamster kidneys are normally the size, color, and shape of kidney beans. The kidneys are
located on the left and right posterior walls of the peritoneal cavity and are visualized by
moving the loops of intestine to one side or the other. Note any areas of hemorrhage or
heterogeneity. Cut the vascular structures with scissors. To avoid crush artifacts, remove
the organ by grasping only the hilum with tweezers, rather than the organ itself. One
kidney should be cultured and the other submitted for histopathology.
7. Consider collecting other tissues (lung or spleen) depending on the goals of the study.
The lungs are found in the chest cavities and appear as collapsed white structures after
the chest cavity has been opened. Note any areas of hemorrhage. Remove the lungs by
cutting the hilum with scissors. Examination of the lung is of interest because pulmonary
hemorrhage is an important cause of morbidity and mortality in human disease.
The spleen is a dark, red, ribbon-like structure in the left, upper peritoneal cavity and is
visualized by moving the loops of intestine to the hamsters right.
BASIC
PROTOCOL 3
Materials
Blood and organs from Leptospira-infected hamster (and blood from uninfected
hamster as control in serological procedure)
Semisolid Leptospira medium with 5-fluorouracil (see recipe)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Culture of late logarithmic growthphase Leptospira (UNIT 12E.1)
Neutral buffered formalin (e.g., Fisher)
70% (v/v) ethanol
Steiner-Steiner silver staining kit (optional; e.g., Sigma)
Stomacher bags (e.g., Thomas Scientific)
Microscope with dark-field optics (UNIT 2A.1)
Dedicated histopathology facility (optional)
Additional reagents and equipment for paraffin-embedding and sectioning of
tissues (Zeller, 1989), hematoxylin/eosin staining (Zeller and Rogers, 1993), and
Steiner-Steiner silver staining (Steiner and Steiner, 1944; reagents available as
kit from Sigma and other histology suppliers)
NOTE: All reagents and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.
2a. Inoculate tissue sample as follows. Place a 1 1cm segment of liver tissue or one
kidney in 1 ml EMJH liquid medium in a sterile Stomacher bag. Homogenize, then
inoculate into semisolid medium with s-fluorouracil at dilutions of 1/100 (by adding
0.1 ml homogenized tissue to 10 ml semisolid medium) and 1/1000 (by adding 1.0
ml of the 1/100 dilution to 10 ml semisolid medium).
Spirochetes
12E.2.5
Current Protocols in Microbiology
Supplement 2
A simple way to homogenize tissue is to place the tissue and medium in a Stomacher
bag, fold over the open end of the bag, and role a 100-ml cylindrical flask over the bag,
forcing the contents between the flask and the hard surface of the counter top inside the
biological safety cabinet.
3a. Examine a sample from each culture biweekly by dark-field microscopy for growth
of leptospires.
Hamster Model of
Leptospirosis
Figure 12E.2.1 Silver stain of hamster kidney tissue obtained 28 days after challenge. Arrows
show positively-staining aggregates of leptospires lining the epithelial surface of several adjacent
renal tubules. For the color version of this gure go to http://www.currentprotocols.com.
12E.2.6
Supplement 2
3c. Stain sections with hematoxylin and eosin (Zeller and Rogers, 1993) to characterize
the histopathology of the organs. Perform silver staining using the technique of
Steiner and Steiner (1944) to detect organisms.
Steiner-Steiner silver staining kits are available from Sigma and other suppliers.
4c. Have the sections analyzed by a person trained in histopathology to identify the
organisms and characterize the extent and severity of lesions, including assessment
and grading of the location, number, and types of inflammatory cells present.
Figure 12E.2.1 shows a silver stain of hamster kidney tissue obtained 28 days after
challenge showing leptospires (arrows) lining the epithelial surface of several adjacent
renal tubules.
ALTERNATE
PROTOCOL
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12E.2.7
Current Protocols in Microbiology
Supplement 2
2. Extract total DNA from the blood and tissues of challenged hamsters using the
QIAamp Blood Kit (Qiagen) and the DNeasy Tissue Kit (Qiagen), respectively.
3. For each sample DNA, negative control DNA, and positive control DNA dilution,
prepare a PCR reaction mix (total volume, 50 l; prepare all samples and controls
in duplicate) in a PCR plate well as follows:
a. First prepare 45 l TaqMan Universal PCR Master Mix containing:
3 pmol/l Lepto F PCR primer
3 pmol/l Lepto R PCR primer
2 pmol/l TaqMan probe
b. Add 5 l template DNAi.e., sample DNA, negative control (L. biflexa) DNA,
or positive control DNA dilutionto the corresponding wells.
c. Add 5 l TE buffer to the designated wells in place of the DNA for a no-template
negative control.
4. Conduct amplification and fluorescence detection in the ABI Prism 7700 dedicated
thermal cycler using the following program, collecting the data throughout:
40 cycles:
15 sec
1 min
95 C
60 C.
5. Using the dedicated real-time PCR software supplied with the ABI Prism 7700
Sequence Detection System, quantify the leptospiral DNA in the samples using the
positive control dilution series of DNA from the Leptospira challenge strain as a
standard curve.
UNIT 1D.3
Several vendors supply satisfactory BSA, fraction V, for use in media preparation. Before
purchasing a large amount of BSA from an individual vendor, it is critical to test it by
preparing the complete BSA supplement and using it to prepare the medium for a pilot
experiment.
Hamster Model of
Leptospirosis
12E.2.8
Supplement 2
COMMENTARY
Background Information
The Golden Syrian hamster model of leptospirosis is well characterized and has a number of important advantages, including excellent reproducibility and susceptibility to a
broad range of pathogenic strains (Stavitsky,
Spirochetes
12E.2.9
Current Protocols in Microbiology
Supplement 2
Hamster Model of
Leptospirosis
Critical Parameters
Based on animal husbandry issues and the
reproducibility of the model, hamsters are the
animal model of choice for leptospiral vaccine
Figure 12E.2.2 Time from challenge to 50% mortality. 3-week-old Golden Syrian hamsters in
groups of four were challenged by intraperitoneal inoculation with serial 10-fold dilutions of Leptospira kirschneri serovar Grippotyphosa, strain RM52. Each dose was administered to hamsters
in groups of four. Each point represents the time to death of the second of four hamsters. The
LD50 of this strain is <10 organisms.
12E.2.10
Supplement 2
Time Considerations
Literature Cited
Troubleshooting
Leptospires rapidly lose virulence during
in vitro cultivation. Loss of the ability to produce lethal infection in hamsters can occur
in as few as ten passages in culture medium.
Stocks of primary cultures isolated from animals should be stored in liquid nitrogen, as
described in UNIT 12E.1, and thawed out when
needed for animal-challenge studies. Culture
attenuation can also be minimized by passaging cultures frequently enough to avoid
onset of stationary phase. More highly passaged strains and strains of unknown passage number may be reisolated from hamsters to obtain a challenge strain with improved
virulence.
Anticipated Results
Spirochetes
12E.2.11
Current Protocols in Microbiology
Supplement 2
and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo. Am. J. Vet.
Res. 62:995-1000.
Donovan, J. and Brown, P. 2006a. Handling and
restraint. In Current Protocols in Immunology
(J.E. Coligan, B. Bierer, D.H. Margulies, and
W. Strober, eds.) pp. 1.3.1-1.3.6. John Wiley &
Sons, Hoboken, N.J.
Hamster Model of
Leptospirosis
van den Ingh, T.S., and Hartman, E.G. 1986. Pathology of acute Leptospira interrogans serotype
icterohaemorrhagiae infection in the Syrian
hamster. Vet. Microbiol. 12:367-376.
Wilson, K. 1997. Preparation of genomic DNA from
bacteria. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K.
Struhl, eds.) pp. 2.4.1-2.4.5. John Wiley & Sons,
Hoboken, N.J.
Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. In
Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. 14.1.1-14.1.8. John Wiley & Sons, Hoboken,
N.J.
Zeller R. and Rogers, M. 1993. Counterstaining and
mounting of autoradiographed in situ hybridization slides. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K.
Struhl, eds.) pp. 14.5.1-14.5.5. John Wiley &
Sons, Hoboken, N.J.
Key References
Faine et al., 1999. See above.
This monograph contains a wealth of detailed information and useful commentary and is an invaluable
resource that should be on hand in any laboratory
that works with animal models of leptospirosis.
Richmond, J.Y. and McKinney, W. 1999. Biosafety
in Microbiological and Biomedical Laboratories, 4th ed. US Government Printing Office,
Washington, D.C.
Printed version of important safety document, also
available online (see Internet Resources).
12E.2.12
Supplement 2
Internet Resources
http://www.cdc.gov/od/ohs/biosfty/
bmbl4/bmbl4toc.htm
Web site for the guide to Biosafety in Microbiological and Biomedical Laboratories, 4th Edition
(Richmond and McKinney, 1999; see Key References).
Spirochetes
12E.2.13
Current Protocols in Microbiology
Supplement 2
UNIT 12E.4
The genus Leptospira belongs to the order Spirochaetales and is composed of both
saprophytic and pathogenic members, such as Leptospira biflexa and L. interrogans,
respectively. Both saprophytic and pathogenic Leptospira have been classified into
serovars (more than 220 serovars are recognized as pathogens; Levett, 2001) which
are defined by agglutination cross-absorption, and more recently have been classified
into 17 species based on DNA-DNA hybridization studies. Knowledge of the genetics
of Leptospira remains at a very early stage in comparison to that of other bacterial
species. The lack of genetic tools in pathogenic Leptospira does not allow for the
full-characterization of genes of interest. Only recently has the first evidence of gene
transfer been demonstrated in the pathogen L. interrogans by random transposition of
Himar1, a transposon of eukaryotic origin (Chiang and Rubin, 2002). In contrast, numerous tools for genetic manipulation of saprophytic Leptospira have been developed in
the authors laboratory, as will be described in this unit. These studies enable the use of
L. biflexa as a model bacterium. Among spirochetes, L. biflexa is a relatively fast-growing,
aerobic bacterium: 1 week is necessary to obtain colonies on solid medium. It could
therefore allow the functional analysis of genes from slow- or noncultivable spirochete
members.
In this section, several approaches are described that are useful for genetic manipulation of
the saprophyte L. biflexa. Basic Protocol 1 describes the preparation of electrocompetent
cells of L. biflexa and the procedure for transforming them with plasmid DNA. L. biflexa
can be transformed at a high rate using a replicative plasmid containing a kanamycin- or
spectinomycin-resistance cassette as a selectable marker (see Basic Protocol 2). Replicative plasmids are suitable for heterologous gene expression and complementation studies.
Targeted mutagenesis can be achieved in L. biflexa by using a suicide plasmid delivering
the inactivated allele in the targeted chromosomal gene (see Basic Protocol 3). Use of
the counter-selectable marker rpsL can improve the recovery of allelic exchange (see
Basic Protocol 4). Finally, the authors recently developed an in vivo transposon mutagenesis system in L. biflexa that could allow large scale mutagenesis studies (see Basic
Protocol 5).
CAUTION: Leptospira biflexa is a Biosafety Level 1 (BSL-1) organism. Such organisms
are not known to consistently cause disease in healthy adult humans, and are of minimal
potential hazard to laboratory personnel and the environment. Standard microbiological
practices should be followed when working with these organisms. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for more information.
IMPORTANT NOTE: Since penicillin is the recommended treatment against leptospirosis, antibiotic resistance cassettes such as ampicillin should not be used.
BASIC
PROTOCOL 1
This protocol describes the preparation of electrocompetent cells of L. biflexa and how
to transform them with plasmid DNA. Replicative plasmids are suitable for heterologous
gene expression and complementation (see Basic Protocol 2), while suicide plasmids,
i.e., plasmids that are not replicative in L. biflexa, are currently used to perform allelic
exchange (see Basic Protocol 3) and random transposon mutagenesis (see Basic Protocol
5). Briefly, L. biflexa is grown to exponential phase, and the cells are then washed
Spirochetes
Contributed by Hel`ene Louvel and Mathieu Picardeau
Current Protocols in Microbiology (2007) 12E.4.1-12E.4.12
C 2007 by John Wiley & Sons, Inc.
Copyright
12E.4.1
Supplement 5
and concentrated in water. A mixture of cells and DNA are electroporated, allowing
the DNA to enter into the cells. Finally, transformants are grown and then plated onto
antibiotic-containing (selective) medium.
Materials
L. biflexa serovar Patoc strain Patoc1 (National Reference Center for Leptospira,
Institut Pasteur, Paris, France)
EMJH liquid medium (see recipe)
Sterile water (B. Braun)
Plasmid DNA (see Basic Protocols 2, 3, 4, and 5; Fig. 12E.4.1), salt-free
EMJH agar plates supplemented with the appropriate antibiotic (see recipe)
30 C shaker and incubator
50-ml polypropylene centrifuge tubes (Corning)
0.2-cm electroporation cuvettes (Bio-Rad), chilled
Electroporator (Bio-Rad, Gene Pulser)
15-ml polypropylene tubes (Falcon), sterile
Figure 12E.4.1 Schematic representation of some plasmid vectors used for the transformation of L. biexa.
C9, hyperactive transposase; Ph10, L. interrogans hsp10 promoter; PgBb, B. burgdorferi gB promoter; KmR,
kanamycin-resistance cassette; SpcR, spectinomycin-resistance cassette. Small arrowheads on pSHT represent the borders of the transposon. A suicide vector used to inactivate a L. biexa gene is represented (white
bars anking KmR indicate the sequences of the targeted gene used for homologous recombination). Expected
transformation efciencies are indicated. Plasmids can be requested from the authors (mpicard@pasteur.fr).
Genetic
Manipulation
of L. biflexa
12E.4.2
Supplement 5
2. Transfer the culture to 50-ml centrifuge tubes. Centrifuge 20 min at 4000 g, room
temperature. Aspirate and pour off the supernatant.
Because Leptospira are sensitive to cold temperatures (i.e., <10 C), cells should be
kept at room temperature for all subsequent steps.
3. Add an identical volume of sterile water to the cell pellet and gently resuspend cells
(do not vortex). Centrifuge tubes again as in step 2.
Vortexing may damage the leptospiral cells.
Analyze transformants
9. Following overnight growth, spread 10 to 250 l of the cells on EMJH plates
supplemented with the appropriate selective marker. Incubate the plates at 30 C for
one week.
The spectinomycin- and kanamycin-resistance cassettes are the only two selective markers
currently known to be functional in Leptospira species. Depending of the selective marker
carried by the plasmid vector (Fig. 12E.4.1), EMJH plates should be supplemented with
40 g/ml kanamycin or 40 g/ml spectinomycin. At 30 C, colonies may take 5 to 10 days
to come up on EMJH medium. Plates should be sealed with Parafilm to avoid desiccation.
Not all colonies appear simultaneously, suggesting that several maturation stages may
exist (Fig. 12E.4.2A).
Spirochetes
12E.4.3
Current Protocols in Microbiology
Supplement 5
Figure 12E.4.2 Colony morphology of L. biexa. (A) Colonies on EMJH plates after 1 week of
incubation at 30 C. (B) Top (upper panel) and side (lower panel) views of an L. biexa colony.
Note that Leptospira cells grow into the solid medium, forming subsurface colonies.
10. Pick transformants using a micropipettor and subculture in 5 ml EMJH liquid medium
containing appropriate selection antibiotic. Maintain antibiotic selection during subsequent growth and manipulation of strains.
Leptospira species do not grow on the surface of solid EMJH medium, but in subsurface
colonies (Fig. 12E.4.2B).
BASIC
PROTOCOL 2
BASIC
PROTOCOL 3
Genetic
Manipulation
of L. biflexa
12E.4.4
Supplement 5
Materials
Suicide plasmid (for example, a pGEM7Z-f+ Promega derivative plasmid that
replicates in E. coli but not in Leptospira species) containing the gene of interest
interrupted by a kanamycin or spectinomycin resistance cassette (Fig. 12E.4.1)
TrisCl, pH 8.0 (APPENDIX 2A)
PCR primers flanking the insertion site of the resistance cassette in the targeted
gene (Fig. 12E.4.3)
UV chamber (GS Gene linker, Bio-Rad)
100 C water bath
Additional reagents and equipment for preparation and transformation of
electrocompetent Leptospira biflexa (Basic Protocol 1) and PCR (Kramer and
Coen, 2001)
Grow the strain to be mutagenized and prepare electrocompetent cells
1. Perform Basic Protocol 1 steps 1 to 4.
Induce homologous recombination
2. Subject 100 ng suicide plasmid containing the inactivated allele to 5 to 15 sec UV
treatment (254 nm, 400 W/cm2 ) using a UV chamber.
Alternatively, to induce homologous recombination, plasmid DNA can also be denatured
by alkali treatment. To do this, denature the DNA in a 50-l solution containing 0.2 mM
EDTA/0.2 M NaOH for 15 to 30 min at 37 C, and then purify using Qiaquick minicolumns
(Qiaquick PCR purification kit, Qiagen).
6. Perform PCR (Kramer and Coen, 2001) to amplify the insertion of the resistance
cassette in the target gene.
Use standard PCR conditions for the gene to be amplified. In transformants, the increase
in size of the amplified product in comparison to the wild-type strain is due to the insertion
of the resistance cassette (double cross-over event) into the target locus. Transformants
resulting in single cross-over events (plasmid insertion into the target locus) exhibit
one amplified product identical to the one obtained from the wild-type strain and, when
PCR conditions are optimal for the amplification of large DNA fragments, a larger one
corresponding to the allele interrupted by the resistance cassette (Fig. 12E.4.3).
Spirochetes
12E.4.5
Current Protocols in Microbiology
Supplement 5
Figure 12E.4.3 Schematic diagram of the procedure for targeted mutagenesis by homologous
recombination. (A) The target gene in a suicide plasmid (unable to replicate in Leptospira species)
is interrupted by a kanamycin- or spectinomycin-resistance cassette, shown in grey. The suicide
plasmid is used to transform L. biexa by electroporation. The transformants resulting from homologous recombination are then selected on EMJH supplemented with the appropriate antibiotic.
The genotypes of the transformants are characterized by PCR with primers P1 and P2 anking
the insertion site of the resistance cassette. (B) Gel electrophoresis of amplied products with
primers P1 and P2. An increase in size with respect to the wild-type strain is observed for the
transformants resulting from the integration of the resistance cassette by double cross-over recombination in the target gene. The single cross-over recombinants, resulting from the integration
of the suicide plasmid, were characterized by a wild-type amplied product and a larger product.
BASIC
PROTOCOL 4
Genetic
Manipulation
of L. biflexa
12E.4.6
Supplement 5
Materials
L. biflexa serovar Patoc strain Patoc1 resistant to streptomycin (National Reference
Center for Leptospira, Institut Pasteur, Paris, France)
Primer LpR1 (5 - TACAAAATGTCGCTATCTGG-3 )
Primer LpR2 (5 -TGTATTTAGGATCGCCTTCG-3 )
EMJH agar plates supplemented with 40 g/ml streptomycin and another
appropriate antibiotic (see recipe)
Sterile toothpicks
Additional reagents and equipment for preparing thermolysates (Basic Protocol 3),
PCR (Kramer and Coen, 2001), cloning (Ausubel et al., 2007, Chapter 3), and
transformation of Leptospira (Basic Protocol 1)
Construct plasmid DNA for transformation
1. Prepare thermolysates of the L. biflexa wild-type strain as described in Basic Protocol
3, step 5.
2. Perform PCR (Kramer and Coen, 2001) to amplify the rpsL wild-type allele using
primers LpR1-LpR2 and crude DNA extract of L. biflexa wild-type strain as a
template.
3. Clone the PCR product into the plasmid vector (Ausubel et al., 2007, Chapter 3)
used to deliver the inactivated allele.
After a vector containing a streptomycin-resistant mutant rpsL gene has been produced,
one may chose to use that vector as the basis for all subsequent allelic exchange mutagenesis experiments.
Grow the streptomycin-resistant strain of L. biflexa and transform with plasmid DNA
4. Perform Basic Protocol 1 with the streptomycin-resistant strain of L. biflexa and
transform competent cells with rpsL+ -containing plasmid derivatives.
Counterselect transformants
5. When colonies appear on kanamycin or spectinomycin plates (to 10 days), replica
plate using sterile toothpicks onto EMJH plates supplemented with streptomycin.
6. Restreak colonies growing on plates supplemented with streptomycin on plates
supplemented with streptomycin and kanamycin or spectinomycin to confirm the
streptomycin resistance.
Double cross-over mutants should be resistant to streptomycin.
Transformants can be directly spread on plates supplemented with streptomycin and
kanamycin or spectinomycin. However, the two-step procedure generally enhances the
recovery of double cross-over events. Perform PCR with primers flanking the insertion
site of the resistance cassette to identify double cross-over mutants (Fig. 12E.4.3).
BASIC
PROTOCOL 5
Spirochetes
12E.4.7
Current Protocols in Microbiology
Supplement 5
Materials
pSC189 derivative plasmids carrying both the hyperactive transposase C9 and the
Himar1 transposon containing a kanamycin resistance cassette (Fig. 12E.4.1,
plasmid vectors are available upon request from authors: mpicard@pasteur.fr)
EMJH agar plates supplemented with 40 g/ml kanamycin and 50 M hemin (see
recipe)
Additional reagents and equipment for introducing the plasmid by electroporation
(Basic Protocol 1), replica plating, extraction of genomic DNA (Wilson, 1997),
and ligation-mediated PCR (Prodhom et al., 1998)
Grow the strain to be mutagenized and prepare electrocompetent cells
1. Perform Basic Protocol 1 to introduce the Himar1-delivering plasmid by electroporation.
It has to be noted that due to the presence of a hyperactive transposase, pSC189 derivative
plasmids may not be stable in E. coli. Each plasmid preparation should therefore be
done with freshly transformed E. coli cells. In contrast, since the plasmid vectors are not
replicative in Leptospira species, after transposition of Himar1 into the L. biflexa genome,
the transposase gene which is adjacent to Himar1 but not included into Himar1 is lost
together with the plasmid. This avoids subsequent transposase-mediated events.
3. Screen for auxotrophs by replica plating onto hemin-EMJH and EMJH plates.
Mutants should be purified by streaking for single colonies and retested for their phenotypes.
Albumin supplement
Dissolve 10 g BSA into 50 ml distilled water with stirring (30 min), being careful
to avoid foaming. Add the following:
continued
12E.4.8
Supplement 5
EMJH, base
1 g Na2 HPO4
0.3 g KH2 PO4
1 g NaCl
0.25 g NH4 Cl
0.005 g thiamine
Add H2 O to 1 liter
Base EMJH medium is commercially available in powder form (Leptospira Medium Base
EMJH; BD Biosciences).
12E.4.9
Current Protocols in Microbiology
Supplement 5
COMMENTARY
Background Information
E. coli-L. biflexa shuttle vector
A major breakthrough in Leptospira genetics was the first report of genetic transformation by electroporation of an E.coli-L. biflexa
shuttle vector. It contains the replication origin of the circular replicon of the L. biflexa
LE1 prophage, a kanamycin-resistance cassette from the Gram-positive bacterium Enterococcus faecalis, and an E. coli plasmid
origin (Saint Girons et al., 2000). Replicative vectors containing a gene encoding resistance to spectinomycin can also be used
(Bauby et al., 2003; Bourhy et al., 2005a).
Two types of replicative plasmids are available, plasmids with the minimal replication
origin of LE1 (1-kb in length) and plasmids
containing both the minimal replication origin
of LE1 and the parAB partition operon, therefore conferring higher stability in absence of
selective pressure (Bauby et al., 2003; Bourhy
et al., 2005a).
Targeted mutagenesis
In L. biflexa, homologous recombination
between the inactivated allele and the wildtype allele is greatly enhanced after UV irradiation of the delivery vector (Picardeau
et al., 2001). This method allowed the inactivation of several genes such as flaB, recA,
metX, trpE, and hemH in the saprophytes L.
biflexa and L. meyeri (Picardeau et al., 2001,
2003; Tchamedeu Kameni et al., 2002; Bauby
et al., 2003; Guegan et al., 2003). Single
cross-overs (integrated plasmids) can be easily differentiated from double cross-overs (allelic exchange) by PCR analysis of colonies.
Since the proportion of allelic exchange mutants is low, and single recombination is the
general rule, the use of a plasmid vector containing a counter-selectable marker adjacent
to the inactivated gene should eliminate clones
harboring the plasmid vector and/or clones that
have integrated the vector by a single crossover event. The presence of the rpsL wildtype gene in streptomycin-resistant transformants promotes bacterial death in the presence of streptomycin and facilitates the recovery of double cross-over mutants. Spontaneous
streptomycin resistant colonies appear at a frequency of 105 , which is low enough to permit
the use of rpsL as a counter-selectable marker
(Picardeau et al., 2001).
Genetic
Manipulation
of L. biflexa
12E.4.10
Supplement 5
Anticipated Results
Using Basic Protocol 1, transformation efficiencies can reach 105 bacteria/g plasmid
DNA with pGKBLe24 derivatives. In targeted
mutagenesis, the number of transformants
should range from 20 to 100 transformants/g
DNA, including 10% to 60% allelic exchange
bacteria. However, these results greatly vary
from gene to gene. Using Himar1, transformation efficiency can reach 104 transformants/g
DNA (Fig. 12E.4.1).
Time Considerations
L. biflexa is a slow-growing organism with
a generation time of 4 hr. Typically, strains
should be grown 3 to 4 days in EMJH liquid
medium to reach the exponential phase (see
UNIT 12E.1). Once the culture of the bacterial
cells is ready to be harvested, the cells are
washed and concentrated in 1 hr, then transformed with DNA. Plating transformed cells
occurs the day after. At least 1 week is necessary to obtain colonies on solid medium. If
required, an additional 2 days will be needed to
isolate genomic DNA for PCR, Southern blot
analysis, and/or DNA sequencing reactions.
Literature Cited
Ausubel, F.A., Brent, R., Kingston, R.E., Moore,
D.D., Seidman, J.G., Smith, J.A., and Struhl,
K. (eds.), 2007. Current Protocols in Molecular
Biology, Chapter 3. John Wiley & Sons, Hoboken, N.J.
Bauby, H., Saint Girons, I., and Picardeau, M.
2003. Construction and complementation of
the first auxotrophic mutant in the spirochaete
Leptospira meyeri. Microbiology 149:689-693.
Bourhy, P., Frangeul, L., Couve, E., Glaser, P., Saint
Girons, I., and Picardeau, M. 2005a. Complete
nucleotide sequence of the LE1 prophage from
the spirochete Leptospira biflexa and characterization of its replication and partition functions.
J. Bacteriol. 187:3931-3940.
Bourhy, P., Louvel, H., Saint Girons, I., and
Picardeau, M. 2005b. Random insertional mutagenesis of Leptospira interrogans, the agent
of leptospirosis, using a mariner transposon.
J. Bacteriol. 187:3255-3258.
Chiang, S.L. and Rubin, E.J. 2002. Construction of
a mariner-based transposon for epitope-tagging
and genomic targeting. Gene 296:179-185.
Guegan, R., Camadro, J.M., Saint Girons, I., and
Picardeau, M. 2003. Leptospira spp. possess
a complete heme biosynthetic pathway and
are able to use exogenous heme sources. Mol.
Microbiol. 49:745-754.
Kramer, M.F. and Coen, D.M. 2001. Enzymatic
amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol.
56:15.1.1-15.1.14.
Levett, P.N. 2001. Leptospirosis. Clin. Microbiol.
Rev. 14:296-326.
Louvel, H., Saint Girons, I., and Picardeau, M.
2005. Isolation and characterization of FecAand FeoB-mediated iron acquisition systems of
the spirochete Leptospira biflexa by random insertional mutagenesis. J. Bacteriol. 187:32553258.
Picardeau, M., Brenot, A., and Saint Girons, I.
2001. First evidence for gene replacement in
Leptospira spp. Inactivation of L. biflexa flaB
results in non-motile mutants deficient in endoflagella. Mol. Microbiol. 40:189-199.
Picardeau, M., Bauby, H., and Saint Girons, I.
2003. Genetic evidence for the existence of two
pathways for the biosynthesis of methionine in
Leptospira spp. FEMS Microbiol. Lett. 225:257262.
Prodhom, G., Lagier, B., Pelicic, V., Hance, A.J.,
Gicquel, B., and Guilhot, C. 1998. A reliable
amplification technique for the characterization
Spirochetes
12E.4.11
Current Protocols in Microbiology
Supplement 5
Genetic
Manipulation
of L. biflexa
12E.4.12
Supplement 5
UNIT 13A.1
INTRODUCTION
Fusobacterium are Gram-negative, anaerobic, nonspore-forming bacilli. While some type
strains are fusiform (spindle-shaped), others are not. The bacteria form either speckled,
bread-crumb-like colonies, or smooth colonies. This unit presents the basic techniques
for growing pure cultures of fusobacteria on blood agar plates or in broth (Basic Protocol
1), enriching fusobacteria from a mixed population such as dental plaque (Basic Protocol
2), and storing fusobacteria on a short- or long-term basis (Basic Protocol 3).
CAUTION: Fusobacterium are Biosafety Level 2 (BSL-2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms.
See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: All incubations are performed anaerobically at 37 C in 90% N2 , 5% CO2 , and
5% H2 . Manipulations outside the anaerobic chamber should be limited to <30 min
whenever possible (see Strategic Planning and Critical Parameters).
STRATEGIC PLANNING
Blood agar plates are best prepared 1 to 2 days before use. After the agar solidifies, the
plates can be left closed on the laboratory bench overnight or with lids off in a laminar
flow hood for 10 to 20 min to dry off excessive moisture. The unused plates can then be
stored, well wrapped, at 4 C and used within 1 to 2 weeks. Agar and broth media should
be placed inside the anaerobic chamber one day before use to reduce the oxygen content.
The plates should be unwrapped and the test tube caps loosened to allow for anaerobic
equilibration.
BASIC
PROTOCOL 1
Materials
Columbia blood agar plates (see recipe)
Columbia broth (see recipe)
Fusobacterium cultures (clinical isolates or ATCC strains)
Anaerobic incubator: 37 C anaerobic chamber, desiccant, palladium catalyst
pellets, nitrogen gas tank, gas mix (90% N2 , 5% CO2 , and 5% H2 ) tanke.g.,
Coy or Forma or
Anaerobic jar system (e.g., 2.5-liter Pack-Rectangular Jar, AnaeroPack System;
Mitsubishi Gas Chemical Co.; http://www.mgc-a.com/Pages/anaeropac.html)
Sachets for anaerobic jar system (Mitsubishi Gas Chemical Co.)
Anaerobic indicator strips (Becton Dickinson)
1. Place Columbia blood agar plates and broth inside the 37 C anaerobic chamber or
anaerobic jar one day before use to reduce the oxygen contained in the medium.
2. Using a 3-mm inoculating loop, inoculate fusobacteria by streaking onto the blood
agar plates or by mixing into the broth.
It is important when preparing broth cultures that four to five colonies instead of only one
are inoculated into the broth to ensure sufficient inoculum.
Other
Eubacteria
Contributed by Yiping W. Han
Current Protocols in Microbiology (2006) 13A.1.1-13A.1.8
C 2006 by John Wiley & Sons, Inc.
Copyright
13A.1.1
3a. If using an anaerobic chamber: Transfer the plates and tubes inside the chamber;
place plates upside down and loosen the caps of the tubes.
To ensure anaerobicity, the desiccant and catalysts in the anaerobic chamber need to
be recycled frequently. The anaerobicity should be checked periodically with anaerobic
indicator strips.
3b. If using anaerobic jar system: Place plates and tubes in the jar along with a newly
opened sachet; place plates upside down and loosen the caps of the tubes. Close the
lid and place the anaerobic jar in a regular 37 C incubator.
The sachet must be replaced each time the jar is opened.
Materials
Reduced transport fluid (RTF; see recipe)
FEA or CVE plates (see recipes)
Dental plaque (provided by a dental care professional)
Anaerobic incubator: 37 C anaerobic chamber, desiccant, palladium catalyst
pellets, nitrogen gas tank, gas mix (90% N2 , 5% CO2 , and 5% H2 ) tanke.g.,
Coy or Forma or
Anaerobic jar system (e.g., 2.5-liter Pack-Rectangular Jar, AnaeroPack System;
Mitsubishi Gas Chemical Co.; http://www.mgc-a.com/Pages/anaeropac.html)
Sachets for anaerobic jar system (Mitsubishi Gas Chemical Co.)
Anaerobic indicator strips (Becton Dickinson)
1. Place FEA or CVE plates inside the 37 C anaerobic chamber or jar one day before
use to reduce the oxygen contained in the medium.
2. Suspend dental plaque (from either individual sites or pooled from different sites in
the same patient) into 1 ml RTF. Vortex 60 sec.
3. Plate 10- or 100-fold serial dilutions of the RTF suspension onto prereduced FEA or
CVE plates from step 1.
4. Incubate anaerobically at 37 C for 5 to 7 days.
CVE permits almost total recovery of pure cultures of fusobacteria when compared to
nonselective medium and suppresses pure cultures of non-fusobacteria by six orders of
magnitude. Crystal violet inhibits Gram-positive organisms. Fusobacteria are relatively
resistant to erythromycin, which can inhibit a number of other oral bacteria at low
concentrations.
FEA yields no suppression of F. nucleatum relative to nonselective medium and may
allow growth of fewer contaminating organisms than CVE because the combination of
vancomycin, neomycin, and josamycin inhibits an even broader spectrum of oral bacteria.
The differential susceptibility of Fusobacterium and Bacteroides to josamycin is greater
than erythromycin. In addition, the egg yolk in FEA allows for differentiation of F.
necropherum from other fusobacteria due to its lipase activity.
For more information see Walker et al. (1979) and Morgenstein et al. (1981).
Laboratory
Maintenance of
Fusobacteria
13A.1.2
Current Protocols in Microbiology
STORAGE OF FUSOBACTERIA
Fusobacteria grown on plates or in broth should be stored anaerobically, either in an
anaerobic chamber or an anaerobic jar at room temperature. The cultures on plates
should be passaged every 2 weeks. The broth cultures can be passaged three to four
times. For long-term storage, glycerol stocks should be made and maintained at 80 C,
as described below.
BASIC
PROTOCOL 3
Materials
Fusobacterium cultures
Columbia broth (see recipe)
Glycerol (autoclaved)
Anaerobic chamber or anaerobic jar (see Basic Protocol 1)
1.5-ml microcentrifuge tubes, sterile
1. Grow fusobacteria in Columbia broth as in Basic Protocol 1 to early stationary phase
(OD600 1).
At a doubling time of 5 to 6 hr during growth, it may take 24 to 72 hours to reach this
density, depending on the inoculum.
2. Place Columbia broth and glycerol inside the 37 C anaerobic chamber or anaerobic
jar one day before use to reduce the oxygen contained in the medium.
3. Mix 500 l fusobacterial culture with 500 l prereduced 50% glycerol in Columbia
broth in a sterile 1.5-ml microcentrifuge tube. Invert the tube several times to mix
well and store immediately at 80 C.
Columbia broth
Suspend 35 g Difco Columbia broth base (see recipe) in 1 liter of water, autoclave
in 100-ml aliquots 15 min at 121 C. As needed, dispense 5 ml into sterile 15-ml
polypropylene screw-cap tubes. Store up to 6 months at room temperature.
Columbia broth base
28.57% (w/w) pancreatic digest of Casein (10 g)
14.29% (w/w) yeast extract (5 g)
14.29% (w/w) proteose peptone #3 (5 g)
8.57% (w/w) tryptic digest of beef heart (3 g)
0.29% (w/w) L-cysteine HCl (0.1 g)
7.14% (w/w) glucose (2.5 g)
14.29% NaCl (w/w) (5 g)
Current Protocols in Microbiology
Other
Eubacteria
13A.1.3
CVE plates
Dissolve the following in 1 liter H2 O with mixing:
10 g trypticase base (e.g., Becton Dickinson)
5 g yeast extract (e.g., Fisher)
5 g NaCl
2 g glucose
0.2 g tryptophan
5 mg crystal violet
Autoclave 15 min at 121 C. Cool to 50 C. Add 4 mg erythromycin and 50 ml
defibrinated whole sheep blood, prewarmed to room temperature. Pour into sterile
100 15mm Petri plates (30 ml per plate). After the agar solidifies, leave the
plates on the laboratory bench overnight or in a laminar flow hood with lids off for
10 to 20 min to dry excess moisture. Store, well wrapped, up to 2 weeks at 4 C.
Refer to Walker et al. (1979) for more information.
FEA plates
Dissolve the following into 1 liter H2 O with mixing:
41 g Brucella agar base (BBL)
5 g Na2 HPO4
1 g K2 HPO4
0.1g MgSO4
5 mg hemin (Sigma)
1 ml polysorbate 80 (Sigma)
Adjust to pH 7.6 with NaOH, autoclave at 121 C for 15 min, and cool to 50 C.
Add 5 mg vancomycin, 100 mg neomycin, 3 mg josamycin (Sigma), and 25 ml
sterile egg yolk suspension (Fisher). Pour into sterile 100 15mm Petri plates
(30 ml per plate). After the agar solidifies, leave the plates on the laboratory bench
overnight or in a laminar flow hood with lids off for 10 to 20 min to dry excess
moisture. Store, well wrapped, up to 2 weeks at 4 C.
Refer to Morgenstein et al. (1981) for more information.
Laboratory
Maintenance of
Fusobacteria
13A.1.4
Current Protocols in Microbiology
Final concentrations are 0.04% Na2 CO3 , 0.001 M EDTA, 0.02% dithiothreitol, 0.045%
K2 HPO4 , 0.045% KH2 PO4 , 0.09% NaCl, 0.09% (NH4 )2 SO4 , and 0.018% MgSO4
Refer to Loesche et al. (1972) for more information.
preterm labor (Hill, 1993, 1998). When intravenously injected into pregnant mice to
mimic transient bacteremia during periodontal infection, the organism colonized and
proliferated specifically in the placenta and induced preterm and term stillbirths (Han et al.,
2004).
F. nucleatum is known as an adhesive
organism. It adheres to a variety of host
mammalian cells, including epithelial and endothelial cells, polymorphonuclear leukocytes
(PMNs), monocytes, erythrocytes, fibroblasts,
and HeLa cells, as well as salivary macromolecules, extracellular matrix proteins, and
human IgG (Winkler et al., 1987; Ozaki et al.,
1990; Tuttle and Mangan, 1990; Xie et al.,
1991; Babu et al., 1995; Han et al., 2000,
2004;). It also coaggregates with all oral bacterial species tested so far and, thus, is a key
player in dental plaque formation (Kolenbrander and Andersen, 1989; Kolenbrander et al.,
1989; George and Falkler, 1992; Kolenbrander et al., 1995; Grimaudo and Nesbitt, 1997;
Andersen et al., 1998; Bradshaw et al., 1998;
Jabra-Rizk et al., 1999; Metzger et al., 2001;
Rosen et al., 2003). It has been suggested
that F. nucleatum possesses multiple adhesins
including both lectin-like and nonlectin-like
types (Murray et al., 1988; Mangan et al.,
1989; Tuttle et al., 1992; Takemoto et al., 1995;
Shaniztki et al., 1998; Weiss et al., 2000). Investigation of the adherence properties of F.
nucleatum is a major focus of fusobacterial
research. Three components, a 40- to 42-kDa
major outer membrane porin protein, FomA, a
39.5-kDa polypeptide, and a 30-kDa polypeptide, have been suggested as possible adhesins
from F. nucleatum involved in inter-bacterial
coaggregation (Kaufman and DiRienzo, 1989;
Kinder and Holt, 1993; Shaniztki et al., 1997).
FomA was also found to bind to the human
IgG Fc fragment (Guo et al., 2000). A high
Other
Eubacteria
13A.1.5
Current Protocols in Microbiology
Critical Parameters
Literature Cited
Andersen, R.N., Ganeshkumar, N., and Kolenbrander, P.E. 1998. Helicobacter pylori adheres selectively to Fusobacterium spp. Oral Microbiol.
Immunol. 13:51-54.
Troubleshooting
Laboratory
Maintenance of
Fusobacteria
If the bacterial culture does not grow as expected, the temperature and the anaerobicity in
the chamber should be examined. The anaerobicity can be tested using a BBL disposable
anaerobic indicator strip (Becton Dickinson).
Alternatively, the oxygen and hydrogen levels
in the chamber can be monitored by an oxygen and hydrogen analyzer (Coy Laboratory
Products). If oxygen is detected, the air in the
chamber needs to be recycled. The presence
of oxygen may indicate a leak in the chamber,
which can be detected using a leakage detector
(Coy Laboratory Products). The rubber gloves
are more prone to leakage than other parts of
the chamber.
Anticipated Results
Most F. nucleatum cultures can reach an
OD600 of >2 when grown in broth. When using selective media such as CVE or FEA, contaminating species will also grow, although
most oral species will be inhibited 106 - to
108 -fold. Frozen stocks of fusobacteria can
usually last >10 years if stored properly
at 80 C, although loss of viability may occasionally occur with some clinical isolates.
Time Considerations
Depending on the strain, it usually takes
2 to 7 days for fusobacteria to grow up on
blood agar plates or in broth. When subculturing from broth to broth, however, the bacteria
may reach the stationary phase within less than
24 hr if the dilution factor is low enough (e.g.,
1:5 or 1:10).
Babu, J.P., Dean, J.W., and Pabst, M.J. 1995. Attachment of Fusobacterium nucleatum to fibronectin immobilized on gingival epithelial
cells or glass coverslips. J. Periodontol. 66:285290.
Bennett, K.W. and Eley, A. 1993. Fusobacteria:
New taxonomy and related diseases. J. Med. Microbiol. 39:246-254.
Bradshaw, D.J., Marsh, P.D., Watson, G.K., and Allison, C. 1998. Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in
planktonic and biofilm oral microbial communities during aeration. Infect. Immun. 66:47294732.
Darveau, R.P., Belton, C.M., Reife, R.A., and Lamont, R.J. 1998. Local chemokine paralysis:
A novel pathogenic mechanism for Porphyromonas gingivalis. Infect. Immun. 66:16601665.
Dzink, J.L., Sheenan, M.T., and Socransky, S.S.
1990. Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium
nucleatum subsp. nucleatum subsp. nov., comb.
nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp.
nov., nom. rev., comb. nov. Int. J. Syst. Bacteriol.
40:74-78.
Garrity, G.M. (ed.) 2001. Bergeys Manual of Systematic Bacteriology, Vol. 1, 2nd ed. SpringerVerlag, New York.
13A.1.6
Current Protocols in Microbiology
George, K.S. and Falkler, W.A. Jr. 1992. Coaggregation studies of the Eubacterium species. Oral
Microbiol. Immunol. 7:285-290.
Grimaudo, N.J. and Nesbitt, W.E. 1997. Coaggregation of Candida albicans with oral Fusobacterium species. Oral Microbiol. Immunol.
12:168-173.
Kolenbrander, P.E. and Andersen, R.N. 1989. Inhibition of coaggregation between Fusobacterium
nucleatum and Porphyromonas (Bacteroides)
gingivalis by lactose and related sugars. Infect.
Immun. 57:3204-3209.
Other
Eubacteria
13A.1.7
Current Protocols in Microbiology
Tuttle, R.S., Strubel, N.A., Mourad, J., and Mangan, D.F. 1992. A non-lectin-like mechanism by
which Fusobacterium nucleatum 10953 adheres
to and activates human lymphocytes. Oral Microbiol. Immunol. 7:78-83.
Walker, C.B., Ratliff, D., Muller, D., Mandell, R.,
and Socransky, S.S. 1979. Medium for selective isolation of Fusobacterium nucleatum from
human periodontal pockets. J. Clin. Microbiol.
10:844-849.
Weiss, E.I., Shaniztki, B., Dotan, M., Ganeshkumar, N., Kolenbrander, P.E., and Metzger, Z.
2000. Attachment of Fusobacterium nucleatum
PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin.
Oral Microbiol. Immunol. 15:371-377.
Winkler, J.R., John, S.R., Kramer, R.H., Hoover,
C.I., and Murray, P. A. 1987. Attachment of oral
bacteria to a basement-membrane-like matrix
and to purified matrix proteins. Infect. Immun.
55:2721-2726.
Xie, H., Gibbons, R.J., and Hay, D.I. 1991. Adhesive properties of strains of Fusobacterium
nucleatum of the subspecies nucleatum, vincentii and polymorphum. Oral Microbiol. Immunol.
6:257-263.
Laboratory
Maintenance of
Fusobacteria
13A.1.8
Current Protocols in Microbiology
Laboratory Maintenance of
Flavobacterium psychrophilum and
Flavobacterium columnare
UNIT 13B.1
ABSTRACT
Bacteria in the genus Flavobacterium are highly heterogeneous and are comprised of
pathogenic and non-pathogenic species. They are found in a wide variety of environments
that include fresh and marine water, soil, and ocean sediments. This unit focuses on laboratory maintenance of two important fish pathogens within this genus. Flavobacterium
psychrophilum and F. columnare are fastidious, slow-growing organisms that require
specialized medium for optimal growth in the laboratory. Methods are described for the
preparation of media, broth and agar culture, cryopreservation, and enumeration of viable
cells for these bacteria. In addition, the diseases caused by these pathogens are described
along with methods of diagnosis and treatments. This unit serves as a general guide for
the cultivation of F. psychrophilum and F. columnare and discusses some of the difficulties that have been encountered for each. Curr. Protoc. Microbiol. 6:13B.1.1-13B.1.12.
C 2007 by John Wiley & Sons, Inc.
Keywords: Flavobacterium r bacterial culture r pathogen r soil bacteria r
aquatic bacteria
INTRODUCTION
The genus Flavobacterium is a highly heterogeneous and diverse group of Gram-negative
bacteria found everywhere from a variety of soil and water environments to plants, food
stuffs, and even distilled water lines. This unit presents general protocols for the laboratory maintenance of Flavobacterium species, with an emphasis on the fish pathogens
F. psychrophilum and F. columnare, which greatly impact fisheries and aquaculture
worldwide. Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), while F. columnare is the
etiological agent of columnaris disease.
Flavobacterium psychrophilum and F. columnare achieve optimal growth in vitro on
specialized selective low-nutrient medium. Tryptone yeast extract salts (TYES) is one
medium that promotes good growth for both species (see Basic Protocol 1) and is
routinely used in many laboratories. Several other media formulations have been shown
to support good growth for these species (see Reagents and Solutions). The process
of culturing these bacteria is described from frozen and lyophilized stocks as well as
routine maintenance of cultures in broth or agar medium (see Basic Protocol 1). The
procedure is identical for F. psychrophilum and F. columnare with the exception of
temperature. Flavobacterium psychrophilum grows best at 15 C, while the optimum
temperature for F. columnare is 25 C. Cryopreservation of Flavobacterium species can
be achieved by preparing glycerol stocks and storing at 80 C (see Basic Protocol
2); using this method, bacteria can remain viable for over 30 years. Enumeration of
viable cells for Flavobacterium can be achieved using a spread plate method (see Basic
Protocol 3). However, characteristics can differ between isolates and some may clump
or auto-agglutinate during growth, making this method of enumeration difficult. In such
Other
Eubacteria
13B.1.1
Supplement 6
cases, direct counting of cells by microscopy, which is briefly described in the Critical
Parameters and Troubleshooting section, can be used to estimate the number of cells
present but will not provide viable cell counts.
CAUTION: Flavobacterium psychrophilum and Flavobacterium columnare are Biosafety
Level 1 (BSL-1) organisms. Such organisms are not known to consistently cause disease
in healthy adult humans, and are of minimal potential hazard to laboratory personnel and
the environment. Standard microbiological practices should be followed when working
with these organisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more
information.
STRATEGIC PLANNING
TYES Medium
Flavobacterium psychrophilum and F. columnare do not grow effectively on standard
bacteriological medium such as tryptic soy agar (TSA; APPENDIX 2C); therefore, several
specialized low-nutrient media have been developed. One of the most common is TYES
(Holt et al., 1993), which has proven to be effective for the cultivation of numerous
isolates in the authors laboratory. The procedure for the preparation and sterilization
of 1 liter of TYES broth or agar is described in Reagents and Solutions. Other media
commonly used for the cultivation of these species is discussed below, with formulations
also provided in Reagents and Solutions.
Alternative Media: Anacker and Ordal, Hsu-Shotts, Shieh, and MAT
There have been numerous media and enhancements proposed for the growth of
F. psychrophilum and F. columnare. Three alternative media that are commonly used
include Anacker and Ordal (Anacker and Ordal, 1955), Hsu-Shotts (Bullock et al., 1986),
and Shieh (Shieh, 1980). It may be possible to obtain better growth of F. psychrophilum
and F. columnare using these media. Additionally, the use of MAT medium (Crump
et al., 2001) has been useful for reducing agglutination during broth culture (see Critical
Parameters and Troubleshooting). It is recommended that the growth of the isolate of
interest be analyzed in each of these and TYES to determine which medium is optimal
for the isolate being used. See Reagents and Solutions for formulations of media.
Avoiding contamination
The most common type of contamination during the culture of Flavobacterium species
is contamination from environmental bacteria present in the laboratory. The best way to
avoid contamination is prevention. When preparing culture media and other solutions for
use with bacteria, it is essential that they be sterilized prior to use. The use of sterilization
monitors during autoclaving is a good practice to ensure sterility of the solutions (see
Critical Parameters and Troubleshooting).
When preparing liquid media, it is best to sterilize the liquid medium in the culture flasks,
tubes, or other containers in which the bacteria will be grown. This prevents the possibility
of contaminating the medium while transferring it from a medium bottle into a sterile
culture flask or tube. When pouring petri plates for solid medium, it is best to pour the
plates in a biological safety cabinet or near a Bunsen burner. The use of a Bunsen burner
creates a draft of air that rises, which will reduce the possibility of bacterial contamination.
Laboratory
Maintenance of
F. psychrophilum
and F. columnare
The use of aseptic technique is critical when working with bacteria (see Basic Protocols
1, 2, and 3). All materials that will be used need to be sterile and it is good practice to
keep the area in which the work will be performed as sterile as possible by cleaning the
surface with 10% bleach or 70% ethanol. Passage of bacteria can be conducted near a
Bunsen burner as described above or in a biological safety cabinet. The utilization of
13B.1.2
Supplement 6
sterile medium and solutions, aseptic technique, and being cautious at all times when
working with bacteria will greatly reduce the potential for contamination.
BASIC
PROTOCOL 1
Materials
Lyophilized or frozen stock of F. psychrophilum or F. columnare (see Basic
Protocol 2)
Sterile KPBS (APPENDIX 2A)
TYES agar plates and broth (or alternative medium broth and plates; see recipe for
TYES agar and broth)
Sterile inoculation loops
Sterile 1-ml syringes and needles
Parafilm
Low-temperature incubator (15 to 25 C)
15-ml tubes
Additional reagents and equipment for Gram staining (APPENDIX 3C) and light
microscopy (UNIT 2A.1)
1a. For frozen stocks: Remove frozen stock of Flavobacterium isolate, aseptically remove cap, and gently scrape the frozen stock with a sterilized inoculation loop.
Aseptically streak for isolation on a petri plate containing TYES agar (or alternative
medium), use duplicate plates per isolate.
Keep the frozen stock cold by placing in an ice bucket when not in use.
1b. For lyophilized stocks: If the stock of Flavobacterium is lyophilized in a glass vial,
aseptically reconstitute the bacteria by injecting 1 ml sterile KPBS into the vial using
a sterile 1-ml syringe and needle. Remove the reconstituted bacteria and aseptically
pipet 30 l onto a petri plate containing TYES agar (or alternative medium) and
streak to isolate colonies, use duplicate plates per isolate.
2. Seal the agar plates with Parafilm, and incubate at 15 C for F. psychrophilum or
25 C for F. columnare.
The growth of F. psychrophilum and F. columnare isolates vary; however, growing for
4 to 7 days should be sufficient to obtain growth for most isolates.
3. Following incubation, observe the plates for isolated colonies typical of F. psychrophilum or F. columnare.
Colonies of F. psychrophilum are generally characterized as bright yellow, shiny, nonadhering, raised convex colonies with a thin spreading irregular margin, sometimes
referred to as a fried-egg appearance. However, some strains of the bacterium may
produce convex yellow colonies with smooth margins or a mixture of the two types
of colonies (Fig. 13B.1.1). Flavobacterium columnare colonies are characterized as
yellowish, strongly adhering, flat rhizoid colonies with an irregular margin, however,
some isolates may exhibit a smooth margin (Fig. 13B.1.1).
Other
Eubacteria
13B.1.3
Current Protocols in Microbiology
Supplement 6
Figure 13B.1.1 Morphology of F. psychrophilum colonies exhibiting a smooth margin (A), F. psychrophilum colonies
exhibiting thin spreading margins (fried-egg) (B), F. columnare colony exhibiting a characteristic at rhizoid colony with
an irregular margin (C), and F. columnare colonies exhibiting smooth margins (D). For a color version of this gure, see
http://www.currentprotocols.com
4. Aseptically pick an isolated colony using a sterilized inoculation loop and inoculate
a sterile 15-ml culture tube containing 15 ml TYES broth (or alternative medium).
Lightly cap the culture tube and gently vortex.
Due to the adherent nature of F. columnare colonies, it may be necessary to excise or
scrape colonies from agar using the sterile inoculation loop.
5. Incubate the inoculated broth statically or on an orbital shaker for 3 days at 15 C for
F. psychrophilum or 25 C for F. columnare.
6. Confirm the purity of the culture using Gram staining (APPENDIX 3C) and light microscopy (UNIT 2A.1) and/or aseptically plating a loop of culture for isolation onto
TYES agar (or alternative medium; APPENDIX 4A) and incubate for 4 to 7 days at 15 C
for F. psychrophilum or 25 C for F. columnare.
Laboratory
Maintenance of
F. psychrophilum
and F. columnare
13B.1.4
Supplement 6
Figure 13B.1.2 Cell morphology of F. psychrophilum (A) and F. columnare (B) following Gram staining and light microscopy. For a color version of this gure, see http://www.currentprotocols.com
Figure 13B.1.3 Growth of F. psychrophilum strain ATCC #49418 in TYES broth for 72 hr at
15 C. Duplicate 500-ml asks containing 200 ml TYES were inoculated with 0.5 ml of a 72-hr
culture (1.25 107 cfu ml1 ) of F. psychrophilum and cultured either statically (open squares) or
with the use of an orbital shaker at 100 rotations min1 (closed squares).
7. Aseptically obtain a loop of 3-day culture using a sterilized inoculation loop and
inoculate a sterilized culture tube or flask containing the desired volume of TYES
broth (or alternative medium).
The volume of broth required will vary depending on the desired use of the cultured
bacteria. For routine maintenance of the bacteria, it is recommended to use 15 ml broth.
If larger quantities of bacteria are needed, it is recommended to use 200 to 250 ml broth
in a 500-ml culture flask and inoculated with a larger volume of culture.
13B.1.5
Current Protocols in Microbiology
Supplement 6
9. Once log-phase growth is achieved, confirm the purity of the culture (see step 6). If
the culture is confirmed to be pure, then use for subsequent analyses.
The optical density of the Flavobacterium cultures can be determined using a spectrophotometer at a wavelength of 525 nm. Sterile TYES or an alternative medium broth should be
used as a blank. It should be noted that for some strains of F. columnare, OD525 readings
may be inaccurate if significant auto-agglutination is observed.
BASIC
PROTOCOL 2
CRYOPRESERVATION OF FLAVOBACTERIUM
Flavobacterium species can be cryopreserved in glycerol with good viability. Log-phase
Flavobacterium culture is mixed with sterile glycerol stock and 1-ml aliquots are frozen
at 80 C in cryovials.
Materials
Sterile glycerol stock (see recipe)
Log-phase growth of Flavobacterium (see Basic Protocol 1)
Sterile 15-ml polypropylene tubes
1.0-ml cryovials
Vortex
Cryovial storage box
1. Label a 15-ml polypropylene tube with the isolate number and ten 1.0-ml cryovials
with the isolate number, date, and any other information deemed necessary.
2. Aseptically add 4 ml sterile glycerol stock to the tube.
3. Aseptically add 6 ml log-phase growth of Flavobacterium to the 15-ml polypropylene
tube.
This procedure will give a final concentration of 20% glycerol.
Laboratory
Maintenance of
F. psychrophilum
and F. columnare
13B.1.6
Supplement 6
Materials
Petri plates with TYES or alternative medium agar (see recipe)
Sterile KPBS (APPENDIX 2A)
Flavobacterium culture (see Basic Protocol 1)
95% ethanol
Sterile culture tubes (13 100mm)
Cell spreader
Parafilm
Current Protocols in Microbiology
1. Label nine sterile 13 100mm culture tubes from 101 to 109 . Also label petri
dishes containing TYES agar (or alternative medium) in an identical manner and
include isolate number, date, and any other information deemed necessary.
It is recommended that duplicate or triplicate agar plates be used per dilution to accurately
determine the viable cell count.
5. Gently vortex the 101 dilution, aseptically remove 50 l, and dispense the liquid
onto the TYES agar (or alternative medium) plate labeled 101 .
6. Sterilize a cell spreader by immersing in 95% ethanol and flaming. Allow to cool, and
then spread the liquid over the entire surface of the plate. Repeat with the duplicate
(or triplicate) plates and remaining Flavobacterium dilutions.
7. Seal the agar plates with Parafilm, invert, and incubate 3 to 7 days at the optimum
temperature for the Flavobacterium species being used (see Basic Protocol 1).
8. Once observable colonies have grown, count the number of colonies on each plate
and average the duplicates (or triplicates) for each dilution.
The use of a dissecting microscope or colony counter will allow for accurate colony
counts. It is also suggested that only plates containing between 30 and 300 colonies be
used for enumeration to obtain statistically relevant numbers.
9. Estimate the number of viable cells in the original bacterial culture by using the
following equation for the dilution(s) that resulted in the growth of between 30 and
300 colonies:
Other
Eubacteria
continued
13B.1.7
Supplement 6
13B.1.8
Supplement 6
COMMENTARY
Background Information
Protocols vary greatly for the laboratory
maintenance of Flavobacterium species due
to the heterogeneous nature of this genus.
For example, the American Type Culture Collection (ATCC) suggests the use of nutrient
broth for F. pectinovorum and lactose medium
for F. hibernum. Jooste and Hugo (1999) reviewed cultivation methods for the genera
of the family Flavobacteriaceae. The culture
of the fish pathogens, F. psychrophilum and
F. columnare, requires a specialized medium
such as TYES (see Strategic Planning) due to
their fastidious nature. Other medium formulations are available (see Strategic Planning)
and numerous improvements to these standard
medium have been recommended (Song et al.,
1988; Lorenzen, 1993; Daskalov et al., 1999;
Michel et al., 1999; Cepeda et al., 2004). A
growth analysis should be performed utilizing
the medium described in this unit to obtain
optimal growth of the species and isolate of
interest.
The preparation of glycerol stocks is a
standard method for the cryopreservation of
bacteria. Bacteria preserved in this manner
remain viable for over 30 years. Another
method of cryopreservation commonly used
for Flavobacterium species is lyophilization.
This also results in a high level of viability following preservation. Several methods of lyophilization and suspending medium
have been tested for F. psychrophilum and
Other
Eubacteria
13B.1.9
Current Protocols in Microbiology
Supplement 6
Laboratory
Maintenance of
F. psychrophilum
and F. columnare
Flavobacterium columnare
Flavobacterium columnare is the etiological agent of columnaris disease. Most
species of freshwater fish are susceptible to
F. columnare, and susceptibility is greatly increased if fish have been exposed to predisposing conditions such as stress. Columnaris
disease is the second leading cause of mortality in the channel catfish (Ictalurus punc-
13B.1.10
Supplement 6
of cells may be the best way to quantify certain isolates. The method consists of staining
suspensions of bacteria with crystal violet and
counting cells in a Petroff-Hauser bacterial
counting chamber. It is important to realize
that this method provides only total numbers
of cells and not viable cell counts. However,
a possibility is to couple direct counting with
the use of bacterial viability kits to estimate
the number of viable cells by direct enumeration. Such methods have been used with F. psychrophilum (Michel et al., 1999; Vastos et al.,
2003).
Anticipated Results
The growth of different isolates of F. psychrophilum and F. columnare can vary greatly
and growth curves should be generated for the
isolate being used. A typical growth curve for
F. psychrophilum grown statically or on an orbital shaker is illustrated in Figure 13B.1.3.
The methods of cryopreservation described
in this unit should yield viable stocks for
greater than 30 years. Enumeration of cells via
the spread plate method will allow for quantification of viable cells from most cultures of
Flavobacterium.
Time Considerations
Preparing medium for Flavobacterium
should take 10 min for solid and liquid media. Sterilization of the medium by autoclaving
should take 30 min, depending on the type
of autoclave being used. Cooling medium to a
useable temperature for broth (room temperature) and agar medium (60 C for pouring)
will vary depending on the ambient temperature of the laboratory.
The entire process of obtaining a culture of
Flavobacterium from a frozen or lyophilized
stock will take 12 to 18 days. Preparing
streaks of bacteria from frozen or lyophilized
stocks will take 15 min, followed by 4 to
7 days of incubation to allow for isolated
colonies to grow. Inoculating the obtained isolated colonies to broth medium will take <15
min, followed by 3 days of incubation. Confirmation of the purity of the culture will take
20 min for Gram staining (APPENDIX 3C) and
microscopic evaluation, or 4 to 7 days for plating bacteria and examining for colonies with
the phenotype of the species being cultivated.
Inoculating the desired volume of medium
with the 3-day culture will take <15 min, followed by 2 to 7 days of incubation to obtain
log-phase cultures that can be used for subsequent analyses. At this point the cultures
should be examined for purity. The time required for this would be the same as previously
described.
Cryopreserving an isolate of Flavobacterium as glycerol stocks will take 15 min.
Labeling tubes and cryovials will take 5 min,
mixing culture and glycerol stock will take
5 min, and dispensing aliquot stocks and
freezing will take <5 min.
Enumerating viable cells by the spread
plate method will take 8 days. Set-up, tube
and plate labeling, and serial dilutions will take
30 min. Aseptically plating bacterial dilutions will take 30 min. Inoculated plates need
to be incubated for up to 7 days or until sufficient growth is achieved to visualize colonies.
Following incubation, counting colonies and
calculating cfu ml1 will require 30 min
depending on the number of plates requiring
counts.
Literature Cited
AFS-FHS (American Fisheries Society-Fish Health
Section). 2006. FHS Blue Book: Suggested Procedures for the Detection and Identification of
Certain Finfish and Shellfish Pathogens, 2006
Edition. AFS-FHS, Bethesda, Maryland.
Anacker, R.L. and Ordal, E.J. 1955. Study of a bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 70:738-741.
Bernardet, J.-F., Segers, P., Vancanneyt, M., Berthe,
F., Kersters, K., and Vandamme, P. 1996. Cutting
a gordian knot: Emended classification and description of the genus Flavobacterium, emended
description of the family Flavobacteriaceae,
and proposal of Flavobacterium hydatis nom.
Nov. (Basonym, Cytophaga aquatilis Strohl and
Tait 1978). Int. J. Syst. Evol. Microbiol. 46:128148.
Bullock, G.L., Hsu, T.C., and Shotts, E.B. 1986.
Columnaris disease of fishes. U.S. Fish and
Wildlife Service, Fish Disease Leaflet 72.
National Fisheries Research Center-Leetown,
National Fish Health Research Laboratory,
Kearneysville, West Virginia.
Cepeda, C., Garca-Marquez, S., and Santos, Y.
2004. Improved growth of Flavobacterium psychrophilum using a new culture medium. Aquaculture 238:75-82.
Crump, E.M., Perry, M.B., Clouthier, S.C., and
Kay, W.W. 2001. Antigenic characterization
of the fish pathogen Flavobacterium psychrophilum. Appl. Environ. Microbiol. 67:750759.
Daskalov, H., Austin, D.A., and Austin, B. 1999. An
improved growth medium for Flavobacterium
psychrophilum. Lett. Appl. Microbiol. 28:297299.
Desolme, B. and Bernardet, J.-F. 1996. Freezedrying
of
Flavobacterium
columnare,
Flavobacterium psychrophilum and Flexibacter
maritimus. Dis. Aquat. Org. 27:77-80.
Other
Eubacteria
13B.1.11
Current Protocols in Microbiology
Supplement 6
Evenson, O. and Lorenzen, E. 1996. An immunohistochemical study of Flexibacter psychrophilus infection in experimentally and naturally infected rainbow trout (Oncorhynchus
mykiss) fry. Dis. Aquat. Org. 25:53-61.
Holt, R.A., Rohovec, J.S., and Fryer, J.L. 1993.
Bacterial coldwater disease. In Bacterial diseases of fish (V. Inglis, R.J. Roberts, and N.R.
Bromage, eds.) pp. 3-23. Blackwell Scientific
Publications, Oxford.
Jooste, P.J. and Hugo, C.J. 1999. The taxonomy,
ecology and cultivation of bacterial genera belonging to the family Flavobacteriaceae. Int. J.
Food Microbiol. 53:81-94.
Lorenzen, E. 1993. The importance of the brand
of the beef extract in relation to the growth of
Flexibacter psychrophilus in Anacker & Ordal
medium. Bull. Eur. Assoc. Fish Pathol. 13:6465.
Michel, C., Antonio, D., and Hedrick, R.P. 1999.
Production of viable cultures of Flavobacterium
psychrophilum: Approach and control. Res. Microbiol. 150:351-358.
Michel, C. and Garcia, C. 2003. Virulence stability
in Flavobacterium psychrophilum after storage
and preservation according to different procedures. Vet. Res. 34:127-132.
Plumb, J.A. 1999. Health Maintenance and Principal Microbial Diseases of Cultured Fishes. Iowa
State University Press, Ames, Iowa.
Schmidtke, L.M. and Carson, J. 1995. Characteristics of Flexibacter psychrophilus isolated from
Atlantic salmon in Australia. Dis. Aquat. Org.
21:157-161.
Internet Resources
http://aquanic.org/publicat/usda rac/efs/srac/
479bfs.pdf
Website link to an extension bulletin of the Southern Regional Aquaculture Center (SRAC) on columnaris disease.
http://www.fish.washington.edu/wrac/
Coldwater bacterial disease.pdf
Website link to an extension bulletin of the Western
Regional Aquaculture Center (WRAC) on coldwater
disease.
Laboratory
Maintenance of
F. psychrophilum
and F. columnare
13B.1.12
Supplement 6
UNIT 13C.1
ABSTRACT
Bacteroides species are Gram-negative, obligate anaerobic bacteria. They are the predominant indigenous bacterial species in the human intestinal tract, where they play an
important role in the normal physiology of the host, but they can also be significant
opportunistic pathogens. The fact that these are obligate anaerobes is the prevailing
feature that affects the methodology used for their cultivation. Several techniques are
described for anaerobic culturing; the anaerobic glove box and anaerobic jars are the
two techniques most adaptable to a range of research needs. Straightforward methods
are presented for propagation on solid media and in broth cultures, and for the long-term
storage and maintenance of stock cultures. The Bacteroides species are saccharolytic,
which is the second feature of their physiology that impacts cultivation methodology.
Several flexible media formulations, including a defined minimal media, are provided
that allow the researcher to choose the carbon source best suited for his or her work.
C 2008 by John Wiley & Sons, Inc.
Curr. Protoc. Microbiol. 9:13C.1.1-13C.1.21.
Keywords: anaerobic culturing r Bacteroides r anaerobic jars r anaerobic chambers
INTRODUCTION
This unit describes methods for the culturing, storage, and maintenance of the Gramnegative, anaerobic Bacteroides species. Members of the genus Bacteroides play important roles in human and animal health (Smith et al., 2006). They are the predominant
members of the indigenous intestinal flora in humans where they contribute to normal
intestinal development, physiology, and function. Largely due to their proximity to the
human host, several Bacteroides species can be opportunistic pathogens and they are
frequently isolated from a range of anaerobic infections including intraabdominal and
pelvic abscesses, soft tissue, female genital tract, and bacteremia. Their ability to successfully colonize mammals is due in part to their extended aerotolerance, simple nutritional
requirements, and ability to utilize diverse carbohydrate substrates for carbon and energy.
All of these factors are important considerations when culturing these organisms. Due
to a combination of innate and acquired mechanisms the Bacteroides species are resistant to a wide array of commonly used antibiotics which can complicate antimicrobial
therapy of anaerobic infections. Although there has been considerable interest in the
mechanisms responsible for the transmissible, acquired antibiotic resistance, it is their
innate resistance to aminoglycosides and vancomycin that has proven to be important for
development of selective media for their isolation (see Smith et al., 2006).
The single most important consideration when culturing the Bacteroides is that they are
obligate anaerobes and they will not divide in the presence of oxygen. That said, they
are one of the most aerotolerant anaerobic species known and a recent report (Baughn
and Malamy, 2004) has suggested that they may actually slowly divide in the presence of
nanomolar concentrations of oxygen. Thus, while it is appropriate that this unit include
a discussion of anaerobic culturing techniques, the methods discussed here may not
be suitable for many other anaerobic bacteria since they are not as rigorous in their
Current Protocols in Microbiology 13C.1.1-13C.1.21, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc13c01s9
C 2008 John Wiley & Sons, Inc.
Copyright
Other
Eubacteria
13C.1.1
Supplement 9
requirement for exclusion of oxygen at all stages of media preparation and inoculation.
In this regard, it is important to note that anaerobic culturing requires some specialized
equipment. It is our intention to describe sufficient equipment options so that those
new to the field will be able to easily initiate Bacteroides culturing without a large
initial investment in expensive equipment. However, this unit is not intended to be a
comprehensive treatise on anaerobic culturing methods and the reader will be directed to
several excellent reviews and manuals for detailed descriptions of anaerobic techniques.
CAUTION: Bacteroides are considered Biosafety Level 1 (BSL-1) organisms in the USA
although in some European countries they are BSL-2. These organisms are not known to
consistently cause disease in healthy adult humans, and are of minimal potential hazard
to laboratory personnel and the environment. However, given that some procedures
described in this unit require the use of syringes for the transfer of cells, it is recommended
that the standards for BSL-2 be followed. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.
STRATEGIC PLANNING
Culturing Bacteroides in Liquid Media
Bacteroides cultures must be incubated anaerobically to allow growth, but since they are
aerotolerant many manipulations can be done conveniently on the benchtop. The choice
of whether to work on the benchtop or in an anaerobic chamber/glove box depends on
the needs of the experiment. Culturing performed entirely in the glove box will result in
more consistent growth patterns with shorter lag periods, which are necessary for some
physiological studies but may be of no consequence when simply inoculating a series of
overnight cultures for DNA isolation.
Another important consideration when growing cultures in broth is the type of tube or
growth vessel to be used. For experiments in which the culture will remain in the anaerobic
chamber or jar, any type of closure that maintains sterility will suffice. However, it often
is desirable to grow cultures outside of the chamber due to limited space or the need
to frequently monitor growth. In these situations tubes or flasks which can be tightly
sealed with a butyl rubber stopper are used. Thus, for long term incubation of culture
tubes outside of the glove box the culture tubes must be inoculated in the chamber
and then sealed with a sterile rubber stopper (#00 for 13 100mm tubes and #1 for
16 150mm tubes). Screw-cap tubes with a rubber cap liner are useful culture tubes
and will maintain an anaerobic atmosphere for a short time when removed from the
anaerobic chamber. Another type of tube suitable for anaerobic culturing outside of the
glove box is the Hungate tube (Bellco Biotechnology; see Internet Resources). These are
standard screw-cap tubes that have a specialized cap (with a 9-mm opening) that is used
to hold a flanged, septum-type butyl rubber stopper in place in the top of the tube. When
using Hungate tubes inoculation and sampling are done directly through the stopper with
a syringe or the cap is taken off and the rubber stopper removed with a hemostat. Another
advantage of Hungate tubes is that they can be autoclaved with the caps securely fastened
(see Support Protocol 5).
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
13C.1.2
Supplement 9
Other
Eubacteria
13C.1.3
Current Protocols in Microbiology
Supplement 9
Materials
Supplemented brain heart infusion medium (BHIS; see recipe) or Tryptone yeast
extract glucose medium (TYG; see recipe), 5 ml in 13 100mm Hungate
screw-cap tubes
Chopped meat stock culture of Bacteroides strain in chopped meat medium (see
Basic Protocol 3)
Glove box (Support Protocol 3)
Neoprene gloves
Disposable antiseptic wipes (Allegiance Antiseptic Towelettes)
6 hemostat
1-ml disposable sterile pipet
1. Prepare a clean work space in the glove box and gather the materials together.
It is good practice to bring all glassware, plasticware, pipets, and other materials into the
glove box at least 4 hr (preferably overnight) prior to use. This is especially important for
plasticware which tends to bind oxygen and releases it slowly, which can lead to oxidation
of the media.
2. Arrange two tubes of BHIS medium in a rack and fully loosen the caps. Make sure
the tubes are well separated in the rack so that there is room to maneuver with the
neoprene gloves.
TYG medium is a versatile, complex medium that can be substituted for BHIS in situations
where glucose is not desirable (also see Critical Parameters and Troubleshooting).
3. Gently mix the contents of the chopped meat culture in the Hungate tube, then swab
the top of the tube with an antiseptic wipe. Take off the screw cap and carefully remove
the stopper with the hemostat. Place the stopper upside down in a clean location.
4. Using a 1-ml pipet remove 0.6 ml from the culture, then lift the cap to one BHIS
tube, dispense 0.4 ml of the culture, and then repeat with the second BHIS tube
adding 0.2 ml to it.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
Growth of cultures from chopped meat stocks can vary depending on the age of the culture,
types of antibiotics present in the media (Table 13C.1.1), or other factors. Thus, usually
two amounts of inoculum are used to optimize the results. If equal growth is observed in
both tubes, then the one that received the lower inoculum volume is used for subsequent
operations.
13C.1.4
Supplement 9
Resistance
gene
Working
concentration (g/ml)
Cefoxitin
cfxA
20-25
Chloramphenicol
cat
25 in 95% ethanol
25
Clindamycin
ermF
Erythromycin
ermF
50 in 95% ethanol
10-20
Tetracyclinea
tetQ
10 in 70% ethanol
4-10
Gentamicin
50-200
Nalidixic acidb
20 in 0.1 M NaOH
40-50
Rifampicina
20 in 100% methanol
20
Fusidic acid
25 in 95% ethanol
50
Trimethoprim
100
Antibiotic
Antibiotics used in plasmid
and transposon vectors
An alternate method to prepare cultures from chopped meat stocks in Hungate tubes is to
simply wipe the top of the tube with an antiseptic wipe and then insert a 1-ml syringe (20-G
needle) into the tube. Remove the indicated amount of culture to transfer to the new tubes.
One also can inoculate broth cultures from plates. The only difference is that disposable
inoculating loops are used to pick colonies and are then swirled in the tubes to dislodge
the cells. These tubes can be vortexed to further break up the cell clumps and ensure even
growth in the tube.
5. Screw the caps into place and then replace the stopper on the chopped meat culture
making sure that it is well seated in the tube.
6. Place the inoculated tubes at 37 C in the glove box incubator.
Dense growth (A550 > 0.8) of the cultures should be observed following overnight
incubation.
Growth will be slower if antibiotics (Table 13C.1.1) are added to the medium and the
generation time varies greatly with different media.
BASIC
PROTOCOL 2
The following is a typical protocol for starting cultures from a petri plate followed by
incubation in an anaerobic jar.
Materials
BHIS medium (see recipe) or TYG medium (see recipe), 5 ml in 13 100mm
screw cap tubes in rack
Bacteroides strain streaked on BHIS or TYG plate (see Strategic Planning)
Sterile culture tubes
Inoculating loop
Bunsen burner
Anaerobic jar (Support Protocols 1 and 2)
37 C incubator
Other
Eubacteria
13C.1.5
Current Protocols in Microbiology
Supplement 9
1. Dispense freshly prepared BHIS medium into sterile culture tubes. Gather other
materials and cultures.
2. Sterilize loop, allow to cool, and pick up cells from a large colony.
3. Remove cap from culture tube, flame lip of tube, and swirl loop in medium.
4. Replace cap on tube, mix well, and place in round test tube rack.
Ensure that the caps on the culture tubes are sufficiently loose to allow exchange of gasses.
5. Place rack in jar and then process jar as described below using GasPak gas-generating
envelopes or as described for the vented jars.
6. Place jar upright in a 37 C incubator.
Materials
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
Palladium catalyst (Coy Laboratories; in wire mesh basket attached to jar lid)
Cultures (rack of culture tubes or streaked petri plates)
10 ml tap or dH2 O
Polycarbonate GasPak jar or equivalent with lid
GasPak anaerobic system envelope
13C.1.6
Supplement 9
2. Cut off the corner of a GasPak envelope and place it upright in the jar together with
the rack of petri plates or tubes containing the cultures. Add 10 ml of water to the
gas generation packet with a pipet or syringe.
When placing any liquid cultures in the jar, the caps of the culture tubes must be loose to
allow adequate circulation of the atmosphere. Also, avoid stacking petri plates directly
under the catalyst basket since extreme heat is generated during the reaction.
Two jar sizes are available. The smaller 2.5 liter jar can accommodate one rack of 12
standard petri plates and requires one GasPak envelope. The 9.5 liter jar uses three
envelopes and can accommodate 36 standard petri plates.
3. Open an anaerobic indicator strip and place it in the jar. The strip is saturated with
methylene blue and turns from blue to colorless in the absence of oxygen.
The strip should begin to turn colorless within 60 min of closing the jar and it should be
completely colorless within a few hours.
4. Quickly place the lid on top of the jar and secure with the lid clamp. Place the jar in
a 37 C incubator for 24 to 48 hr. The jar must remain upright at all times in order to
avoid spillage of the water.
Within an hour of incubation the user can check progress of the anaerobic system by
examination of the indicator strip visible through the clear polycarbonate jar and by the
appearance of condensation on the inside of the jar indicating the formation of water
vapor as the free oxygen is removed. The use of the clear polycarbonate jars also is
advantageous since cultures can be inspected periodically without having to open the jar.
SUPPORT
PROTOCOL 2
Other
Eubacteria
13C.1.7
Current Protocols in Microbiology
Supplement 9
supply costs remain relatively low since it does not require purchase of the gas-generating
envelopes.
Materials
Palladium catalyst (in wire mesh basket attached to jar lid)
Cultures (rack of culture tubes or petri plates)
Polycarbonate GasPak jar or equivalent with lid
GasPak anaerobic indicator
Standard vacuum pump with gauge, tubing, and three-way stopcock (Fisher
Scientific)
Gas Mixture cylinder (85% N2 , 10% CO2 , and 5% H2 ) and regulator
37 C incubator
NOTE: An automatic evacuation/replacement system, Anoxomat (see Internet
Resources), that precisely evacuates and fills jars by a microprocessor controlled pumping unit is now available. The standard Anoxomat configuration has one gas connection
and one jar connection but options are available for connecting up to 5 jars at one time.
1. Fill the wire mesh basket with 2 to 3 g of palladium catalyst pellets and attach it to
the jar lid.
The same considerations for care and maintenance of the palladium catalyst exist as
stated in Support Protocol 1.
2. Place all cultures in the jar, open an anaerobic indicator strip, place it in the jar, close
the lid and secure with the lid clamp.
The jars used in this method are essentially the same as those described in Support
Protocol 1 except that they are vented with a hose nipple to which is attached a short
length of rubber tubing and a screw clamp to control air flow. Commercially available
jars all have a vent option.
3. Attach the jar to a vacuum pump via the vent nipple and ensure the screw clamp
is fully open. Evacuate the jar to 25 Hg. Using the stopcock direct the air flow
away from the vacuum pump to the gas cylinder and fill the jar slowly with gas being
careful not to overfill the jar. Repeat the evacuation and fill again.
The anaerobe jars are designed to hold a vacuum but cannot hold positive pressure. Thus,
there is little danger of overfilling a jar to the point where it will burst; however, close
attention must be paid to the gas replacement step so that the jar lid does not become
damaged.
It is generally convenient to construct a small gassing manifold which has the stopcock
valve and tubing fixed to a wooden platform together with a vacuum gauge to monitor
pressure in the jar (Fig. 13C.1.1). The idea is to design the manifold so that the user can
control direction of the air flow at the proper time between the vacuum pump and the
gas cylinder. The vacuum gauge should be connected to the manifold by a T connector
and should be located between the jar and the first stopcock valve. The manifold greatly
simplifies the evacuation and filling process and ensures that the jars will not be overfilled
with gas.
4. Finally, evacuate one more time and then use the stopcock to direct airflow to the
gas mix cylinder and proceed to fill the jar with the anaerobic gas mixture. Close the
screw clamp and disconnect the jar from the vacuum pump assembly.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
13C.1.8
Supplement 9
Figure 13C.1.1 Typical gassing manifold for the lling and evacuation of vented anaerobic jars
using a standard vacuum pump and gas mixture.
Anaerobe Chamber
The anaerobic chamber or anaerobic glove box is a self-contained, closed system providing the investigator with an anaerobic laboratory bench where nearly any experiment
can be performed under anaerobic conditions. The only disadvantage of the anaerobic
chamber is in the initial cost of the investment but the experimental benefits far outweigh
the cost for a laboratory routinely engaged in anaerobic microbiology. Anaerobic chambers accommodate an incubator, allowing the culture of plates, flasks, and tubes without
the need for jars. There also is shelving space to allow the storage of media and other
materials, thus eliminating the need to freshly prepare media before use. Cultures also
can be stored in the anaerobic chamber on plates but care must be taken to ensure that
they do not dry out.
SUPPORT
PROTOCOL 3
As with the vented anaerobic jars, the glove box atmosphere contains 5% to 10% CO2 ,
5% to 10% H2 , and the balance will be nitrogen or argon. An anaerobic atmosphere
with <5 ppm of O2 is maintained in the glove box by circulation through wire screen
filters containing palladium catalyst. All materials (e.g., cultures, media, and tubes) enter
the chamber through a double door airlock which is purged with anaerobic gas and thus
functions to limit the amount of oxygen that enters the glove box. Most modern anaerobic
chambers possess an automated airlock that simplifies their use and makes if difficult to
accidentally introduce large amounts of air into the chamber by inappropriate opening of
the airlock doors. Once one becomes at ease with the inconvenience of working with the
neoprene gloves they will find the anaerobic chamber to be a convenient, efficient way
to culture the Bacteroides. There are several types of chambers available, from rigid to
flexible, and gloved to gloveless (see Internet Resources). By far the most adaptable and
cost effective are flexible vinyl glove boxes manufactured by Coy Laboratory Products
and based on the original design by Aranki and Freter (Aranki et al., 1969). Only use of
the flexible glove box will be considered in this unit.
Other
Eubacteria
13C.1.9
Current Protocols in Microbiology
Supplement 9
Materials
Palladium catalyst, 4 packs (wire screen enclosure containing 165 g catalyst)
Silica gel desiccant type IV, 2 trays each containing 2.5 kg desiccant (Sigma
Chemical)
Bacterial cultures
Coy flexible glove box (2 person) with 37 C forced air incubator (Coy
Laboratories)
Oxygen/hydrogen meter (Coy Laboratories)
Nitrogen gas cylinder and regulator (Coy Laboratories)
Gas mixture cylinder (80% N2 , 10% CO2 , and 10% H2 ) and regulator (Coy
Laboratories)
1. Gather materials together and open the outer airlock door. Place the catalyst, trays
of desiccant, and other materials inside the airlock. Close the outer airlock door and
initiate the purging cycle to lower the oxygen level in the entry lock.
CAUTION: Hot liquids should not be brought through the airlock since they will boil
over during the vacuum cycle. Allow them to cool to at least 60 C.
2. The purging cycle will evacuate and replace the airlock atmosphere with N2 twice
and then following a final evacuation the air lock is filled with the anaerobic gas
mixture. When the cycle is complete, check the front panel of the airlock to determine
that the anaerobic indicator light is on and then open the inner airlock door. Remove
all items from the airlock and then place in any outgoing materials before closing
the inner door.
The anaerobic indicator light is used to signal the status of the airlock and is activated
following the purging cycle and remains lit until the outer airlock door is opened. The
indicator light should always be checked prior to opening the inner airlock door in order
to prevent accidental contamination of the chamber atmosphere with oxygen.
3. Place the catalyst packs into the two catalyst fan boxes and set the trays of desiccant
on the shelving unit. Check the oxygen/hydrogen meter to ensure safe, efficient
operation of the glove box.
Four catalyst packs (two in each fan box) are needed in very active chambers and they
should be changed about twice a week. Humidity can get high in active chambers due
to oxygen removal so the silica gel desiccant should be changed twice a week or more
according to the level of humidity as shown by the indicator in the desiccant.
To rejuvenate the catalyst, heat in an oven at 160 C for 2 hr, then store with a desiccant
until needed. The life expectancy of the catalyst pellets is 1 year but can vary depending
of the level of usage and exposure to hydrogen sulfide which poisons the catalyst. Refer
to Critical Parameters and Troubleshooting for instructions on how to determine if the
catalyst is still active. Assessing changes in the color indicator present in the media is
not a useful indicator of the catalyst state as there are many possible causes to such a
change. Desiccant is dried before use in a 110 C oven and stored in an airtight container.
A chamber using a 10% hydrogen gas mixture should maintain internal levels of hydrogen
between 4% to 6%. Higher hydrogen levels suggest a decrease in catalyst efficiency and
lower hydrogen levels indicate a recent exposure to oxygen or infrequent use of the
chamber. The hydrogen in the chamber atmosphere is only replaced during entry through
the airlock and the levels can become low over time in an inactive chamber. If hydrogen
levels fall < 1% to 2% additional gas mixture should be added through the airlock.
4. Place bacterial cultures in the incubator and other materials on the shelving unit.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
13C.1.10
Supplement 9
When first bringing cultures into the glove box loosen the caps to allow circulation of
the anaerobic atmosphere in the tubes or flasks. Likewise, fresh media should be stored
overnight with the caps loosened to ensure that all oxygen diffuses out of the media and
that there is full exchange of the head space of the media vessels with the anaerobic
atmosphere.
Current Protocols in Microbiology
5. Open the outer door and remove items from air lock. Recycle the airlock leaving it
in the anaerobic state if not planning additional use of the glove box.
SUPPORT
PROTOCOL 4
The basic use of a gassing cannula is described here in order to support other protocols
described in this unit. The reader is directed to Holdeman et al. (1977), Bryant (1972), and
Breznak and Costilow (1994) for more detailed descriptions of using gassing cannulas
and the preparation of prereduced media.
Materials
Tube of chopped meat medium (Support Protocol 5)
Bacteroides strain streaked on BHIS or TYG plate (see Strategic Planning)
Gas mixture cylinder (90% N2 , 10% CO2 , 10% H2 ) and regulator (Coy
Laboratories)
Gassing cannula with 6 , 16-G blunted needle bent at 90 (Popper & Sons)
Bunsen burner
Test tube rack
Inoculating loop
6 hemostat
37 C incubator
1. Open the regulator for the gas mixture and adjust the gas flow so that a gentle stream
of gas flows from the end of the cannula with a force great enough to put a small
dent into the Bunsen burner flame.
The gassing cannula is a 2-ml Luer-Lok glass syringe. The syringe barrel is filled with
cotton and then sterilized. The syringe is then attached to the gas source via 1/4 rubber
tubing.
It is not desirable to have such a strong flow of gas from the cannula that the culture
media will splatter or bubble up from the vessel.
2. Flame the top of the chopped meat medium tube and quickly remove the rubber
stopper with the hemostat, being careful to not contaminate the rubber stopper. Place
the tube in the rack.
3. Quickly sterilize the cannula by heating the bent needle in the Bunsen burner until it
is red, then place it in the tube of medium so that it rests on the lip of the tube.
4. Sterilize the inoculating loop and then pick a colony from the plate. Transfer the
colony to the tube of medium by gently stirring the loop in the medium.
Antibiotics (Table 13C.1.1) and other additions to the medium can be made while the
tubes are under the flow of gas.
Other
Eubacteria
13C.1.11
Current Protocols in Microbiology
Supplement 9
5. Retrieve the rubber stopper, briefly flame it, and reseal the tubes quickly by pulling
up on the cannula as the stopper is being pushed down in place. Once the tube is
closed twist the stopper to ensure a good seal and the turn off the gas.
Care must be taken when closing tubes and other culture vessels with the rubber stoppers.
Do not use too much force or push down excessively since the tubes will break and injury
can result. Use a twisting motion to ensure a good seal with minimal force.
Materials
Chopped meat medium stock cultures (Support Protocol 5)
Bacteroides strain streaked on BHIS or TYG plate (see Strategic Planning)
37 C incubator
1. Inoculate chopped meat medium stock cultures with a single-well isolated colony
from an agar plate.
2. Incubate the culture 36 to 48 hr at 37 C. Store the cultures in the dark at room
temperature.
Due to the inherent turbidity of the chopped meat medium it is not always easy to determine
if adequate growth has occurred; thus, cultures are usually incubated for the prescribed
length of time to ensure adequate growth. Cultures grown in this way will remain viable
for 10 years.
The inoculation of chopped meat cultures can be done as described above in the gassing
cannula protocol or they can be inoculated from plates in the anaerobic chamber as
described under the inoculation of liquid cultures protocol.
The growth in chopped meat cultures is not as vigorous as in some other media due
to the low levels of carbohydrate present in the medium, but it is this very fact that
makes chopped meat a good storage medium. The low carbohydrates result in low acid
production which consequently does not stress the cells.
SUPPORT
PROTOCOL 5
Materials
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
13C.1.12
Supplement 9
2-liter beaker
Stir bar
Bunsen burner
Aluminum foil
Cheese cloth
Graduated cylinder
2-liter flask
Gassing cannula (Support Protocol 4)
Gas cylinder (either a gas mix or 100% N2 ) and regulator (Coy Laboratories)
Laboratory tape
Anaerobe chamber
Hungate tubes (Bellco Biotechnology), 16 125mm with septum stoppers and
screw caps
1. In a 2-liter beaker put one liter dH2 O, a stir bar, 500 g extra lean ground beef, and
25 ml of 1 M NaOH. Bring to a boil with slow stirring and let boil for 5 min.
2. Cover the beaker with aluminum foil and place at 4 C overnight or until fat solidifies.
Remove ALL of the fat and discard it.
The fat comes to the surface and is easily removed.
3. Strain the meat broth through several layers of cheese cloth, retain both the broth
and the meat particles. Spoon off any remaining fat floating on the surface of the
strained broth.
4. Pour the broth into a graduated cylinder and bring up volume to one liter with dH2 O.
5. Pour the broth into a 2-liter flask and add 30 g tryptone, 5 g yeast extract, 2.5 g
KH2 PO4 , 2.5 K2 HPO4 , 10 ml hemin solution, and 1 ml resazurin solution.
6. Using the gassing cannula, bring the broth to a boil under a stream of N2 or N2 +
CO2 gas and boil for 2 min. Cool the broth on ice while still gassing. When cooled
stopper flask tightly, secure the stopper with laboratory tape, and then put the flask
into the anaerobe chamber.
Boiling the medium under a stream of oxygen-free gas will drive off all oxygen from the
medium and as it cools under the stream of anaerobic gas there will be little or no oxygen
contamination. This mixture is high in organic material and can rapidly boil over if care
is not taken to adjust the flame as the boil begins.
7. While the broth is cooling prepare about 100 Hungate culture tubes in racks by
adding ground beef meat particles so that they are evenly distributed among the
tubes. Place chopped meat tubes, rubber stoppers, and screw caps into the anaerobe
chamber and allow them to equilibrate for a short time.
Placing the meat into the tubes can be facilitated by pressing the tubes into the meat and
then using a 1-ml disposable pipet or glass rod to push the meat to the bottom of the tube.
8. Add 1.0 g of cysteine and 20 ml of NaHCO3 (10% solution) to the meat broth and
mix by swirling.
9. When cysteine is dissolved dispense 10 ml of broth into each tube, then secure a
stopper into place, and screw on the cap. Ensure that the cap is tightened to prevent
leakage of the anaerobic atmosphere during autoclaving.
10. Remove tubes from chamber and autoclave for 30 min.
The sealed Hungate tubes with the tightened cap are designed to be autoclaved directly in
the test tube racks. These tubes will easily withstand the internal pressure resulting from
the autoclave temperatures and they will maintain their anaerobic atmosphere.
Other
Eubacteria
13C.1.13
Current Protocols in Microbiology
Supplement 9
BASIC
PROTOCOL 4
Materials
BHIS medium and plate (see recipe)
Glycerol
Overnight culture (Basic Protocol 1)
125-ml bottle
Glass rod or rubber spatula
2-ml Cryogenic vials (Nalgene)
Dry ice/ethanol bath
1. Prepare 70 ml of BHIS medium without cysteine, resazurin, or NaHCO3 . Add 30 ml
of glycerol, filter sterilize, and store in a 125-ml bottle.
2. Inoculate a BHIS plate containing the appropriate antibiotics (Table 13C.1.1) with
0.1 ml of an overnight culture. Incubate overnight or up to 48 hr at 37 C.
3. Label cryogenic vials and then cover label with clear tape.
4. Pipet 2 ml of BHIS/glycerol onto the surface of the plate, harvest cells from plate
using a glass rod or rubber spatula and place in cryogenic vial.
5. Flash freeze in a dry ice/ethanol bath and store at 70 C.
BASIC
PROTOCOL 5
Materials
Bacto skim milk (Difco)
BHIS plate containing the appropriate antibiotics (see recipe)
Overnight culture (Basic Protocol 1)
Glass rod or rubber spatula
2 ml Cryogenic vial (Nalgene)
Dry ice/ethanol bath
1. Add 150 g Bacto skim milk to 1 liter dH2 O. Stir 30 min or until powder is dissolved.
Autoclave for 10 min (longer sterilization times will result in curdled milk).
2. Store medium at 4 C until needed.
3. Inoculate a BHIS plate containing the appropriate antibiotics (Table 13C.1.1) with
0.1 ml of an overnight culture. Incubate overnight and up to 48 hr at 37 C.
4. Label cryogenic vial and then cover label with clear tape.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
5. Pipet 2 ml of skim milk medium on to the lawn of cells and harvest with a glass rod
or rubber spatula.
6. Place cell mixture into the cryogenic tube.
7. Flash freeze in a dry ice/ethanol bath and store at 70 C.
13C.1.14
Supplement 9
BASIC
PROTOCOL 6
It is possible to perform growth rate measurements with a variety of culture tubes using
this protocol. The only stipulation is that if screw-cap tubes are to be used they must be
returned to the incubator inside of the anaerobic glove box between OD readings. This is
necessary since screw-cap tubes will not maintain an anaerobic atmosphere for extended
periods.
Materials
100 ml bottle of defined minimal medium (see recipe)
Chopped meat stock culture of Bacteroides in chopped meat medium (see Basic
Protocol 3)
Anaerobic glove box
13 100mm sterile screw cap tubes in rack
Antiseptic wipes
Hemostat
37 C incubator
125-ml side arm flask (sterile, aluminum foil cap)
1- and 10-ml pipets, disposable
Sterile #3 rubber stopper (in covered beaker)
Spectrophotometer
1. Gather materials together in the anaerobic glove box and prepare work space by
placing all items within easy reach.
2. Arrange four tubes in the rack, completely loosen caps, and dispense 5 ml of defined
minimal medium into the tubes immediately replacing the caps.
3. Gently mix the chopped meat culture, clean the top with an antiseptic wipe, unscrew
cap, and remove the stopper with a hemostat.
4. Remove 1 ml of broth from the chopped meat culture and add 0.5 ml, 0.3 ml, and
0.2 ml respectively to the tubes containing medium. Tighten the caps and mix the
tube. Place in the 37 C incubator overnight.
The fourth tube of medium will be used to zero the spectrophotometer during the growth
curve determination.
Initial growth of the inoculum in defined medium can be variable when coming from a
stock culture thus it is necessary to use several inoculum amounts to ensure adequate
growth.
5. Carefully remove the foil from the top of the 125-ml flask and place upside down in
a clean location. Add 50 ml of medium using a 10-ml pipet, replace the foil cap and
place in the incubator.
6. The following day remove flask and tubes from incubator and inspect tubes for
growth.
Other
Eubacteria
13C.1.15
Current Protocols in Microbiology
Supplement 9
7. Choose the tube with the most growth (most turbid). If all tubes have equal growth
then pick the tube with the lowest initial inoculum size and place it in a rack. Loosen
the cap, remove 1 ml and add to the flask.
8. Using a hemostat, carefully grasp a sterile #3 stopper from the beaker and seal the
flask. Twist the stopper to ensure a tight seal and then remove from the anaerobic
chamber together with the tube of sterile medium.
9. Set the spectrophotometer to A550 and zero with the tube of sterile medium. Record
the initial OD from the side arm and then place the flask in a 37 C water bath.
10. Continue to record OD readings as needed until growth has completed 20 to 30 hr.
50 ml
25 ml (0.5%, 28 mM)
1g
10 ml
10 ml
1.5 ml
20 ml
885 ml
15 g
Mix together components except for NaHCO3 and adjust the pH to 7.1 using 1 N
NaOH. Autoclave for 20 min. Cool in water bath at 50 to 55 C before adding
20 ml of sterile 10% NaHCO3 .
Other carbohydrates can be substituted for glucose and 1 ml of 0.01% vitamin B12 solution
can be substituted for methionine.
FeSO4 solution
For 100 ml:
Dissolve 0.278 g of FeSO4 7H2 O in 100 ml distilled water. Add two drops of
concentrated HCl.
Hemin solution
For 200 ml:
Dissolve 100 mg of hemin in 2 ml of 1 M NaOH and then bring up volume to
200 ml with dH2 O. Store in an amber bottle up to 1 year at 4 C.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
Mineral 3B solution
Ingredients per liter:
KH2 PO4
NaCl
MgCl2 6H2 O
CaCl2 2H2 O
CoCl2 6H2 O
MnCl2 4H2 O
NH4 Cl
Na2 SO4
18 g
18 g
0.4 g
0.52 g
0.02 g
0.2 g
10.0 g
5.0 g
Dissolve salts in 800 ml dH2 O, then bring volume up to 1 liter. Autoclave for
20 min.
13C.1.16
Supplement 9
NaHCO3 , 10%
Dissolve 50 g into 500 ml H2 O, filter sterilize.
Resazurin, 0.1%
Dissolve 0.1 g of resazurin (Sigma Chemical) in 100 ml distilled water.
Salts solution A
Ingredients per liter:
CaCl2 2H2 O
MgSO4 7H2 O
KH2 PO4
K2 HPO4
NaCl
0.26 g
0.48 g
1g
1g
2g
Mix CaCl2 2H2 O and MgSO4 7H2 O in 300 ml H2 O until dissolved. Then add
500 ml H2 O and add remaining salts. When dissolved add 200 ml H2 O and store
indefinitely at 4 C.
37 g
1.0 g
10 ml
1 ml
20 ml
970 ml
15 g
Add all dry components to a flask containing the water, swirl until dissolved and then
add the hemin and resazurin solutions. Aliquot the medium to 125-ml bottles (or
any appropriate size bottle) and autoclave immediately for 20 min. When medium
has cooled to 60 C place in the anaerobic chamber for storage.
Add 2 ml of NaHCO3 solution to each 100 ml of medium prior to use and then
aliquot medium into sterile screw-cap tubes as needed. Do not add NaHCO3 prior
to autoclaving as it will be converted to CO2 and lost from the medium. Antibiotics
(Table 13C.1.1) that are required must be added just prior to inoculation of tubes.
For solid medium prepare as above, autoclave, and then place in a 55 C water bath.
When the medium is cool, add the sterile NaHCO3 and antibiotics if needed. Pour
plates on the benchtop or in a Biosafety hood. Allow the plates to dry for several
hours and then use immediately or place in the anaerobic chamber for storage.
20 g
10 g
5g
1g
40 ml
Other
Eubacteria
continued
13C.1.17
Current Protocols in Microbiology
Supplement 9
10 ml
1 ml
15 g
20 ml
All of the components except NaHCO3 are added to 930 ml of H2 O. Then adjust
pH to 7.0 and autoclave for 20 min. When medium has cooled to 55 C add sodium
bicarbonate solution.
COMMENTARY
Background Information
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
also are present, indicating that the gut atmosphere is the product of a complex anaerobic
metabolism. This is a highly reducing environment and the redox potential usually runs
in the range of 150 to 350 mV. The pH
varies along the length of the tract from slightly
acidic along the ascending colon to neutral or
mildly alkaline conditions at the distal colon.
The temperature is constant at 37 C and
there is a constant influx of nutrients. It is not
surprising that this is a highly competitive environment and home to some 500 species of
bacteria. The Bacteroides have proven to be
the most successful microbes in the human gut
and the renewed interest in their interactions
with the host promises to reveal new insight
into their roles in human health. For example, a colonic microflora with increased numbers of Firmicutes and a smaller population of
Bacteroides has been associated with obesity
in animal models (Turnbaugh et al., 2006).
13C.1.18
Supplement 9
is this catalyst that is the source of most problems. The catalyst must be heated regularly as
described in the protocols above in order to
drive off the moisture on the catalyst surface.
It also is important to replace the catalyst on
a regular schedule (every 9 months to a year)
since it eventually becomes permanently poisoned from volatile sulfides produced by bacterial metabolism. Although the Bacteroides
do not produce large amounts of H2 S it is
still useful to have in the glove box a solution
of 90 mM lead acetate or trays of activated
charcoal to adsorb the sulfides.
A simple test will determine if the catalyst
is still active. Rejuvenate the catalyst by heating at 160 C for 2 hr and let cool. Then using
the gassing cannula direct a stream of an anaerobic gas mix containing H2 over the surface of
the catalyst. The catalyst pellets should immediately become hot and if the stream of gas is
maintained the pellets will begin to glow red
from the heat. If the pellets do not heat up then
the catalyst must be replaced.
A second common source of oxygen contamination results from air leaks in the culturing equipment. The rubber gaskets that seal
anaerobic jar lids to the jar must be inspected
frequently to make sure they are free of defects or debris. Likewise the gasket around the
airlock entry of an anaerobic chamber must be
intact to maintain anaerobiosis.
Anaerobic glove box
The anaerobic glove box has become the
method of choice for cultivation of anaerobes
in most research laboratories so it may be beneficial to review some of the important features
and maintenance requirements. The clear, flexible, vinyl glove box is the most practical
for the average laboratory and unless space
is of paramount importance always choose a
2-person (4 glove) chamber for the extra storage space and convenience of having an extra
pair of hands for some procedures. The flexible
style is preferred for a number of reasons but
the fact that these chambers are always under a
slight positive pressure is a great advantage for
detecting leaks and maintaining anaerobiosis
when there is a leak. Leaks are immediately
detected whenever the chamber deflates more
quickly than usual.
Two required accessories for the glove box
are a 37 C incubator and an oxygen/hydrogen
gas meter. The incubator will tend to limit the
number of entries and exits from the chamber
since cultures can be readily monitored. However, if a smaller glove box is purchased and
one does not wish to give up the bench space
Other
Eubacteria
13C.1.19
Current Protocols in Microbiology
Supplement 9
Time Considerations
The Bacteroides are robust organisms and
can double at a relatively high rate under optimal conditions. Generation times for
B. fragilis and B. thetaiotaomicron in BHIS
media is 40 to 60 min but the presence of
antibiotics can have a significant affect even in
resistant organisms. In defined medium growth
rate will increase from 1.75 to 2 hr when glucose is the carbon/energy source. When inoculating a BHIS plate with a frozen or chopped
meat stock of Bacteroides, one should expect
to see colonies within 36 hr.
Acknowledgement
Much of the work described here has been
supported by PHS grant AI40588 to CJS.
Laboratory
Maintenance and
Cultivation of
Bacteroides
Species
Literature Cited
Aranki, A., Syed, S.A., Kenney, E.B., and Freter,
R. 1969. Isolation of anaerobic bacteria from
human gingiva and mouse cecum by means of
Internet Resources
http://www.anoxomat.com/english/standardpage.
php[?ArtikelID=46
http://www.anaerobesystems.com
http://www.bellcoglass.com/product detail.php?product id=276
The above three Web sites contain information
about anaerobic systems.
http://www.bd.com/ds/productCenter/BblGaspak
ProductsAndAccessories.asp
13C.1.20
Supplement 9
http://www.mgc-a.com/Pages/anaeropac.html
http://www.oxoid.com/us/index.asp?mpage=ipsearch
&c=US
The above three Web sites provide information on
commercially available gas packs.
http://www.coylab.com/anaerobic chamber.html
http://www.shellab.com/bactron.html
http://www.biotrace.com/content.php?hID=2&nhID
=75
http://www.800ezmicro.com/productImages.asp?
mb=01&ez=9
The above four Web sites provide information on
anaerobic chambers.
Other
Eubacteria
13C.1.21
Current Protocols in Microbiology
Supplement 9
UNIT 13C.2
This unit describes current molecular genetic approaches using the suicide vector
pPR-UF1 (see Basic Protocol 2) and the transposon Tn4351 (see Basic Protocol 6 and
Alternate Protocol 2) to perform mutant construction in Porphyromonas gingivalis. Plasmid pPR-UF1 is a modified form of plasmid pVA3000, a suicide vector previously used
for construction of single cross-over mutations. Plasmid pPR-UF1 contains two multiple
cloning restriction sites into which inserts from the target gene can be cloned. Techniques
using MgCl2 to prepare P. gingivalis competent cells for electroporation purposes (see
Basic Protocol 1) and conjugation protocols (see Basic Protocol 3 and Alternate Protocol
1) are described in this unit. While a mutation using a suicide vector is specific to a gene
of interest, transposon mutagenesis is more or less random. Plasmid R751::Tn43514 is
used to introduce transposon Tn4351 into P. gingivalis. This plasmid carries two copies
of Tn4351 and can be transferred from E. coli to P. gingivalis via conjugation. Since
R751::Tn43514 is unable to replicate in P. gingivalis cells, any erythromycin-resistant
(Emr ) P. gingivalis transconjugants should be generated by transposition of Tn4351 from
R751::Tn43514 to the P. gingivalis chromosome, as described in Basic Protocol 1. In
order to rule out polar mutation effects during phenotype studies of a particular gene,
complementation should be performed; a technique to restore the wild-type phenotype
of the mutant by complementation in trans using shuttle vector pT-COW is provided
(see Basic Protocol 4). Finally, a description of a noninvasive reporter system using the
xylosidase/arabinosidase enzyme to study gene expression and regulation in P. gingivalis
(see Basic Protocol 5) completes this unit on genetic manipulations of P. gingivalis.
CAUTION: Porphyromonas gingivalis and Escherichia coli are Biosafety Level 2 (BSL2) pathogens. Follow all appropriate guidelines and regulations for the use and handling
of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B)
for more information.
CAUTION: Potentially hazardous chemicals will be used throughout the experiments
described in this unit. Readers should refer to UNIT 1A.3 for proper handling and disposal
of chemical hazards.
NOTE: Because P. gingivalis has special nutrient requirements, hemin and vitamin K1
need to be added to TSB. Therefore, throughout this unit, supplemented TSB as described
in this recipe is used for P. gingivalis cultivation. Supplemented TSB can be prereduced
and prewarmed by incubating a bottle of fresh medium in the anaerobic chamber overnight
before using it (also see Reagents and Solutions).
NOTE: Refer to UNIT 12B.1 for information about equipment needed to maintain anaerobic
conditions.
BASIC
PROTOCOL 1
Materials
Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
3- to 4-day blood agar plate culture of P. gingivalis W83 (ATCC #BAA-308)
Electroporation (EP) buffer (see recipe), cold
Contributed by Myriam Belanger, Paulo Rodrigues, and Ann Progulske-Fox
Current Protocols in Microbiology (2007) 13C.2.1-13C.2.24
C 2007 by John Wiley & Sons, Inc.
Copyright
Other
Eubacteria
13C.2.1
Supplement 5
13. Spread the entire 1.0 ml from the transformation onto ten blood agar plates (100 l
per plate) containing appropriate antibiotics (see Basic Protocol 2).
13C.2.2
Supplement 5
14. Incubate anaerobically until P. gingivalis transconjugant colonies are visible (up to
10 days).
15. Confirm the putative transformant by Southern blot (UNIT 14B.1; also see Brown, 1993,
1999) or colony PCR analysis.
BASIC
PROTOCOL 2
Plasmid pVA3000 has been previously used as a suicide vector for mutant construction
(Wu et al., 2002). Plasmid pVA3000 (Lee et al., 1996) was created by a modification
in the Bacteroides-E. coli shuttle vector pVA2198 (Fletcher et al.,1995). The origin
of replication for Bacteroides in pVA2198 was removed by KpnI digestion and the
remaining 5.3-kb fragment was self-ligated, giving rise to pVA3000. This plasmid carries
two erythromycin markers (ErmAM for E. coli and ErmF for P. gingivalis) and a small
multiple cloning site (only five restriction enzyme sites). Recently, the authors have made
improvements to plasmid pVA3000. New restriction enzyme sites were designed and
cloned into the SalI/SphI double-digestion site in the previously existing multiple cloning
site. Also, a new multiple cloning site was designed and inserted into the PvuI/KpnI
double-digestion site upstream of the ErmAM-F markers, giving rise to plasmid pPRUF1 (Fig. 13C.2.1). Using plasmid pPR-UF1, it is possible to construct double cross-over
mutations and, therefore, achieve a stable disruption of the target gene. In addition, to
select for double cross-over mutants, it is important to linearize the constructed plasmid
by restriction enzyme digestion before introducing it into the recipient microorganism. In
this case, the linearized plasmid is introduced by electroporation. This step is necessary
to avoid selection of single cross-over mutants. Examples of restriction enzymes that can
be used to linearize recombinant pPR-UF1 are shown in Figure 13C.2.1.
To perform a double cross-over mutation, a fragment located at the 5 end and another
fragment located at the 3 end (see Fig. 13C.2.2) of the target gene need to be amplified
by PCR and then cloned into multiple cloning sites (MCS) 1 and 2 of plasmid pPRUF1 (see Figs. 13C.2.1 and 13C.2.2). Each of the two resulting amplified fragments
will be cut by restriction enzymes that are unique to the targeted gene and that are also
present in the specified MCS of the plasmid. Therefore, each fragment must include two
restriction enzyme sites to enable it to be inserted correctly into the plasmid. Furthermore, the amplified fragments should not contain sites for restriction enzymes that will
be used to linearize the plasmid prior to electroporation. See Critical Parameters and
Troubleshooting for more information.
Materials
Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
3- to 4-day blood agar plate culture of P. gingivalis W83 (ATCC #BAA-308) or
other P. gingivalis strain of interest
Wizard Genomic DNA Purification Kit (Promega; use manufacturers Isolation of
Genomic DNA from Gram Positive and Gram Negative protocol)
Nuclease-free distilled deionized H2 O
PCR primers designed to amplify fragments A and B of the gene of interest
QIAquick PCR Purification Kit (Qiagen)
Restriction enzymes to cut fragments (inserts) A and B of the gene of interest
(Fig. 13C.2.1)
LigaFast Rapid DNA Ligation System (Promega)
Competent E. coli cells (e.g., DH5, Invitrogen)
Plasmid pPR-UF1 (can be obtained from the authors laboratory;
apfox@dental.ufl.edu)
Other
Eubacteria
13C.2.3
Current Protocols in Microbiology
Supplement 5
Figure 13C.2.1 Partial restriction map of plasmid pPR-UF1, the suicide vector used to construct
mutants in P. gingivalis. Restriction enzymes AfeI, PciI, DrdI, ApaLI, AlwNI, and Eco571 can be
used to linearize the plasmid before transformation of P. gingivalis by electroporation. Restriction
enzyme HindIII should not be used for cloning an insert. The ErmF cassette of pPR-UF1 also
confers clindamycin resistance in P. gingivalis; therefore, 5.0 g/ml clindamycin may be used
instead of erythromycin to select P. gingivalis transconjugants.
LB plates (APPENDIX 4A) containing 300 g/ml erythromycin (add from 300 mg/ml
erythromycin stock; see recipe)
QIAprep Spin Miniprep Kit (Qiagen)
1% agarose gel (Voytas, 2000)
Restriction enzyme HindIII for screening to confirm the presence of inserts in
recombinant plasmids pPR-UF1A and pPR-UF1AB
Restriction enzyme AfeI, PciL, DrdI, ApaLI, AlwNI, or Eco57I (Fig. 13C.2.1) for
linearizing the recombinant plasmid, pPR-UF1AB
Electrocompetent P. gingivalis cells (see Basic Protocol 1, steps 1 to 7)
Blood agar plates (see recipe) containing 50 g/ml gentamicin (add from 50 mg/ml
stock; see recipe) and 5.0 g/ml erythromycin (add from 5.0 mg/ml stock; see
recipe)
Genetic
Manipulation of
P. gingivalis
Refrigerated centrifuge
DNase-free pipet tips and tubes
13C.2.4
Supplement 5
Figure 13C.2.2 Construction of P. gingivalis mutant by allelic or gene replacement (double crossover mutant). Two fragments from the target gene are amplied by PCR, digested with restriction
enzymes, and cloned into MCS 1 and MCS 2 of plasmid pPR-UF1.
Additional reagents and equipment for DNA purification (Moore and Dowhan,
2002), determining DNA concentration (Gallagher and Desjardins, 2006), the
polymerase chain reaction (PCR; Kramer and Coen, 2001), restriction digestion
(Bloch and Grossman, 1995), transformation of E. coli (Seidman et al., 1997),
agarose gel electrophoresis (Voytas, 2000), transformation of P. gingivalis by
electroporation (Basic Protocol 1), and Southern blotting (UNIT 14B.1; also see
Brown, 1993, 1999)
Other
Eubacteria
13C.2.5
Current Protocols in Microbiology
Supplement 5
6. Purify both PCR products (inserts A and B) from the PCR reaction using a QIAquick PCR purification kit or other standard purification technique (e.g., Moore
and Dowhan, 2002). If using the QIAquick kit, elute DNA with 30 l of the elution
buffer (EB) provided with the kit.
7. Digest purified PCR fragment A using the two restriction enzymes chosen to cut this
fragment (Bloch and Grossman, 1995).
If the chosen enzymes function under the same buffer conditions, a double digest can be
performed as described above. Otherwise, two separate enzymatic digests will needed.
8. Purify the digested fragment from the enzymatic reaction (using the QIAquick
PCR Purification Kit; also see Moore and Dowhan, 2002) and determine the DNA
concentration (Gallagher and Desjardins, 2006).
Genetic
Manipulation of
P. gingivalis
Various molar ratios of vector:insert can be used to enhance the efficiency of the ligation.
A 1:3 vector to insert molar ratio and a starting quantity of 0.12 g of plasmid pPR-UF1
is typically used in ligation reactions in the authors laboratory. To determine the amount
of insert necessary to add to a ligation reaction with plasmid pPR-UF1, see Critical
Parameters and Troubleshooting.
11. Transform the ligation reaction into chemically or electrocompetent E. coli cells
such as DH5 (Seidman et al., 1997).
13C.2.6
Supplement 5
15. Before proceeding to the electroporation of P. gingivalis, linearize 1 g recombinant plasmid per transformation using AfeI, PciL, DrdI, ApaLI, AlwNI, or Eco57I
(Fig. 13C.2.1) to avoid generation of single cross-over mutants. Use 1 g of linearized DNA per transformation.
The authors experience is that the use of the enzymes (described above) to linearize the
recombinant plasmid will increase the number of putative mutants recovered.
21. Incubate anaerobically up to 10 days until P. gingivalis colonies are visible. Confirm
mutants by Southern blot (UNIT 14B.1; also see Brown, 1993, 1999) or colony PCR
analysis.
Other
Eubacteria
13C.2.7
Current Protocols in Microbiology
Supplement 5
BASIC
PROTOCOL 3
Materials
Recipient P. gingivalis strain W83 (ATCC #BAA-308) or other P. gingivalis strain
of interest
Blood agar plates (see recipe) without antibiotics
Donor E. coli strain (S17-1; not available commercially; can be obtained from
authors laboratory, apfox@dental.ufl.edu)
LB plates (APPENDIX 4A) containing appropriate antibiotics (see Critical Parameters
and Troubleshooting)
Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
Blood agar plates (see recipe) containing 150 g/ml gentamicin and appropriate
antibiotic to select for transconjugants (see Critical Parameters and
Troubleshooting)
Blood agar plates (see recipe) containing 50 g/ml gentamicin and appropriate
antibiotic to ensure antibiotic resistance and purity of transconjugants (see
Critical Parameters and Troubleshooting)
1. Grow the recipient P. gingivalis strain on a blood agar plate without antibiotics (or
containing 50 g/ml gentamicin) at 37 C anaerobically for 2 to 3 days.
2. Grow the donor E. coli containing the recombinant plasmid aerobically on a LB agar
plate with the appropriate antibiotic (see Critical Parameters and Troubleshooting)
at 37 C overnight.
In conjugation experiments, the authors prefer to use the mobilizing E. coli strain S17-1
as a donor strain for P. gingivalis.
Steps 3 to 5 of this protocol can be modified as described by Maley et al. (1992) and by
Park and McBride (1993). See Alternate Protocol 1.
3. Using a sterile loop, scrape E. coli and P. gingivalis from plates obtained in steps 1
and 2, and mix on a 3- to 4-cm2 area of a blood agar plate containing no antibiotics.
4. Incubate at 37 C anaerobically overnight.
5. Collect the mixed cells and resuspend in 1.0 ml supplemented TSB.
6. Spread 0.1 ml cell suspension onto blood agar plates containing 150 g/ml gentamicin and the appropriate antibiotic to select for transconjugants (see Critical
Parameters and Troubleshooting).
Genetic
Manipulation of
P. gingivalis
13C.2.8
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8. Streak several colonies onto fresh blood agar plates containing 50 g/ml of gentamicin and the appropriate antibiotic to ensure antibiotic resistance and purity (see
Critical Parameters and Troubleshooting).
ALTERNATE
PROTOCOL 1
This protocol is an adaptation of the methods described by Maley et al. (1992) and by
Park and McBride (1993).
BASIC
PROTOCOL 4
An undesirable feature of pT-COW is that there are no multiple cloning sites and few
restriction enzyme sites available (Fig. 13C.2.3). It is important to choose restriction sites
that are not located within critical DNA regions of the plasmid, e.g., the replication and
mobilization (rep/mob) regions. However, restriction enzyme sites that exist in one of the
antibiotic resistance markers for E. coli can be used. Later, when selecting E. coli cells
carrying the recombinant plasmid (pT-COW plus gene), use the selective antibiotic with
a complete copy of its gene. In Figure 13C.2.3, all known unique restriction enzyme sites
are shown.
Refer to Basic Protocol 2 and its Critical Parameters and Troubleshooting section for
information on designing primers that introduce restriction sites upon amplification.
Other
Eubacteria
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Figure 13C.2.3 Partial restriction map of plasmid pT-COW, shuttle vector used for complementation of P. gingivalis mutants and reporter gene analysis. When using pT-COW, grow E. coli in
media containing 50 g/ml carbenicillin or 5.0 g/ml tetracycline, and grow P. gingivalis in media
containing 1.0 g/ml of tetracycline.
Materials
PCR primers designed to amplify the gene of interest
P. gingivalis chromosomal DNA, purified (see Basic Protocol 2)
Restriction enzymes appropriate for cutting the insert and for cutting the plasmid
pT-COW (see Bloch and Grossman, 1995; also see Fig. 13C.2.3)
QIAquick PCR Purification Kit (Qiagen)
Plasmid pT-COW (Shoemaker et al., 1986; not available commercially; contact
author at apfox@dental.ufl.edu for more information)
LigaFast Rapid DNA Ligation System (Promega)
Electrocompetent E. coli strain S17-1 cells (can be obtained from the authors
laboratory; apfox@dental.ufl.edu)
LB agar plates (APPENDIX 4A) containing 50 g/ml carbenicillin (add from 50 mg/ml
stock; see recipe) or 5.0 g/ml tetracycline (add from 10 mg/ml stock; see
recipe)
QIAprep Spin Miniprep Kit (Qiagen)
1% agarose gel (Voytas, 2000)
Additional reagents and equipment for the polymerase chain reaction (PCR;
Kramer and Coen, 2001), DNA purification (Moore and Dowhan, 2002),
determining DNA concentration (Gallagher and Desjardins, 2006), the
polymerase chain reaction (PCR; Kramer and Coen, 2001), restriction digestion
(Bloch and Grossman, 1995), transformation of E. coli (Seidman et al., 1997),
agarose gel electrophoresis (Voytas, 2000), transformation of P. gingivalis by
conjugation (Basic Protocol 3 or Alternate Protocol 1), large-scale preparation
of plasmid DNA (Heilig et al., 1998), and Southern blotting (UNIT 14B.1; also see
Brown, 1993, 1999)
Genetic
Manipulation of
P. gingivalis
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2. Double digest the purified insert using the restriction enzyme(s) chosen for this
insert (Bloch and Grossman, 1995). Purify the digested fragment from the enzymatic
reaction using the QIAquick PCR Purification Kit (also see, e.g., Moore and Dowhan,
2002), and determine the DNA concentration (Gallagher and Desjardins, 2006).
5. Transform E. coli strain S17-1 with the ligation reaction (Seidman et al., 1997).
Other E. coli strains with greater transformation efficiency may be used to propagate
the recombinant plasmid (e.g., DH5). However, before proceeding to transformation of
P. gingivalis, it is necessary to transform the E. coli strain S17-1 with the recombinant
plasmid.
6. Spread 100 l from the transformation onto an LB agar plate containing the appropriate antibiotic (50 g/ml carbenicillin or 5.0 g/ml tetracycline).
The antibiotic selection should take in consideration which antibiotic resistance gene for
E. coli is still intact in the recombinant plasmid (see introduction Basic Protocol 5).
The remaining transformation mixture may be stored at 4 C and plated out the next day
if necessary.
7. Incubate at 37 C overnight.
8. Pick several colonies and streak them onto LB agar plates containing the appropriate
antibiotic (50 g/ml of carbenicillin or 5.0 g/ml of tetracycline). Incubate the plate
at 37 C overnight and then keep at 4 C as the master plate.
9. Analyze colonies from the master plate for desired recombinant plasmid by either
colony PCR or isolation of plasmid (using the QIAprep Spin Miniprep Kit; also
see Moore and Dowhan, 2002) followed by digestion with an appropriate restriction
endonuclease (Bloch and Grossman, 1995). Run digested product on 1% agarose gel
(Voytas, 2000).
Other
Eubacteria
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Materials
PCR primers designed to amplify the gene of interest
P. gingivalis chromosomal DNA, purified (see Basic Protocol 2)
High-fidelity Taq DNA polymerase
QIAquick PCR Purification Kit (Qiagen)
Restriction enzymes appropriate for cutting the insert and for cutting the plasmid
pT-COW (see Bloch and Grossman, 1995; also see Fig. 13C.2.4)
QIAquick Gel Extraction Kit (Qiagen)
Plasmid pXA1 (Whitehead, 1997)
Restriction enzymes EcoRI and BamHI for cutting plasmid pXA1 (also see Bloch
and Grossman, 1995)
Plasmid pT-COW (Shoemaker et al., 1986; not available commercially; contact
author at apfox@dental.ufl.edu for more information)
1% agarose gels (see Voytas, 2000)
LigaFastTM Rapid DNA Ligation System (Promega)Nuclease-free H2 O
Electrocompetent E. coli strain S17-1 cells (can be obtained from the authors
laboratory; apfox@dental.ufl.edu)
QIAprep Miniprep Kit (Qiagen)
25 C water bath
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Manipulation of
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Additional reagents and equipment for the polymerase chain reaction (PCR;
Kramer and Coen, 2001), DNA purification (Moore and Dowhan, 2002),
restriction digestion (Bloch and Grossman, 1995), agarose gel electrophoresis
(Voytas, 2000), transformation of E. coli (Seidman et al., 1997), transformation
of P. gingivalis by conjugation (Basic Protocol 3 or Alternate Protocol 1), and
large-scale preparation of plasmid DNA (Heilig et al., 1998)
Current Protocols in Microbiology
Figure 13C.2.4 Example of reporter gene system construction using the xa gene as the reporter gene and plasmid
pT-COW as the shuttle vector.
Other
Eubacteria
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1. Perform PCR (Kramer and Coen, 2001) using primers specific for the desired locus
with purified P. gingivalis chromosomal DNA as the template, under the appropriate
conditions. Purify as described in Basic Protocol 2 (using the QIAquick PCR Purification Kit) or using another appropriate purification technique (e.g., Moore and
Dowhan, 2002).
It is important to emphasize the use of a high-fidelity DNA polymerase when amplifying
the insert. The PCR experiment should be done according to the DNA polymerase manufacturers instructions. Restriction enzyme sites may be incorporated into the primers
used to amplify the TRE regions to ensure proper orientation upon ligation. PCR products may be purified using an appropriate technique. The authors routinely purify PCR
amplification products using the QIAquick PCR Purification Kit (Qiagen).
2. Digest the purified P. gingivalis PCR product using the restriction enzymes chosen
for this insert (Bloch and Grossman, 1995). Purify the digested fragment from the
enzymatic reaction using the QIAquick Gel Extraction Kit or other appropriate
technique (e.g., Moore and Dowhan, 2002). If using the QIAquick kit, elute DNA
with 30 l of the elution buffer (EB) provided with the kit.
3. Remove the xa gene from plasmid pXA1 by restriction enzymatic digestion with
restriction enzymes EcoRI and BamHI. Run the entire reaction in a 1% agarose gel
(Voytas, 2000). Purify the xa gene band (1.4 kb) using the QIAquick Gel Extraction
Kit and determine the DNA concentration (Gallagher and Desjardins, 2006).
4. Digest pT-COW using the same restriction enzymes chosen to cut the 5 region of
the TRE and to cut the 3 region of the xa gene. Purify the digested plasmid from
the enzymatic reaction using the QIAquick PCR Purification Kit and determine the
DNA concentration (Gallagher and Desjardins, 2006).
An undesirable feature of pT-COW is that there are no multiple cloning sites and only a
few unique restriction enzyme sites are available (Fig. 13C.2.3). For cloning purposes,
use restriction enzyme sites that are not located in critical DNA regions of the plasmid,
e.g., replication and mobilization (rep/mob) regions. However, restriction enzyme sites
that exist in one of the antibiotic resistance markers for E. coli can be chosen. Later, when
selecting E. coli cells carrying the recombinant plasmid, use the selective antibiotic with
a complete copy of its gene. In Fig. 13C.2.3, all known unique restriction enzyme sites
are shown.
The ligation of each fragment into the plasmid can be done separately. However, the
ligation may be performed together if using directional cloning, as the digested TRE
insert and digested xa gene will have different compatible cohesive ends.
13C.2.14
Supplement 5
6. Transform E. coli strain S17-1 with the recombinant plasmid (Seidman et al., 1997).
7. Confirm the presence of the inserts in the plasmid by colony PCR or by purification
of plasmids using the QIAprep Spin Miniprep Kit (also see Moore and Dowhan,
2002) followed by cutting with an appropriate restriction enzyme (e.g., HindIII; also
see Bloch and Grossman, 1995), and electrophoretic analysis on a 1% agarose gel
(Voytas, 2000).
Other E. coli strains with greater transformation efficiency may be used to propagate
the recombinant plasmid (e.g., DH5). However, before proceeding to the next step, it is
necessary to transform the E. coli strain S17-1 with the recombinant plasmid.
BASIC
PROTOCOL 6
Transposon mutagenesis produces a transposon library containing thousands of mutants. The transposon insertions are relatively random; thus, it is usually necessary to
screen hundreds of mutants to find the transconjugant carrying the phenotype of interest.
Transposon Tn4351 can be introduced into P. gingivalis using plasmid R751::Tn43514
(Shoemaker et al., 1986). This plasmid carries two copies of Tn4351 and can be transferred from E. coli to P. gingivalis via conjugation. Tn4351 contains two antibiotic
resistance genesone that confers resistance to erythromycin (Emr ) in P. gingivalis and
another that confers resistance to tetracycline (Tcr ) in aerobically grown E. coli. Note that
Em selection will work in P. gingivalis and Tc selection in E. coli; however, the reverse
is not the case. Since R751::Tn43514 is unable to replicate in P. gingivalis cells, any
Emr P. gingivalis transconjugant should be generated by transposition of Tn4351 from
R751::Tn43514 to the P. gingivalis chromosome.
This protocol is an adaptation of the method described by Progulske-Fox et al. (1989).
Materials
Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
3- to 4-day blood agar plate culture of P. gingivalis W83 (ATCC #BAA-308)
E. coli strain HB101 containing plasmid R751::Tn43514 (not available
commercially; see Shoemaker et al., 1986): culture overnight
LB liquid medium (APPENDIX 4A) containing 300 g/ml erythromycin (add from 300
mg/ml stock; see recipe)
Blood agar plates (see recipe), antibiotic free
Blood agar plates (see recipe) containing 150 g/ml gentamicin (add from 50
mg/ml stock; see recipe) and 5 g/ml of erythromycin (add from 300 mg/ml
stock; see recipe)
37 C shaking incubator
Additional reagents and equipment for Southern blotting (UNIT 14B.1; also see
Brown, 1993, 1999)
1. Inoculate 3.0 ml supplemented TSB with P. gingivalis from a 3- to 4-day old blood
agar plate and incubate at 37 C anaerobically overnight.
Other
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5. Spread 0.1 ml cell suspension onto antibiotic-free blood agar plates; incubate aerobically at 37 C for 1 hr, then under an anaerobic atmosphere at 37 C overnight.
6. Harvest all growing cells with an inoculation loop and resuspend in 1.0 ml prereduced, prewarmed, supplemented TSB.
7. Spread 0.1 ml cell suspension onto blood agar plates containing 150 g/ml of
gentamicin and 5 g/ml of erythromycin. Incubate anaerobically until P. gingivalis
transconjugant colonies are visible (up to 10 days).
8. Screen for the desired phenotype, e.g., via animal model or hemagglutination activity.
9. Confirm that the transposon-generated mutant has a single insertion by Southern blot
analysis (UNIT 14B.1; also see Brown, 1993, 1999).
ALTERNATE
PROTOCOL 2
5. Harvest all growing cells with an inoculation loop and suspend in 1.0 ml PBS.
13C.2.16
Supplement 5
6. Spread onto blood agar plates containing 150 g/ml gentamicin and 5 g/ml erythromycin.
7. Incubate anaerobically until P. gingivalis transconjugant colonies are visible (up to
10 days).
8. Screen for the desired phenotype, e.g., via animal model or hemagglutination activity.
9. Confirm that the transposon-generated mutant has a single insertion by Southern blot
analysis (UNIT 14B.1; also see Brown, 1993, 1999).
Other
Eubacteria
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COMMENTARY
Background Information
In order to determine and study the function of a particular gene, mutant construction is a critical step. Studies with mutant
strains carrying the disrupted gene can elucidate important processes such as biochemical
pathways, bacteria-bacteria interactions, and
bacteria-host interactions.
Multiple techniques to construct mutants in
various bacterial species are described in the
literature. For studies with P. gingivalis, three
methods to disrupt genes are routinely used:
transposon mutagenesis, targeted insertional
inactivation, and allelic replacement.
Genetic
Manipulation of
P. gingivalis
Transposon mutagenesis
In transposon mutagenesis, mutant construction can be achieved using the Bacteroides fragilis transposon Tn4351 (Shoemaker et al., 1986). Usually, a clean knockout phenotype is obtained with transposons.
Two desirable features of transposons are that
they carry a selectable marker and that they
can be used as a target for cloning the interrupted gene. Transposons are typically introduced into P. gingivalis by conjugation using
a suicide plasmid.
Progulske-Fox et al. (1989) reported that
plasmid pE5-2, a chimeric shuttle vector that
replicates in E. coli and in some Bacteroides
13C.2.18
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Other
Eubacteria
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Supplement 5
Genetic
Manipulation of
P. gingivalis
13C.2.20
Supplement 5
Anticipated Results
The technique described for isolating P.
gingivalis genomic DNA should result in good
yield and high-quality DNA.
When starting with 3.0 ml culture, high
isolation (5.0 to 9.0 g) of plasmid pPRUF1 should be obtained with the protocol detailed in this unit. In mutant construction using
plasmid pPR-UF1, expect few transconjugant
colonies. Transformation efficiency of electrocompetent P. gingivalis cells is not nearly as
high as in E. coli, and the recombination rate
in P. gingivalis is low.
On the contrary, expect thousands of mutants when using transposon Tn4351. However, screening to find the desired phenotype
will be more tedious than if allelic replacement
were used to generate the mutation.
The transformation efficiency of P. gingivalis has not yet been determined.
The yield of plasmid pT-COW is low (2.0
g from a starting culture of 10 ml in LB
broth). Larger cultures of the E. coli strain
carrying plasmid pT-COW are necessary to
achieve higher concentrations of the plasmid.
For this purpose, the authors suggest following
the protocols detailed in Heilig et al. (1998).
Usually <50 P. gingivalis transconjugant
colonies are obtained per conjugation procedure.
Time Considerations
Constructing a mutation by gene replacement is time consuming, and may take longer
than a month to obtain and confirm. P. gingivalis usually grows overnight in TSB cultures; however, it takes 3 to 4 days to grow
on blood agar plates. E. coli growth to isolate both plasmid pPR-UF1 and recombinant
Other
Eubacteria
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Current Protocols in Microbiology
Supplement 5
plasmids require overnight cultures. PCR reactions are typically 3 to 5 hr long. It takes 7
to 10 days to visualize P. gingivalis transconjugants on blood agar plates after transformation by electroporation. Finally, Southern blot
analysis may take 2 to 3 days to be completed.
Constructing a transposon library can also
be time consuming. The growth of P. gingivalis
transconjugant colonies may take 7 to 10 days.
The screening of the putative mutants for the
desired phenotype is variable. Southern blot
analysis may take 2 to 3 days to complete. In
addition, PCR reactions and DNA sequencing
analysis are necessary to confirm disruption of
the gene of interest.
It takes 1 week to prepare electrocompetent P. gingivalis cells. It is necessary to
cultivate P. gingivalis on a blood agar plate
for 3 to 4 days before inoculation of TSB. An
overnight growth period in TSB is necessary,
followed by a full day for preparation of the
electrocompetent cells.
The conjugation protocols may take longer
than a week. Fresh cultures of P. gingivalis
and E. coli are required. Overnight cultures
for both bacterial species are required in Basic
Protocol 3 and Alternate Protocol 1. Moreover,
after the conjugation is performed, it might
be necessary to wait up to 10 days until P.
gingivalis transconjugants are visible on blood
agar plates.
It should take 1 month to obtain and to
confirm a mutation. P. gingivalis usually grows
overnight in TSB cultures; however, it takes 3
to 4 days to grow on blood agar plates. E. coli
growth to isolate both plasmid pT-COW and
recombinant plasmid require overnight cultures. PCR reactions typically require 3 to 5 hr.
Finally, it takes up to 10 days to visualize P.
gingivalis transconjugants on blood agar plates
after transformation by conjugation.
Introduction and confirmation of a reporter
gene in P. gingivalis cells should not take
longer than 1 month.
Acknowledgements
Literature Cited
Genetic
Manipulation of
P. gingivalis
13C.2.22
Supplement 5
Hoover, C.I. and Yoshimura, F. 1994. Transposoninduced pigment-deficient mutants of Porphyromonas gingivalis. FEMS Microbiol Lett.
124:43-48.
Kramer, M.F. and Coen, D.M. 2001. Enzymatic
amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol.
56:15.1.1-15.1.14.
Lee, S.W., Hillman, J.D., and Progulske-Fox, A.
1996. The hemagglutinin genes hagB and hagC
of Porphyromonas gingivalis are transcribed in
vivo as shown by use of a new expression vector.
Infect. Immun. 64:4802-4810.
Liu, Y., Abaibou, H., and Fletcher, H.M. 2000. Development of a noninvasive reporter system for
gene expression in Porphyromonas gingivalis.
Plasmid 44:250-261.
Lu, B. and McBride, B.C. 1998. Expression of the
tpr protease gene of Porphyromonas gingivalis
is regulated by peptide nutrients. Infect. Immun.
66:5147-5156.
Malek, R., Fisher, J.G., Caleca, A., Stinson, M.,
van Oss, C.J., Lee, J. Y., Cho, M.I., Genco,
R.J., Evans, R.T., and Dyer, D.W. 1994. Inactivation of the Porphyromonas gingivalis fimA
gene blocks periodontal damage in gnotobiotic
rats. J. Bacteriol. 176:1052-1059.
Maley, J., Shoemaker, N.B., and Roberts, I.S.
1992. The introduction of colonic-Bacteroides
shuttle plasmids into Porphyromonas gingivalis: Identification of a putative P. gingivalis
insertion-sequence element. FEMS Microbiol.
Lett. 72:75-81.
Miller, V.L. and Mekalanos, J.J. 1988. A novel
suicide vector and its use in construction of
insertion mutations: Osmoregulation of outer
membrane proteins and virulence determinants
in Vibrio cholerae requires toxR. J. Bacteriol.
170:2575-2583.
Moore, D. and Dowhan, D. 2002. Purification and
concentration of DNA from aqueous solutions.
Curr. Protoc. Mol. Biol. 59:2.1.1-2.1.10.
Nakayama, K. 1994. Rapid viability loss on exposure to air in a superoxide dismutase-deficient
mutant of Porphyromonas gingivalis. J. Bacteriol. 176:1939-1943.
OBrien-Simpson, N.M., Paolini, R.A., Hoffmann,
B., Slakeski, N., Dashper, S.G., and Reynolds,
E.C. 2001. Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model. Infect. Immun. 69:7527-7534.
Onoe, T., Hoover, C.I., Nakayama, K., Ideka, T.,
Nakamura, H., and Yoshimura, F. 1995. Identification of Porphyromonas gingivalis prefimbrilin possessing a long leader peptide: possible
involvement of trypsin-like protease in fimbrilin
maturation. Microb. Pathogenesis 19:351-364.
Park, Y., Lu, B., Mazur, C., and McBride, B.C.
1997. Inducible expression of a Porphyromonas
gingivalis W83 membrane-associated protease.
Infect. Immun. 65:1101-1104.
Park, Y. and McBride, B.C. 1993. Characterization of the tpr gene product and isolation
Other
Eubacteria
13C.2.23
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Supplement 5
Key Reference
Kuramitsu, H. K. 2003. Molecular genetic analysis
of the virulence of oral bacterial pathogens: An
historical perspective. Crit. Rev. Oral Biol. Med.
14:331-344.
Describes genetic manipulations that can be performed with various bacteria, including P. gingivalis.
Lamont, R.J. and Jenkinson, H.F. 1998. Life below
the gum line: Pathogenic mechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol.
Rev. 62:1244-1263.
Comprehensive review article describing the various virulence factors of P. gingivalis.
Internet Resources
http://www.tigr.org/tigr-scripts/CMR2/
GenomePage3.spl?database=gpg
Access to whole genome sequence of Porphyromonas gingivalis strain W83 as well as genomic
analysis tools.
http://www.tigr.org
The Institute for Genomic Research (TIGR) provides structural, functional and comparative analysis of genomes and gene products.
Genetic
Manipulation of
P. gingivalis
13C.2.24
Supplement 5
UNIT 14B.1
This unit describes an assay (see Basic Protocol) to assess the functionality of papillomavirus origins of replication during transient transfection of cultured cells. Papillomavirus genomes generally will not replicate autonomously in cultured cells because
of poor endogenous expression of viral replication proteins (E1 and E2). Consequently,
replication competence is typically evaluated after transient cotransfection of the cloned
genome (or a cloned genome fragment) with expression vectors for the E1 and E2 proteins
(Fig. 14B.1.1). This assay is widely used to map the genomic location and boundaries
of the functional origin, to test replication activity of origin mutants, and to determine
replicative capacity of E1 and/or E2 mutants.
CAUTION: Human papillomaviruses are Biosafety Level 2 (BSL-2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic
microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more
information.
NOTE: All reagents and buffers used should be of the highest available molecular-biology
grade and free of nucleases. Disposable gloves should be worn at all times when handling
samples and gels to avoid nuclease contamination from the skin.
BASIC
PROTOCOL
The plasmid vector used to clone the papillomavirus origin fragment should be devoid of
eukaryotic cis regulatory sequences, such as transcriptional control elements or origins of
replication that would function in mammalian cells, as either of these types of sequences
could inuence replication from the cloned papillomavirus origin. For studies in the authors laboratory, the pUC18 vector is used, though many similar vectors should function
adequately in this assay. The choice of vector for expression of the papillomavirus E1 and
E2 proteins is less constrained. Typically, all that is required for the expression vector
is a strong, constitutive promoter that will function in the cell type being transfected.
The pcDNA-series vectors (Invitrogen) have a cytomegalovirus (CMV) promoter that is
widely active in many cell types, and is usually suitable for E1 and E2 expression. Note
that the pcDNA vectors themselves will not replicate (except in COS cells which supply
T antigen), though they will persist sufciently during the time course of the assay to
provide adequate E1 and E2 levels.
The cloned papillomavirus genomic or origin DNA must be prepared from dam+ E. coli
to allow methylation at adenine residues. Most commonly used strains of E. coli are
dam+ , but the published genotypes should be consulted to ensure that a dam mutation
is not present. The authors laboratory typically uses the TB1 strain which is available from New England Biolabs. Puried DNA is transfected into an appropriate cell
line, and at 24-hr intervals after transfection, the low-molecular-weight episomal DNA
is extracted (see Support Protocol 1), restriction digested with DpnI, and analyzed by
Southern blotting (see Support Protocol 2). DpnI cleaves at the sequence 5 -GATC-3
only when the A is methylated, so residual, unreplicated input DNA will be digested
Animal DNA
Viruses
Contributed by Van G. Wilson
Current Protocols in Microbiology (2005) 14B.1.1-14B.1.17
C 2005 by John Wiley & Sons, Inc.
Copyright
14B.1.1
Figure 14B.1.1 Schematic overview of the transient replication assay. The input DNAs for the
co-transfection consist of a papillomavirus origin-containing test vector (pBOR) and two expression
vectors, pE1 and pE2, that provide the viral E1 and E2 proteins, respectively. All the input plasmid
DNAs will normally be methylated at specic adenine residues due to the endogenous dam methylation system in E. coli. After cotransfection of the three plasmids into an appropriate cell line, the
presence of a functional papillomavirus origin will allow E1- and E2-dependent replication of the
test vector. During replication, the adenine methylation will be lost, as metazoan cells lack this
activity. Subsequent Hirt extraction isolates the small nucleic acids, including the replicated and
unreplicated plasmid DNAs. The input, methylated plasmid DNA is susceptible to DpnI digestion
and will be cleaved into small fragments. In contrast, replicated plasmid DNA will be unmethylated
and, therefore, resistant to DpnI cleavage. A simultaneous second digestion with a single-cut restriction enzyme (e.g. HindIII) facilitates subsequent quantitation by converting supercoiled and
open circular forms of the plasmid into a single linear species. The replicated and unreplicated
DNAs are separated by agarose gel electrophoresis and visualized by Southern blotting with a
probe specic for the test vector. Details of this overall process are described in the text.
into small fragments. Since higher eukaryotic cells lack adenine methylation, papillomaviral DNA that replicates after transfection will produce progeny molecules that are
unmethylated at adenine residues. This material will be resistant to DpnI cleavage, resulting in a replication product the same size as the input DNA. The amount of replicated
DNA can be quantitated and should increase over time. This assay can be used to conrm replication activity or to compare replication efciency of mutant versus wild-type
origins.
Materials
Cell Culture
Assay for
Papillomaviruses
14B.1.2
Current Protocols in Microbiology
Prepare cells
1. Grow Chinese hamster ovary (CHO) cells in Hams F12 medium supplemented with
10% FBS.
The number of cells needed depends on the number of samples to be transfected. In a
75-cm2 ask at 50% to 70% conuency, there are 6 106 CHO cells; each transfected
sample will require 1 106 cells. Other rodent or primate cell lines can also be used, as
replication of papillomavirus origin-containing DNA is not species restricted, as long as
the E1 and E2 proteins are expressed.
2. On the afternoon prior to transfection (day 1), harvest the subconuent cells by
trypsinization using tissue-culture-grade trypsin. Pool the harvested cells and quantitate with a hemacytometer (Strober, 1997).
3. Prepare new 60-mm tissue culture plates with 5 ml Hams F12 medium/10% FBS
per plate.
There should be one plate for each sample to be transfected, though preparing a few extras
is advisable to account for contamination or other loss.
If antibiotics have routinely been included in the culture medium, they should be excluded from steps 3 to 11, as antibiotics will cause cell death during Lipofectamine2000
transfection.
4. Add 1 106 cells per plate, swirl gently to distribute evenly, and incubate overnight.
Current Protocols in Microbiology
Animal DNA
Viruses
14B.1.3
Sample
pE1
pE2
pUC
pBOR
pcDNA3.1
Transfect cells
5. The next morning (day 2), check each plate for uniform cell distribution, cell health,
and possible contamination.
Conuency should be 50% to 80%.
6. Approximately 1 hr before transfection, aspirate the medium from each plate and
replace with 3 ml fresh Hams F12/10% FBS. Return plates to incubator until
step 11.
7. Prepare the samples for transfection by mixing combinations of the appropriate DNA
stock solutions in microcentrifuge tubes as indicated in Table 14B.1.1.
Minimally, each reaction requires three DNAs: (1) the plasmid to be tested for replication
activity (either a negative control plasmid or the cloned papillomavirus genomic or origin
fragment); (2) an E1 expression vector; and (3) an E2 expression vector. While there is
some degree of cross-species functionality of the origins and replication proteins, maximum activity is observed with a cognate origin/replication proteins mixture. The example
provided is for the bovine papillomavirus (BPV)cloned origin and E1+E2 proteins. The
ratios of these three DNAs have been optimized for the BPV system, and ratios of plasmid
DNAs for other papillomavirus origins with their cognate replication protein expression
vectors should be empirically optimized by systematically varying the amounts of the three
plasmids. The nal quantity of total DNA per sample should be equal for all samples and
should be between 2 to 3 g.
Samples 1, 3, and 4 in Table 14B.1.1 are negative controls with nonorigin DNA (pUC),
no E1, and no E2, respectively. True replication should only occur with sample 2, which
contains origin DNA (pBOR) and both the E1 and E2 replication factors. The pcDNA3.1
DNA, which is the parental vector for pE1 and pE2, is used to adjust each reaction to a
constant total amount of DNA, since the DNA quantity affects the transfection efciency.
8. Add 250 l Hams F12 medium without FBS to each tube of DNA and stir gently
with the pipet tip.
9. Prepare sufcient master mix for all samples by combining 8 l Lipofectamine2000
and 250 l Hams F12 without FBS for each sample. Stir thoroughly with a pipet tip
and incubate 5 min at room temperature.
For example, for ten samples, combine 80 l Lipofectamine 2000 and 250 l Hams F12
without FBS. It is usually advisable to prepare enough volume for one extra sample, to
account for pipetting losses.
10. Add 250 l of the master mix to each DNA sample prepared in step 7. Stir gently
and incubate 20 min at room temperature.
11. When incubation is complete, add all of each DNA sample to a separate plate from
step 6. Swirl plates gently and return to incubator.
Cell Culture
Assay for
Papillomaviruses
12. The next morning (day 3), prepare three 100-mm tissue culture plates for each original
sample. Add 10 ml of Hams F12 medium/10% FBS to each plate and place in an
incubator for at least 1 hr before step 13.
14B.1.4
Current Protocols in Microbiology
13. At 24 hr post-transfection (see step 11), aspirate the medium from each transfected
plate and harvest the cells by standard trypsinization. Divide the cells from each
original plate equally into the three 100-mm plates prepared in step 12. Swirl gently
to distribute the cells and return all the plates to the incubator.
22. Redissolve each nal pellet in 10 l of TE buffer, pH 7.0, and pool the A and B tubes
for each sample.
23. Add 1.6 l of 500 g/ml RNase and incubate 1 hr at 68 C.
After the RNase digestion, the samples can be stored for several days at 4 C or indenitely
at 20 C. Typically, the samples from each time point (48, 72, and 96 hr) are stored until
all of the time-point samples have been processed. Subsequent steps are performed in
parallel on samples from each time point.
Animal DNA
Viruses
14B.1.5
Current Protocols in Microbiology
Figure 14B.1.2 Ethidium bromidestained agarose gel before and after transfer of the DNA to
the GeneScreen membrane. The gel was photographed under UV illumination. Numbers above
the lanes indicate the hour post-transfection at which the DNA samples were harvested.
25. Incubate reactions at least 5 hr at 37 C, then add an additional 5 U DpnI and continue
incubation overnight.
Extensive overdigestion with DpnI is critical, since any undigested input DNA would
falsely contribute to the replicated DNA signal on the Southern blot. The control samples
(1, 3, and 4; see step 7) should exhibit no signal at the position of linear, full-length plasmid.
26. At the end of the incubation (day 9), add 2 l of 10 agarose sample buffer to each
sample and store at 4 C until the agarose gel is run.
28. Load 15 l of each sample (see step 26) on the gel. Also load 10 l of each DNA
quantitation standard.
29. Electrophorese at 20 mA (Voytas, 1992). Use the migration of the bromphenol blue
in the sample buffer to monitor run time.
Cell Culture
Assay for
Papillomaviruses
Since it is necessary to retain the small DpnI digestion products on the gel, electrophoresis
is typically stopped before bromophenol blue reaches the bottom of the gel. Bromophenol
blue runs at 400 to 500 bp in this gel.
14B.1.6
Current Protocols in Microbiology
14B.1.7
Current Protocols in Microbiology
The sheet protector contains the radioactivity so the membrane will not contaminate either
the intensier or phosphor imager screen. The protector also prevents the membrane from
sticking to X-ray lm and damaging the emulsion. Typically these blots require at least
overnight exposure.
Samples 1, 3, and 4 (negative controls) should have only small fragments (DpnI digestion
products), while sample 2 should have both small fragments (residual input DNA) and a
band at the position of the linear pBOR DNA (replicated product).
SUPPORT
PROTOCOL 1
HIRT PRECIPITATION
This is a standard protocol for extraction of low-molecular-weight nucleic acids, e.g.,
plasmids or small DNA virus genomes, from cultured mammalian cells. The procedure
comprises four distinct operations: (1) cell lysis; (2) high-salt precipitation of cell debris
and large, chromosomal DNA fragments; (3) removal of protein from the low-molecularweight DNA fraction; and (4) concentration and recovery of the low-molecular-weight
DNAs by alcohol precipitation. Direct lysis of the plated cells in an SDS-containing buffer
inhibits cellular DNase activity and preserves DNA integrity while facilitating efcient
recovery of all the cellular components. The subsequent addition of NaCl to the lysis buffer
selectively precipitates the cellular debris and large chromosomal DNA fragments, while
the smaller nucleic acids remain in solution. Contaminating cellular proteins remaining
in the soluble fraction after the NaCl-precipitation step are removed by a combination
of proteinase K digestion followed by phenol-chloroform extraction. Lastly, the plasmid
DNA in the deproteinated soluble fraction is concentrated and recovered by alcohol
precipitation. Note that this nal fraction also includes other small nucleic acids such
as RNAs and smaller chromosomal fragments that may be problematic for downstream
applications other than Southern blotting.
Materials
Plates containing cells to be lysed (see Basic Protocol, step 16)
Hirt lysis buffer (see recipe)
5.0 M NaCl (sterilized by autoclaving)
20 mg/ml proteinase K stock in sterile H2 O (store up to 1 year at 20 C)
25:24:1 phenol/chloroform/isoamyl alcohol (Ambion)
Chloroform
TE buffer, pH 7.0 (APPENDIX 2A)
Isopropanol
50 C water bath or heating block
Lyse cells
1. Dispense 800 l of Hirt lysis buffer into each plate and swirl gently for 2 min.
The cell/buffer suspension should become viscous during this swirling process. It is important to ensure that the buffer covers the plate thoroughly, so that all cells are lysed and
harvested.
2. Tilt the plates and incubate 10 min at room temperature to collect the lysed cell
suspension.
3. Pour the suspension directly from each plate into a 1.5-ml microcentrifuge tube.
14B.1.8
Current Protocols in Microbiology
6. At the end of the incubation, centrifuge the tubes 30 min at 12,000 g, 4 C. Transfer
the supernatants to new tubes using a 1-ml automatic pipettor.
After the centrifugation there should be a large, whitish, solid pellet. If the pellet is unstable
or if there is unpelleted debris, then recentrifuge until the supernatant is free from debris.
When removing the supernatant, it is better to leave a small amount of liquid behind than
to transfer any of the debris material.
SOUTHERN BLOTTING
Southern blotting is commonly used to visualize and quantitate the plasmid DNAs that are
extracted from the cells at intervals post-transfection. Hybridization with a probe specic
for the target DNA allows selective detection of the target DNAs amidst an enormous
background of residual cellular DNA fragments. There are numerous variations of the gel
transfer procedure and the hybridization conditions for Southern blotting. The downward
transfer protocol described here is simple, effective, and reliable, but any other standard
transfer procedure would likely be suitable as well. Hybridization conditions may need
to be optimized for the specic plasmid DNAs and probes employed.
SUPPORT
PROTOCOL 2
Materials
Agarose gel containing separated proteins (see Basic Protocol, step 29)
0.25 M HCl
0.4 M NaOH
20 SSC (APPENDIX 2A)
0.4 mg/ml ethidium bromide
Probe DNA stock: prepare linearized pBOR as described for DNA quantitation
standards (see recipe), but resuspend at 1 g/ml in TE buffer, pH 7.0
Animal DNA
Viruses
14B.1.9
Current Protocols in Microbiology
b. Cut six pieces of Whatman 3MM paper to the same dimensions as the membrane.
c. Cut paper towels into pieces the same dimension as the membrane.
A stack of these pieces that is at least 3- to 4-in. high will be needed.
d. Cut two wicks from the Whatman 3MM paper of a width equal to the height of
the trimmed gel (i.e. from the wells to the bottom of the gel) and 2 feet in length
(these can be shortened later as necessary).
4. After 15 min, remove the gel and gel tray from the HCl. Drain the excess HCl from
the surface of the gel by tipping the gel tray on a paper towel. Hold the gel gently
while tipping the tray to prevent it from sliding off the tray.
Cell Culture
Assay for
Papillomaviruses
5. Submerge the gel in 0.4 M NaOH for 1 to 2 min to neutralize HCl, then remove the
gel and let it stand while quickly assembling the following components.
Do not allow the gel to dry. If necessary, return the gel to the NaOH solution to keep it moist.
14B.1.10
Current Protocols in Microbiology
Figure 14B.1.4 Diagram of the organization of the gel transfer stacking setup. Details of the
assembly and transfer are described in the text.
7. Soak the 3MM pieces, the wicks, and an 0.25-in. stack of the paper towels in 0.4
M NaOH until all are thoroughly wetted.
8. Assemble the stack as in Figure 14B.1.4 (transfer is performed at room temperature).
Use a 5- or 10-ml plastic pipet to roll out air bubbles between each layer. The order
of the stack from the bottom up is:
14B.1.11
Current Protocols in Microbiology
Remember that the gel is fragile and is best transferred by sliding it off of the gel tray
onto the membrane. Be sure to orient the gel so that the upper right-hand corner of the
gel coincides with the pencil mark on the membrane.
Lastly, the setup is somewhat messy, so the authors typically assemble the stack on a glass
plate or in a glass dish, though this is not strictly necessary.
14. Place the membrane on a piece of clean lter paper and let stand for several minutes
until the excess liquid is absorbed.
15. Cross-link the DNA to the membrane by performing two cycles of UV irradiation
with a Stratalinker set to full power.
Alternatively, cross-linking can be achieved by baking the membrane for 1 hr at 80 C in
a vacuum oven.
16. Let the membrane air dry at room temperature, then place between two sheets of
fresh lter paper, wrap in aluminum foil, and store at 4 C until hybridization.
21. Remove the DNA from the ice-water bath and microcentrifuge briey at maximum
speed to collect the liquid at the bottom of the tube.
22. Add the following reagents to the tube:
Cell Culture
Assay for
Papillomaviruses
14B.1.12
Current Protocols in Microbiology
24. Add 1 l of DNA polymerase I Klenow fragment stock from the Prime-A-Gene kit
and stir gently to mix components.
25. Incubate 60 min at room temperature.
26. Denature for 2 min at 95 C.
27. Quickly cool by transferring to an ice-water bath. Leave in the bath until step 32.
The probe can be used immediately for hybridization. If not used immediately, it should
be stored at 20 C and always denatured/quick-cooled prior to use.
14B.1.13
Current Protocols in Microbiology
COMMENTARY
Background Information
Cell Culture
Assay for
Papillomaviruses
14B.1.14
Current Protocols in Microbiology
Animal DNA
Viruses
14B.1.15
Current Protocols in Microbiology
Table 14B.1.2 Time Requirements for the Cell Culture Assay for Transient Replication of
Papillomaviruses
Procedure
Growth of cells
Time required
7-14 days
2 days
Protocol step
Basic Protocol, step 1
a
2 hr
Transfection
2 hr
1 hr
Incubation
2-4 days
2-3 days
RNase digestion
1 hr
Restriction digestion
1 day
6 hr
1 hr
Overnight
6 hr
Hybridization
Exposure of membrane to lm or
phosphor imager
1-5 days
a Not described in this unit; see Ausubel et al. (2005) for plasmid purication techniques.
Anticipated Results
Cell Culture
Assay for
Papillomaviruses
Time Considerations
The assay described in this unit is a lengthy
procedure that can take several weeks from
start to nish, depending primarily on how
long it takes to accumulate sufcient cells
to plate for the transfections. For planning
purposes, the durations of individual steps in
the protocol are summarized in Table 14B.1.2.
For all steps where optional overnight incubations are possible (see Basic Protocol for details), the times in the table include these prolonged incubation periods. Fortunately, there
are several stopping points along the way
where the samples can be stored indenitely
if necessary. Nonetheless, strategic planning
is advisable prior to commencing the assay to
ensure that mandatory steps occur at points
where the experimenter has sufcient time
and that needed reagents are available at the
correct times. Blocking out a daily schedule
for the entire protocol in advance of initiating the experiment will prevent unanticipated
delays.
14B.1.16
Current Protocols in Microbiology
Literature Cited
Ausubel, F.M., Brent, R., Kingston, R.E., Moore,
D.D., Seidman, J.G., Smith, J.A., and Struhl, K.
(eds.) 2005. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
Brown, T. 1999. Southern blotting. In Current Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G.
Seidman,J.A. Smith, and K. Struhl, eds.) pp.
2.9.1-2.9.20. John Wiley & Sons, Hoboken, N.J.
Chiang, C.M., Ustav, M., Stenlund, A., Ho, T.F.,
Broker, T.R., and Chow, L.T. 1992. Viral E1 and
E2 proteins support replication of homologous
and heterologous papillomaviral origins. Proc.
Natl. Acad. Sci. U.S.A. 89:5799-5803.
Gopalakrishnan, V. and Khan, S.A. 1994. E1 protein
of human papillomavirus type 1a is sufcient for
initiation of viral DNA replication. Proc. Natl.
Acad. Sci. U.S.A. 91:9597-9601.
Holt, S.E. and Wilson, V.G. 1995. Mutational analysis of the 18-base-pair inverted repeat element
at the bovine papillomavirus origin of replication: Identication of critical sequences for E1
binding and in vivo replication. J. Virol. 69:65256532.
Lin, B.Y., Ma, T.L., Liu, J.S., Kuo, S.R., Jin, G.,
Broker, T.R., Harper, J.W., and Chow, L.T. 2000.
HeLa cells are phenotypically limiting in cyclin
E/CDK2 for efcient human papillomavirus
DNA replication. J. Biol. Chem. 275:6167-6174.
Lu, J.Z., Sun, Y.N., Rose, R.C., Bonnez, W., and
McCance, D.J. 1993. Two E2 binding sites
(E2BS) alone or one E2BS plus an A/T-rich region are minimal requirements for the replication of the human papillomavirus type 11 origin.
J. Virol. 67:7131-7139.
Lusky, M. and Botchan, M.R. 1984. Characterization of the bovine papilloma virus plasmid maintenance sequences. Cell 36:391-401.
Lusky, M. and Botchan, M.R. 1986. Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements. Proc. Natl.
Acad. Sci. U.S.A. 83:3609-3613.
McShan, G.D. and Wilson, V.G. 1997. Reconstitution of a functional bovine Papillomavirus Type
1 origin of replication reveals a modular tripartite replicon with an essential AT-rich element.
Virology 237:198-208.
Peden, K.W.C., Pipas, Y.M., Pearson-White, S., and
Nathans, D. 1980. Isolation of mutants of an animal virus in bacteria. Science 203:1392.
Piirsoo, M., Ustav, E., Mandel, T., Stenlund, A.,
and Ustav, M. 1996. Cis and trans requirements
for stable episomal maintenance of the BPV-1
replicator. EMBO. J. 15:1-11.
Strober, W. 1997. Monitoring cell growth. In Current Protocols in Immunology (J.E Coligan, A.M
Kruisbeek, D.H Margulies, E.M Shevach and W
Strober, eds.) pp. A.3A.1-A.3A.2. John Wiley &
Sons, Hoboken, N.J.
Sverdrup, F. and Khan, S.A. 1994. Replication of
human papillomavirus (HPV) DNAs supported
by the HPV type 18 E1 and E2 proteins. J. Virol.
68:505-509.
Taylor, E.R. and Morgan, I.M. 2003. A novel technique with enhanced detection and quantitation
of HPV-16 E1- and E2-mediated DNA replication. Virology 315:103-109.
Titolo, S., Pelletier, A., Sauve, F., Brault, K.,
Wardrop, E., White, P.W., Amin, A., Cordingley, M.G., and Archambault, J. 1999. Role of
the ATP-binding domain of the human papillomavirus type 11 E1 helicase in E2-dependent
binding to the origin. J. Virol. 73:5282-5293.
Ustav, M. and Stenlund, A. 1991. Transient replication of BPV-1 requires two viral polypeptides
encoded by the E1 and E2 open reading frames.
EMBO. J. 10:449-457.
Ustav, M., Ustav, E., Szymanski, P., and Stenlund,
A. 1991. Identication of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1.
EMBO. J. 10:4321-4329.
Voytas, D. 1992. Agarose gel electrophoresis. In
Current Protocols in Immunology (J.E. Coligan,
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach,
and W. Strober, eds.) pp. 10.4.1-10.4.8. John Wiley & Sons, Hoboken, N.J.
Voytas, D. and Ning, K. 2002. Detection and quantitation of radiolabeled proteins and DNA in gels
and blots. In Current Protocols in Immunology
(J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, and W. Strober, eds.) pp. A.3J.1A.3J.10. John Wiley & Sons, Hoboken, N.J.
Wang, X.M., Jansen, K.U., and McClements, W.L.
2003. DNA replicative functions of highlyexpressed, codon-optimized human papillomavirus proteins E1 and E2. J Virol Methods
108:83-90.
White, P.W., Titolo, S., Brault, K., Thauvette, L.,
Pelletier, A., Welchner, E., Bourgon, L., Doyon,
L., Ogilvie, W.W., Yoakim, C., Cordingley,
M.G., and Archambault, J. 2003. Inhibition of
human papillomavirus DNA replication by small
molecule antagonists of the E1-E2 protein interaction. J. Biol. Chem. 278:26765-26772.
Key References
Ustav and Stenlund, 1991. See above.
This is the rst study to document that the viral E1
and E2 proteins were necessary and sufcient to
support replication of a papillomavirus origin in a
transient transfection assay.
Ustav et al., 1991. See above.
This study correctly identied and mapped the
bovine papillomavirus origin of replication. This
was the rst authentic denition of a functional papillomavirus origin, and documented that a DpnI
assay could be used to successfully detect transient
replication of papillomavirus origincontaining
DNA.
Animal DNA
Viruses
14B.1.17
Current Protocols in Microbiology
UNIT 14C.1
Human adenoviruses (Ads) have proven to be one of the most powerful tools for probing
the molecular and cell biology of eukaryotic cells. Studies using Ad systems as models for
virus replication and cell transformation have been pivotal to understanding transcription
regulation and the mechanisms by which cells regulate their growth (Green, 1978; for
reviews, see Phillipson, 1995 and Shenk, 2001). Thorough understanding of Ad early
gene function has allowed the development of replication-deficient Ad vectors, which
provide important new tools for probing gene function as well as for gene therapy and
cancer treatment. One of the advantages for gene expression analysis and for gene therapy
is that Ads and Ad vectors are able to efficiently infect most human cell types regardless
of their cell cycle state.
Human adenoviruses, of which there are at least 49 distinct types, as well as numerous
Ad expression vectors encoding cloned foreign genes, are easy to propagate, and can be
purified in milligram quantities and to high titers, i.e., 1011 to 1012 infectious units/ml
(Green and Pina, 1963; Pina and Green, 1965; Green et al., 1967a). It is important to
prepare consistent-quality Ads and Ad vectors, and to accurately establish their titers
in order to ensure meaningful and reproducible experimental results. The protocols that
follow describe the propagation and purification of Ad and Ad vectors, as well as their
quantification.
CAUTION: Human Ads are pathogens that can cause upper respiratory infection, gastroenteritis, and conjunctivitis, and therefore must be treated with respect and considered
biohazard agents to some extent. Concentration and purification of Ads and Ad vectors
should be conducted under at least BSL-2 conditions with the use of appropriate personal
protective barriers, including gloves, lab coats, and eye protection. Manipulation of concentrated virus is best conducted in a Class II biosafety cabinet. Institutional Biosafety
Committee review may be required at many institutions prior to work with Ads and Ad
vectors. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
CAUTION: Genetically modified human adenoviruses, especially variants expressing
oncogenes, may under some circumstances be considered BSL-3 agents (refer to NIH
Guidelines for working with recombinant DNA molecules and the local Institutional
Biosafety Committee for further advice). Follow all Centers for Disease Control guidelines for the use and handling of pathogenic microorganisms (http://www.cdc.gov/
od/ohs/biosfty/bmbl/bmbl3toc.htm). Experimental animals receiving Ads and Ad vectors should be handled and disposed of using recommended Animal Biosafety Levels
(ABSL) for infectious agents according to vertebrate animal guidelines. See UNIT 1A.1 and
other pertinent resources (APPENDIX 1B) for more information.
STRATEGIC PLANNING
Safety
Even in view of the abovementioned warnings, working with Ads is considered safe
for nonimmunocompromised workers, especially in view of the extensive amount of
research done worldwide with these viruses. What may be of greater concern is the use of
Ad vectors. Here one should consider what effect the expression of the vector package
will be in inadvertently infected cells of human workers. Another issue to consider in
Animal DNA
Viruses
Contributed by Maurice Green and Paul M. Loewenstein
Current Protocols in Microbiology (2005) 14C.1.1-14C.1.19
C 2005 by John Wiley & Sons, Inc.
Copyright
14C.1.1
Propagation
There are 49 currently known human Ads (Ad types 1 to 49; see Commentary), all of which
are available from the American Type Culture Collection (ATCC; http://www.atcc.org/).
Protocols for the purification and storage of Ads, replication-competent Ad vectors,
and replication-deficient Ad vectors are essentially identical. Protocols for the propagation of Ads and replication-deficient Ad vectors differ only in the cells lines that
can be used for virus propagation. Ads and replication-competent Ad vectors can be
infected into and grown in a wide variety of cells. Two cells lines that are commonly used for this purpose are the human cervical carcinoma cell line HeLa (ATCC
#CCL-2) and the human lung carcinoma cell line A549 (ATCC #CCL-185). Replicationdeficient Ad vectors, on the other hand, must be grown in human 293 cells (ATCC
#CRL-1573) or 911 cells (E1-transformed human embryonic retinal cells; Fallaux et al.,
1996).
The widely used 293 cell line was derived by transformation of primary human embryonic
kidney cells with sheared human Ad type 5 DNA (Graham et al., 1977; Harrison et al.,
1977; Louis et al., 1997). 293 cells express the gene products of the Ad5 early gene 1
(E1A and E1B).
One common characteristic among replication-deficient Ad vectors is the absence or
inactivation of early genes E1A and E1B. Expression of these genes is necessary for
activation of the other Ad genes and is essential for viral replication. E1A and E1B
functions must therefore be provided by the 293 cell in order to propagate replicationdeficient Ad vectors. The protocols in this unit, which refer generically to Ads, are meant
to include both Ad viruses and replication-deficient Ad vectors.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
Contamination
An important consideration while planning and conducting experiments and purification
of Ads is their ability to easily and efficiently infect most cell types. Accidental infection
of normal cell lines and cross-contamination of virus, virus mutants, and Ad vectors
is a concern, and efforts must be made to prevent this from occurring. Common sense
precautions should be observed at all times. Separate biosafety cabinets and incubators
should be used for infected and noninfected cultures, if possible. Biosafety cabinets and
work surfaces should be decontaminated with 70% ethanol after each use. Incubators
should be disinfected as well. It is desirable to use plug-seal type tissue culture flasks
when incubating Ad-infected cultures in a CO2 incubator. The cap can be loosened
when placed in the incubator and tightened after equilibration with the CO2 -enriched
atmosphere. Whenever possible, only one strain of Ad or Ad vector should be prepared
at any given time. Naturally, all tissue culture steps should be conducted using the best
sterile techniques possible. This involves the use of disposable, sterile, plugged tissue
culture pipets fitted with a filtered pipetting aid.
14C.1.2
Current Protocols in Microbiology
BASIC
PROTOCOL 1
One of the following cell lines growing in 75-cm2 tissue culture flasks:
293A cells (Invitrogen #R705-07)
293 cells (ATCC #CRL-1573)
A549 cells (ATCC #CCL-185)
HeLa cells (ATCC #CCL-2)
Phosphate-buffered saline (PBS; see recipe), 4 C and room temperature
Trypsin-EDTA (Invitrogen cat. no. 25300-062)
Complete DMEM medium (APPENDIX 4B) containing 10% FBS(DMEM-10)
0.4% (w/v) trypan blue (Sigma)
Adenovirus (Ad) stock (ATCC; see Strategic Planning and Commentary)
10 mM TrisCl, pH 8.0 (APPENDIX 2A), 4 C
Glycerol (optional)
CsCl, optical grade (UltraPure, Invitrogen)
Tris-buffered saline (TBS; APPENDIX 2A) containing 30% glycerol (see recipe), cold
Inverted tissue culture microscope
150-cm2 plug-seal-capped tissue culture flasks
15- and 50-ml conical polypropylene centrifuge tubes
Refrigerated low-speed centrifuge: e.g., Beckman J6-HC
Beckman Coulter Beckman Coulter Optima-L series ultracentrifuge with NV Ti 65
rotor (or equivalent ultracentrifuge and rotor)
Beckman Coulter OptiSeal ultracentrifuge tubes (Beckman Coulter cat. no.
362181)
Beckman Coulter OptiSeal tube rack assembly for NV Ti 65 rotor (Beckman
Coulter cat. no 360538)
Beckman Coulter OptiSeal spacers (for NV Ti 65; Beckman Coulter cat. no.
362202)
Beckman Coulter OptiSeal vise assembly, wrench, and other necessary tools
18-G, 1-in. (2.5-cm) needles
5-ml syringes
Slide-a-Lyzer dialysis cassettes, 3500 MWCO (Pierce)
2-ml sterile screw-cap tubes with gasket seals (Sarstedt)
Additional reagents and equipment for counting cells with a hemacytometer and
determining cell viability (Strober, 1997a,b)
CAUTION: All procedures should be carried out on absorbable bench paper that can be
autoclaved prior to disposal. All liquid wastes should be treated with bleach to a final
concentration of 10%. Needles and syringes must be placed in an appropriate sharps
container until autoclaved and disposed of. Plasticware should also be autoclaved prior
to disposal.
NOTE: All culture incubations should be performed in a humidified 37 C, 5% CO2
incubator unless otherwise specified.
NOTE: All solutions and equipment coming into contact with live cells must be sterile,
and aseptic technique should be used accordingly.
Animal DNA
Viruses
14C.1.3
Current Protocols in Microbiology
2. When cells become rounded (usually within 5 min), detach from flask surface using
a mild tap of the open hand (be careful not to overtrypsinize 293 cells).
3. Add 16 ml complete DMEM-10 medium to the trypsinized cell suspension.
4. Remove a small aliquot of cell suspension with a 1-ml pipet and combine it with an
equal volume of 0.4% trypan blue. Count the viable (nonstained) cells by loading
10 l on each side of a hemocytometer covered by hemocytometer coverslip and
examining microscopically at 100 magnification (see Strober, 1997a,b).
5. Seed 2 106 viable cells into a 150-cm2 tissue culture flasks containing 30 ml
DMEM-10. Incubate to 50% to 70% confluency.
Moderate amounts of virus are readily produced in monolayer culture grown in 150-cm2
tissue culture flasks. Five infected flasks will yield 2 ml highly purified Ad with a titer of
1 to 3 1011 pfu/ml.
10. Resuspend the cell pellet in 10 ml cold PBS, centrifuge as in step 9, and discard
the supernatant. If multiple flasks have been infected, pool the cell suspensions and
centrifuge again as described in step 9. Store the cell pellet at 70 C until ready for
CsCl purification of virus.
14. Transfer the cell lysate to a 15-ml high-speed centrifuge tube and clarify by centrifuging 10 min at 8000 g, 4 C. Decant the viral supernatant into a 15-ml centrifuge
tube. If crude virus stocks are to be stored for more than several months, add glycerol
to a final concentration of 30% (v/v).
The viral supernatant in the 15-ml centrifuge tube constitutes the crude virus stock.
14C.1.4
Current Protocols in Microbiology
16. Place the needed number of NV Ti 65 OptiSeal tubes into the tube rack provided by
the manufacturer (see Fig. 14C.1.1). Fill each OptiSeal tube to the neck (11.2 ml),
ensuring that the stem is dry. If the sample does not fill the tube to the neck, add more
CsCl solution of the same density (1.34 gm/cm3 in 10 mM TrisCl, pH 8.0).
CAUTION: Tubes must not be underfilled; otherwise there is a danger of tube collapse
during centrifugation.
17. Place an OptiSeal plug into the neck of each tube (Fig. 14C.1.1A). Using the supplied
plug-seating tool, seat the plugs into all of the tubes at the same time (Fig. 14C.1.1B).
Use of the seating tool ensures that equal pressure is used on all plugs.
18. Wipe the outside of each tube with a Kimwipe and insert the tubes into the rotor.
CAUTION: The NV Ti 65 rotor contains eight positions for tubes. It is imperative that
two, four, six, or eight tubes be used, to ensure dynamic balance for centrifugation.
Figure 14C.1.1 (A) Schematic of an OptiSeal NV Ti 65 rotor tube for purification of Ads by CsCl
density gradient equilibrium centrifugation. (B) Tube rack assembly used to seat plugs in the tubes.
Animal DNA
Viruses
14C.1.5
Current Protocols in Microbiology
Figure 14C.1.2 Schematic of an OptiSeal tube after CsCl density gradient centrifugation of an Ad
preparation. Indicated are the expected positions of the virus band and the position of a possible
empty capsid band. Also shown is the optimal position for inserting the syringe needle prior to
harvesting the virus band.
19. Insert an OptiSeal aluminum spacer on the top of each tube to provide support for the
neck and shoulder of the tube during centrifugation. Insert an aluminum rotor plug
(cap) into every one of the eight openings of the rotor, whether it contains a tube or
not.
20. Use the manufacturers supplied hex plug and torque wrench to tighten each cap to
120 in.-lb. while the rotor is held tightly by a rotor vise assembly (or by a strong
colleague).
21. Centrifuge 5 hr (or overnight for convenience) at 321,000 g (63,000 rpm in NV Ti
65 rotor), 22 C.
22. Terminate centrifuge run with minimal or no braking. Remove the rotor from the
centrifuge. Using the supplied extraction tools, carefully remove the cap, spacer, and
centrifuge tube from the rotor. Place the centrifuge tube in the tube rack and carefully
remove the centrifuge tube plug.
The opalescent virus band should be clearly visible near the center of the centrifuge tube
(see Fig. 14C.1.2). Often there is a lighter band near the top of the tube that represents
empty viral capsid.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
Slide-A-Lyzer dialysis cassettes allow for convenient and rapid dialysis of macromolecules
and viruses. They are supplied with foam floats that allow dialysis in a rapidly stirred
beaker.
26. Dialyze the virus against 1 liter of cold TBS/30% glycerol for 3 hr in a 4 C cold
room to remove CsCl.
14C.1.6
Current Protocols in Microbiology
27. Divide the dialyzed virus into 300-l aliquots in 2-ml gasket-containing, sterile
screw cap tubes. Store up to 5 years at 70 C.
The stability of Ads varies according to group serotypes (Green et al., 1967a. In general,
purified, concentrated virus preparations are stable for long periods of time (many years)
when stored in TBS containing 30% glycerol at 35 C to 70 C (Green and Wold, 1979).
Storage in aliquots helps preserve the stock titer from degradation by repeated freeze-thaw
cycles. Valuable stocks should be titered periodically (every few years), passaged, and
repurified if necessary.
ALTERNATE
PROTOCOL 1
HeLa or 293 cells are grown in Joklik-modified MEM supplemented with 10% calf serum
(instead of fetal calf serum) in volumes of 100 ml to 4 liters in Florence flasks that are
loosely sealed with a rubber stopper wrapped with aluminum foil. The stopper and neck of
the Florence flask are covered with a second layer of aluminum foil. Flasks are not filled
beyond three-quarters of capacity, so that sufficient air exchange occurs at the surface
of the medium. Flasks are incubated in a 37 C environmental room and stirred with a
magnetic bar at a rate that prevents cells from settling but that does not result in cell death
or frothing. Cultures adjusted to 5 105 cells/ml will maintain pH values suitable for
exponential growth in the absence of a CO2 -enriched atmosphere.
14C.1.7
Current Protocols in Microbiology
2. Infect with 20 pfu per cell (6 109 total pfu) Ad stock. Transfer to a an aluminumfoil-wrapped 250-ml Florence flask, loosely seal with a foil-wrapped rubber stopper,
then cover the stopper and neck of the flask with additional foil. Incubate with stirring
on magnetic stirrer for 30 min at 37 C.
3. Transfer the infected cells to 950 ml prewarmed complete spinner medium in a 2-liter
Florence flask. Loosely seal and wrap this flask in foil as described for the smaller
flask and incubate at 37 C with stirring for 30 to 40 hr.
When observed with a phase-contrast microscope, HeLa cells infected under these conditions are 2- to 3-fold larger and have prominent nuclei when compared to uninfected
cells. This appearance is due to a lack of cell division and dramatic increases in protein,
DNA, and RNA content (Green, 1962).
4. Transfer suspension to 250-ml centrifuge bottles and harvest infected cells by centrifuging 10 min at 200 g, 4 C, and removing the supernatants. Wash each cell
pellet by adding 15 ml cold PBS, centrifuging again as before, and removing the
supernatant. Store infected cell pellet at 70 C.
Over 90% of the virus remains cell-associated; Green, 1962.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
The ViraPower system involves the cloning of the gene or DNA sequence of interest into an
entry vector (pENTR) followed by transfer from the entry clone to an Ad destination
vector (pDEST). The first step is readily achieved by PCR cloning of the DNA insert
into a topoisomerase-linked entry vector, ensuring highly efficient and rapid directional
cloning into the vector cloning site, which lies between lambda attL recombination sites.
The attL recombination sites allows for the subsequent facile transfer of the DNA insert
into a variety of destination vectors that are designed to accept lambda recombination,
including the ViraPower vector pAd/CMV/V5-DEST, utilized for constructing CMV
promoter-containing Ad vectors. pAd/CMV/V5-DEST, like all replication-deficient Ad
vectors, is deleted in E1A and E1B (see Strategic Planning). Additionally, this vector
is deleted in early region 3 (E3). However, it contains elements and genes required for
production and packaging of replication-deficient Ads.
14C.1.8
Current Protocols in Microbiology
Materials
DNA template of interest
Platinum High Fidelity Taq DNA polymerase (Invitrogen cat. no. 11304-011) or
other good-quality proofreading Taq polymerase
pENTR/SD/D-TOPO cloning kit (Invitrogen cat. no. K2420-20) including:
Salt solution
Topoisomerase-tagged entry vector pENTR/SD/D-TOPO
Competent E. coli TOP10 cells
SOC medium (APPENDIX 4A)
LB agar plates (APPENDIX 4A) with 25 g/ml kanamycin, 37 C
LB liquid medium (APPENDIX 4A) with 25 g/ml kanamycin
QIAprep Spin Miniprep Kit (Qiagen) or equivalent
ViraPower pAd/CMV/V5-Dest System (Invitrogen cat. no. V493-20)
LR Clonase kit (Invitrogen) including:
5 clonase reaction buffer
Clonase enzyme
Proteinase K solution
LB agar plates (APPENDIX 4A) with 100 g/ml ampicillin
LB liquid medium (APPENDIX 4A) with 100 g/ml ampicillin
Plasmid Maxi Kit (Qiagen)
TE buffer, pH 8.0 (APPENDIX 2A), sterile
PacI restriction endonuclease and appropriate buffer (New England Biolabs)
293A cells (Invitrogen) growing in 75-cm2 flask
Complete DMEM medium (APPENDIX 4B) containing 10% FBS (DMEM-10), with
and without antibiotics
Dulbeccos Modified Eagle Medium (DMEM; Invitrogen)
OptiMem I medium (Invitrogen)
Lipofectamine 2000 transfection reagent (Invitrogen)
Trypsin-EDTA (Invitrogen cat. no. 25300-062)
42 water bath
12-ml snap-cap culture tubes, sterile
6-well tissue culture plates
75-cm2 tissue culture flasks
Additional reagents and equipment for PCR (Kramer and Coen, 1997) and
counting viable cells (Strober, 1997b)
NOTE: All culture incubations should be performed in a humidified 37 C, 5% CO2
incubator unless otherwise specified.
NOTE: All solutions and equipment coming into contact with live cells must be sterile,
and aseptic technique should be used accordingly.
Prepare a PCR product encoding the desired gene or DNA sequence of interest
1. Design and synthesize PCR primers appropriate for the template DNA insert.
The forward primer must contain the sequence CACC at the 5 end so that it can be
directionally cloned using the topoisomerase-tagged entry vector. Additionally, it should
contain an ATG in the context of a Kozak consensus sequence. If expression of the V5
tag in the intended Ad expression destination vector, pAdCMV/V5-DEST, is not desired,
then a stop codon must be included in the reverse primer. If the V5 tag is desired, the
absence of a stop codon will allow the expression of the protein containing the V5 peptide
at its C terminus. In this case, the gene should be in frame with the V5 epitope tag after
recombination (see detailed instructions for primer design provided with the ViraPower
kit). The V5 Tag permits the sensitive detection of the protein expressed from the Ad vector
in cells using commercially available antibody.
Animal DNA
Viruses
14C.1.9
Current Protocols in Microbiology
2. Prepare a 50-l PCR reaction containing the DNA template of interest and the desired PCR primer set. Perform PCR (Kramer and Coen, 1997) using a good-quality
proofreading Taq DNA polymerase (e.g., Platinum High Fidelity Taq polymerase) to
ensure that the product is blunt-ended. Refer to the manufacturers instructions for
specific PCR conditions.
Because the PCR primer set is dictated by the sequence of the DNA insert (usually a
complete gene), it is not always possible to avoid the use of difficult, nonoptimal primer
sets. It may be necessary in such cases to alter PCR conditions such as melting temperature
or annealing time. It may also be effective to alter the PCR mixture by the addition of
DMSO or betaine.
Transfer the cloned sequence from the entry vector to pAd/CMV/V5 DEST using the
LR clonase reaction
8. Mix 300 ng of a confirmed entry clone plasmid DNA from step 7 with 300 ng
ViralPower pAd/CMV/V5 DEST and 4 l of 5 clonase reaction buffer (from LR
Clonase reaction kit) to give a final volume of 16 l.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
9. Add 4 l of clonase enzyme (stored at 80 C and thawed on ice) and pipet gently
up and down. Incubate at room temperature for 1 hr (or overnight for convenience).
Add 2 l of the proteinase K solution provided by the manufacturer and incubate
10 min at 37 C. Transform TOP10 (or other suitable E. coli cells) with 1 l of the
digest, as described in step 4, except use LB agar plates containing 100 g/ml of
ampicillin rather than kanamycin.
14C.1.10
Current Protocols in Microbiology
During this reaction, the clonase enzyme mixture catalyzes the 1:1 exchange of the DNA
sequence between the two aatL sites of the entry vector (which include the cloned DNA
insert) with the sequences located between the two aatR sites in the destination vector,
which includes a suicide gene (ccdB) whose expression is toxic to most E. coli cells. The
end result is that unexchanged destination vector will kill cells that it transforms. The
only cells that will grow on an ampicillin-selection plate will be those that contain
the destination vector which has exchanged its suicide gene for the cloned insert from
the entry vector. This reaction is highly efficient. Because there is a direct exchange between the recombination sites of the entry vector and destination vector, theoretically there
can be no alteration in the cloned insert. Therefore, one can proceed with only one or two
clones from this point on. Note that clonase LR is guaranteed by the manufacturer for
6 months when stored at 80 C.
10. Inoculate two of the clones from the ampicillin plate into separate 100-ml quantities
of LB liquid medium containing 100 g/ml ampicillin and incubate overnight at
37 C with shaking.
11. Purify the Ad plasmid DNAs using a Plasmid Maxi Kit. Resuspend the purified Ad
plasmid in sterile TE buffer, pH 8.0. Digest 5 g of Ad plasmid DNA with 10 units
of PacI in the manufacturers recommended buffer at 37 C for 1 hr.
Digestion with PacI removes the Amp gene and the pUC ori from the pAdCMV/V5 DEST
recombinant, thus exposing the Ad 5 and 3 inverted terminal repeats. Transfection of
this linear plasmid into 293 cells will result in self annealing, which allows for Ad DNA
replication and the formation of recombinant Ad vector virus particles containing the
cloned DNA insert.
18. Replace the culture medium every 2 to 3 days until distinct areas of Ad-induced CPE
(also see Basic Protocol 1 and Commentary) are visible. Allow infection to proceed
until >50% of cells show CPE, typically 10 to 13 days post-transfection.
Animal DNA
Viruses
14C.1.11
Current Protocols in Microbiology
BASIC
PROTOCOL 2
Recently, kits have been developed for Ad purification that rely on ion-exchange chromatography, e.g., the AdenoX Virus Purification kit (BD Biosciences Clontech) and
Vivapure adenoPACK purification system (Vivascience). These systems are easy to use
but expensive. Laboratories that routinely prepare several different Ads or Ad vectors
will probably find these systems cost-prohibitive. Use of large-capacity vertical rotors
and CsCl centrifugation as described above (see Basic Protocol 1) is rapid and provides
for high-capacity purification.
It is essential for meaningful studies that the amount of infecting virus in Ad stocks be
accurately known. A plaque assay developed for the quantitation of the commonly used
Ad type 2 (Rouse et al., 1963) has been shown to be effective for most Ad serotypes
(Green et al., 1967a). A plaque is a cluster of infected cells produced by the infection of
a single cell by one virus particle followed by secondary infection of contiguous cells.
Antibody-based assays (see Alternate Protocol 4) rely on the production of viral proteins
(eg., hexon) within cells, and may not adequately reflect infectious units. A disadvantage
of the Ad plaque assay is that it requires from 7 to 11 days to reach a final titer. Described
here is a facile plaque assay method using 293 cells, modified from a protocol described
by Invitrogen. This protocol is very similar to the standard Ad plaque assays which
have been used for many years (Green et al., 1967a). The use of a well suited subclone
of 293 cells (293A) in this assay makes it ideal both for Ads and replication-deficient
Ad vectors.
Materials
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
293A cells (Invitrogen) or 293 cells (ATCC #CRL-1573) growing in 75-cm2 tissue
culture flask
Complete DMEM medium containing 10% FBS (DMEM-10)
Adenovirus (Ad) stock (ATCC; see Strategic Planning and Commentary)
4% agarose stock (see recipe)
0.33% (w/v) Neutral Red stock solution, sterile (Sigma)
5 mg/ml 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT, also
called thiazolyl blue; Sigma cat no. M-2128)
14C.1.12
Current Protocols in Microbiology
Tube no.
Vol. virus
Vol. medium
Resulting dilution
10 l virus stock
990 l
102 dilution
10 l from tube 1
990 l
104 dilution
10 l from tube 2
990 l
106 dilution
900 l
107 dilution
900 l
108 dilution
900 l
109 dilution
Animal DNA
Viruses
14C.1.13
Current Protocols in Microbiology
Figure 14C.1.3 Photograph of one well of a 6-well plaque titration plate showing plaques resulting
from a plaque assay on 293A cells. This well contains 200 l of a 109 dilution of a recombinant
Ad 5 vector at 8 days post-infection.
Count Ad plaques
8. Observe wells at an oblique angle over a light box and count the number of plaques
daily using a marking pen to dot each plaque on the bottom surface of the plate.
Return plate to incubator.
On 293 cells, plaques are visible to the naked eye starting at 8 days post-infection (see
Fig. 14C.1.3 for example).
9. When the plaque count does not increase significantly, make a final count on wells
containing virus dilutions that yield at least 10 plaques (see steps 10a and 10b; count
can be done without staining for 293 cells but is necessary for A549 cells).
Plaque count will stabilize 10 to 12 days post-infection depending upon the Ad serotype.
Wells containing >100 plaques are often too difficult to count.
The size and appearance of plaques vary with the Ad serotype (Green et al., 1967a).
10a. For staining with Neutral Red: Stain cells with Neutral Red (a traditional method)
at 10 days post-infection as follows.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
With A549 cells, it is desirable to stain plates prior to the final plaque count.
14C.1.14
Current Protocols in Microbiology
10b. For staining with MTT: Add 300 l of 5 mg/ml of MTT solution to each well and
incubate at 37 C for 1 hr.
In this case, plaques are visualized as clear areas on a dark background. Staining with
MTT results in cell death and thus can only be used when the plaque assay is terminated.
11. Calculate the final titer by multiplying the plaques per well by the dilution factor
and by five, since only 0.2 ml of the 1 ml dilution was used for the inoculum:
ALTERNATE
PROTOCOL 4
Agarose stock, 4%
4 g UltraPure agarose (Invitrogen cat. no. 15510-019)
100 ml tissue-culture grade H2 O
Autoclave 20 min on liquid cycle
Cool to 65 C prior to use
Store up to 2 months at 4 C
Complete spinner medium
Jokliks Modified Minimal Essential Medium (ICN Biomedicals; prepare per
manufacturers instructions; store up to 3 months at 4 C) containing:
10% calf serum (Invitrogen cat. no. 10371-029)
Cold sterilize under pressure through high-capacity tissue culture filter
(Pall Corporation)
Store up to 3 months at 4 C or up to 1 year at 20 C
Use of fetal calf serum is unnecessary for this medium.
Animal DNA
Viruses
14C.1.15
Current Protocols in Microbiology
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
14C.1.16
Current Protocols in Microbiology
Oncogenic potential
Subgroup
Serotypes
% DNA
homologyb
Percentage of
G + C in DNA
Tumorgenicity in
animals
Transformation in
cell culture
12, 18, 31
48-69/8-20
48-49
High
Yes
89-94/9-20
50-52
Moderate
Yes
1, 2, 5, 6
99-100/10-16
57-59
Low or none
Yes
94-99/4-17
57-61
Low or none
(mammary tumors)
Yes
57-59
Low or none
Yes
41
Unknown
Unknown
Unknown
Unknown
Troubleshooting
Tissue culture problems
Occasional bacterial, yeast, or mold contamination will occur from time to time in any
cell culture laboratory. Strict adherence to sterile technique and the use of biosafety cabinets
will minimize this problem. The inclusion of
antibiotic/antimycotic agents in working cultures is recommended. It is often suggested
that antibiotic/antimycotic agents be avoided
in stock cultures to allow nascent contaminations to become apparent. Stock cultures of
cell lines should be tested on a regular basis
for contamination with mycoplasma.
The use of complete commercial liquid media whenever possible will minimize problems
in cell culture that might occur due to a multitude of reasons, including, significantly, the
quality of the water. Laboratories that prepare
their own cell culture media should ensure that
their cell culture water be pyrogen-free, with
fewer than 10 parts per billion of total organic
content.
Anticipated Results
Basic Protocol 1 and Alternate Protocols 1
and 2 should result in pure stable Ads and Ad
vectors at concentrations, in general, of 1011
pfu/ml or more.
Time Considerations
Basic Protocol 1 and Alternate Protocol 1,
from the time of harvesting Ad infected cells
to the final aliquoting of purified virus, require
only 3 hr of labor, conveniently done over
2 days. This includes centrifugation time and
dialysis. Accurate titrations using plaque assay methods (see Basic Protocol 2) require less
than 2 hr of labor but take up to 12 days for
the final plaque count. The construction and
purification of replication-defective Ad vectors (see Alternate Protocol 2) expressing a
Animal DNA
Viruses
14C.1.17
Current Protocols in Microbiology
Literature Cited
Fallaux, F.J., Kranenburg, O., Cramer, S.J.,
Howeweling, A, Van Ormondt, H, Hoeben, R.C.,
and Van Der Eb, A.J. 1996. Characterization of
911: A new helper cell line for the titration and
propagation of early region 1-deleted adenoviral
vectors. Hum. Gene Ther. 7:215-222.
Graham, F.L., Smiley, J., Russell, W.C., and Nairn,
R. 1977. Characteristics of a human cell line
transformed by DNA from human adenovirus
type 5. J. Gen. Virol. 36:59-74.
Green, M. Studies on the biosynthesis of viral DNA.
1962. Cold Spring Harbor Symp. Quant. Biol.
27:219-235.
Green, M. 1971. Search for adenovirus messenger RNA in cancers of man. In Oncology V
(L. Clark, R.W. Cumley, J.E. McCay, and M.M.
Copeland, eds.) pp. 156-165. Yearbook Medical,
Chicago.
Green, M. 1978. Adenoviruses: Model systems of
virus replication, human cell molecular biology,
and neoplastic transformation. Perspect. Biol.
Med. 21:373-397.
Green, M. and Pina, M. 1963. Biochemical studies
on adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus.
Virology 20:199-207.
Green, M., Pina, M., and Kimes, R.C. 1967a. Biochemical studies on adenovirus multiplication.
XII. Plaquing efficiencies of purified human adenoviruses. Virology 31:562-565.
Human
Adenoviruses:
Propagation,
Purification,
Quantification,
and Storage
14C.1.18
Current Protocols in Microbiology
Internet Resources
http://www.cdc.gov/od/ohs/biosfty/bmbl4/
bmbl4toc.htm
http://www.atcc.org/
American Type Culture Collection.
http://www4.od.nih.gov/oba/rac/guidelines/
guidelines.html
For NIH Guidelines for Research Involving
Recombinant DNA molecules
http://www.geocities.com/CapeCanaveral/
Hangar/2541/
Joe Mymryks home page; an excellent source of
information concerning the Ad5 E1A oncogene, including a large database of Ad5 E1A mutants, E1A
interactive proteins, and E1A resources
Animal DNA
Viruses
14C.1.19
Current Protocols in Microbiology
UNIT 14E.1
Human herpes simplex viruses (HSV) are large, double-stranded DNA viruses that are
enveloped and contain structured capsids and a proteineous layer located between the
envelope and the capsid referred to as the tegument (Roizman and Knipe, 2001). HSV-1
is a neurotropic herpesvirus that causes a variety of infections in humans. It remains latent
in the neurons of its host for life and can be reactivated to cause lesions at or near the
initial site of infection. Recurrent infections result from the lytic replication of the virus
after reactivation from the latent state.
During productive lytic infection in cultured cells, glycoproteins in the viral envelope
interact with receptors on the host cell, including heparan sulfate and the herpesvirus entry
mediators. HSV-1 gene expression proceeds in a tightly regulated cascade, and changes
in its levels during infection are usually the consequence of transcriptional regulation.
The rst viral genes expressed during infection, termed the immediate-early (IE) genes,
are stimulated by incoming virion VP16 and are transcribed in the absence of de novo
viral protein synthesis. The IE gene products 0, 4, 22, and 27 function cooperatively to
regulate the expression of all classes of viral genes. The early genes are expressed next and
encode proteins mainly involved in viral DNA synthesis. The last genes expressed are the
late genes, and they mainly encode virion components such as VP16 and glycoproteins.
The late genes are further divided into the leaky-late and true-late classes of genes.
The true-late genes absolutely require viral DNA synthesis for their production. HSV-1
generally completes its replication cycle within 12 to 18 hr of infection.
This unit describes protocols required for the propagation and quantitation of HSV. These
include splitting of cultures (see Support Protocol 1), freezing cells (see Support Protocol
2), and thawing cells (see Support Protocol 3), as well as preparing virus stocks (see Basic
Protocol 1), determining virus titers (see Basic Protocol 2), and picking virus plaques
(see Basic Protocol 3). Additionally, methods are detailed for preparing whole infected
cell extracts (see Basic Protocol 4), detecting HSV proteins by separation on DATDacrylamide gels (Basic Protocol 5) and subsequent immunoblotting (see Basic Protocol
6), and indirect immunouorescence (see Basic Protocol 7).
These methods may be used for clinical isolates and laboratory strains of both HSV
types 1 and 2. HSV-1 serves as the prototype of the Herpesviridae. There are three main
laboratory strains of HSV-1 that are used throughout the world and are available from
the ATCC. They are HSV-1(17syn+), HSV-1(F), and HSV-1(KOS). Note that HSV1(17syn+) has been completely sequenced and is available in Genbank (Locus: HE1CG;
Accession numbers: X141112, D00317, D00374, S40593).
CAUTION: Human herpes virus is a Biosafety Level 2 (BSL-2) pathogen. Follow all
appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.
NOTE: All culture incubations should be performed in a humidied, 37 C, 5% CO2
incubator unless otherwise specied.
Animal DNA
Viruses
Contributed by John A. Blaho, Elise R. Morton, and Jamie C. Yedowitz
Current Protocols in Microbiology (2005) 14E.1.1-14E.1.23
C 2005 by John Wiley & Sons, Inc.
Copyright
14E.1.1
BASIC
PROTOCOL 1
Materials
Cell line of choice (e.g., Vero; see Table 14E.1.1) grown to conuence in 75-cm2
tissue culture asks (see Support Protocol 1 for culture techniques)
199V medium (see recipe)
HSV stock (ATCC), titered (see Basic Protocol 2)
DMEM/5% NBCS medium (see recipe)
Sterile milk (see recipe)
Freezer box, cardboard (optional)
15-ml conical tubes, sterile
Probe sonicator (e.g., Branson Sonier) with microtip
Screw-capped cryovials
Additional reagents and equipment for titering virus (see Basic Protocol 2)
Infect cells with virus
1. Aspirate medium from a conuent 75-cm2 asks of the appropriate cell type.
2. Prepare 3 ml of 199V medium containing HSV at a multiplicity of infection (MOI)
of 0.01 pfu/cell according to the following formula:
HSV:
Propagation,
Quantication,
and Storage
Cell line
ATCC
Growth conditions
Uses
Vero
(African Green
Monkey Kidney
Cells)
#81-CCL
HEp-2
(Human Epithelial
Carcinoma Cells)
#23-CCL
14E.1.2
Current Protocols in Microbiology
7. Carefully remove the ask from the freezer and carry it into a biosafety hood. Allow
the ask to warm up slowly at room temperature in the hood. Loosen and then tighten
cap to equilibrate the air pressure. Do not allow the asks to warm up too quickly or
to get knocked around. Be very careful not to break the ask.
8. As the ask warms, add 3 ml sterile milk to a fresh 15-ml sterile tube.
The use of milk as a stabilizer is a more effective way of storing HSV, as compared to
storage in medium alone. Powdered milk is cheaper than BSA, and it remains stable
through the autoclaving procedure. Virus titers have been found to decrease dramatically
when stock is stored in medium, while they are signicantly retained with milk storage.
9. As the ice in the ask begins to thaw, shake the virus/cell suspension (often referred
to as a herpes-Slurpee) over the monolayer to resuspend all cellular material in the
ask.
10. Remove the cell suspension from ask using a 5-ml pipet and add it to the 15-ml
tube containing the sterile milk.
11. Gently resuspend cells in the milk. Maintain the virus on ice from this point on.
12. Sonicate three times, each time for 30 sec using a Branson Sonier with microtip at
an output setting of 4 (timer on Hold; % Duty Cycle on Constant; approximate
Power Output of 15%), letting the cell suspension cool on ice for 1 min between
sonications, to free virus particles from the cellular debris.
13. Split virus stock into smaller aliquots and transfer to sterile, screw-capped cryovials.
Label stock with virus name, passage, and date, and store at 70 C or colder.
Dividing the suspension into aliquots prevents excess freezing and thawing of the virus
every time it is removed from the freezer. Label stock by virus name, passage, and date. It
is imperative that virus be stored a temperatures of 70 C or colder. Storage at higher
temperature rapidly leads to loss of infectivity.
BASIC
PROTOCOL 2
Materials
Cell line of choice (e.g., Vero)
DMEM/5% FBS medium (see recipe)
199V medium (see recipe)
Animal DNA
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Current Protocols in Microbiology
2. Add 1 ml 199V medium to each of a series of tubes. Using a sterile pipet, add 10 l
virus stock to the 1 ml of 199V in the rst tube and vortex (this is now a 102
dilution). Transfer 10 l from the 102 tube to the next tube containing 1 ml of 199V
and vortex (this is now a 104 dilution). Transfer 100 l from the 104 tube to the
next tube and vortex to prepare a 105 dilution. Repeat until the 108 dilution has
been reached. Discard 100 l from last tube to keep the volumes constant.
Make serial dilutions of virus at least 106 , 107 , and 108 .
Make sure to use a clean, plugged pipet tip each time. This is crucial. Virus stocks are
routinely of very high titer and repeated usage of a pipet tip for dilutions will affect the
results.
3. Label each 25-cm2 ask of conuent cells (from step 1) with the dilution of virus
that it is to receive. Aspirate the medium. Add 1 ml of each diluted stock to the
corresponding ask of cells.
4. To allow virus to absorb, incubate 2 hr with constant gentle rocking, or swirling
every 30 min. During the incubation, combine 3.38 l of 20 mg/ml pooled human
immunoglobulin with 9 ml DMEM/5% NBCS medium to prepare a solution containing 7.5 g/ml human immunoglobulin.
5. Aspirate the virus inoculum from each ask and add 3 ml of the DMEM/5% NBCS
containing pooled human immunoglobulin prepared in step 4.
6. Incubate 2 days, until plaques are visible.
9. Dilute the KaryoMax Giemsa Stain stock solution 1:10 with distilled water. Aspirate
the methanol and add 2 ml of the diluted Giemsa stain to each ask. Incubate at for
20 min at room temperature.
14E.1.4
Current Protocols in Microbiology
10. Discard the stain down a sink. Rinse cells gently with cold tap water until the liquid
is clear. Dry inverted ask on a paper towel.
11. Count plaques (circle or mark each with a ne-point marking pen) viewing through
an inverted light microscope at low (10) magnication.
While a phase-contrast microscope is not neccesary, it may help some investigators to
more easily distinguish plaques.
To determine titer of the stock, average the duplicate number of plaques counted for a
given dilution. This is the number before the exponent. The dilution is the exponent. For
example, if four plaques are counted on a 108 dish, the titer is 4 108 . Since this is an
infectious center assay, titers are expressed as pfu/ml.
BASIC
PROTOCOL 3
Materials
Virus stock (see Basic Protocol 1)
DMEM/5% NBCS medium (see recipe)
199V medium (see recipe)
20 mg/ml pooled human immunoglobulin (Sigma) in distilled H2 O
Sterile milk (see recipe)
1% (w/v) agarose in KPBS (see recipe for KPBS; store at 4 C)
Phosphate-buffered saline containing potassium (KPBS; see recipe), sterile
25-cm2 tissue culture asks
Plugged, sterile pipet tips
Fine-point marking pen
15-ml snap-cap tubes
Bent Pasteur pipet (see recipe)
Hand-held battery-operated pipetting device (e.g., Pipet-Aid; Drummond
Scientic)
Probe sonicator (e.g., Branson Sonier) with microtip
Additional reagents and equipment for splitting cells (see Support Protocol 1)
Isolate plaques
1. Split Vero cells (Support Protocol 1) appropriately into 25-cm2 tissue culture asks
such that they will be 90% conuent in 24 hr (see Basic Protocol 2, step 1).
2. To ensure that the plaques will be spread out in the dish, dilute the stock down to its
estimated titer by making serial dilutions of virus stock in 199V medium in the same
fashion as for virus titering (see Basic Protocol 2, step 2).
Only 20 to 50 plaques per dish are desirable.
3. Aspirate the medium from each ask of conuent cells prepared in step 1, replace
with 1 ml diluted virus, and incubate 2 hr at 37 C to allow the virus to absorb,
swirling every 30 min. During the incubation, combine 1.125 l of 20 mg/ml pooled
human immunoglobulin with 3 ml DMEM/5% NBCS medium to prepare a solution
containing 7.5 g/ml human immunoglobulin.
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Current Protocols in Microbiology
4. Aspirate medium from each ask and add 3 ml of the DMEM/5% NBCS containing
pooled human immunoglobulin prepared in step 3. Incubate at 37 C until plaques
are seen (usually within 2 days).
5. Choose and circle 5 to 10 plaques, preferably ones with space between them, using
a ne-point marking pen.
As noted below (see Commentary), HSV infection of monolayer cells yields distinctive
phenotypes, including enlargement and rounding of cells. Bear in mind that certain mutant
viruses will have plaque phenotypes that differ slightly from that of wild-type virus.
6. Label one 15-ml snap-cap tube per plaque to be picked, including plaque number,
virus, date, and any other appropriate parameters. Add 2 ml 199V and 2 ml sterile
milk to each tube.
7. Heat 1% agarose in KPBS in a microwave until agarose is completely dissolved.
When the mixture is cool enough to touch (45 C), aspirate the medium from each
dish (step 4) and add 3 ml of the 1% agarose in KPBS.
8. After the agarose has hardened (2 to 5 min), pick plaques using a bent Pasteur pipet
in a hand-held battery-operated pipetting device (e.g., Pipet-Aid) by sucking up the
agarose above the plaque and transferring it to the corresponding tube prepared in step
6, pipetting up and down to release the agar plug. Make sure that it enters the tube.
9. Freeze plaque stocks at 80 C for 15 min.
11. Use 0.5 ml sonicated plaque stock to infect a 90% conuent 25-cm2 ask of Vero
cells (prepared as in step 1). Incubate 2 hr at 37 C to allow the virus to absorb,
swirling every 30 min.
12. Wash monolayer by rst aspirating off the milk and then rinsing with 2 ml prewarmed
sterile KPBS.
13. Aspirate KPBS and add 3 ml DMEM/5% NBCS medium. Incubate until nearly 100%
CPE is observed.
In order to obtain good yields, it is very important that the cells go as far along in the
infection as possible. This can take up to 2 to 3 days.
14. When 100% CPE has been achieved, freeze asks at 80 C for 15 min.
15. Thaw asks in biological safety hood (work with no more than three to four asks at
a time). Swirl the virus/cell suspension (often referred to as herpes-Slurpee) over
the bottom of the ask to resuspend all cellular material.
16. When all of the ice is thawed, transfer the medium to a 15-ml tube containing 3 ml
sterile milk. Mix by pipetting up and down a few times and place the tubes on ice.
Label tubes with appropriate parameters.
HSV:
Propagation,
Quantication,
and Storage
17. Sonicate on ice three times, each time for 30 sec using the same instrument and
settings as described in step 10, letting the virus suspension cool on ice for 30 sec in
between sonications.
18. Store at 80 C or use 0.5 ml directly for infections.
14E.1.6
Current Protocols in Microbiology
SPLITTING CELLS
Both Vero and HEp-2 cells are adherent cell lines that can be grown in almost any
type of culture vessel. Covered dishes must always be kept in a CO2 incubator. Capped
(lterless) asks (75-cm2 or 25-cm2 ) do not require such an incubator but require the
injection of ltered CO2 prior to tightening; these gassed asks may be placed in any
37 C environment, including water baths.
SUPPORT
PROTOCOL 1
Note that it is important to consider the relative conuency of the cell monolayer prior
to splitting. Cells should be split once they reach between 95% to 100% conuency.
The doubling times of some cell lines are signicantly longer than other, standard lines.
Therefore, such cells should be maintained at a higher cell density (e.g., never going
below 60% conuency).
Materials
DMEM/5% FBS medium (see recipe)
Cells: Vero or HEp-2 (Table 14E.1.1)
Phosphate-buffered saline with potassium (KPBS; see recipe), sterile
70% ethanol
Trypsin-EDTA (Invitrogen; also see recipe)
Culture vessels: 75- or 25-cm2 tissue culture asks
Prepare asks
1. Prewarm DMEM/5% FBS medium and sterile KPBS in a 37 C water bath for 10 to
15 min.
In general, DMEM with fetal bovine serum (FBS) is used for maintain cells for passaging,
whereas DMEM with newborn calf serum (NBCS) is used for cells to be infected with
virus.
2. Before use, wipe down all bottles (medium, KPBS, and trypsin-EDTA), aspirator,
pipetting devices, and biological safety hood with 70% ethanol.
3. Using sterile technique, transfer 10 ml DMEM/5% FBS medium into each new
75-cm2 asks into which the cells are to be split or transfer 3 ml of the medium into
each new 25-cm2 ask into which the cells will be split.
If the cell line being passed contains a plasmid encoding a particular gene of interest, it
will often also contain a gene encoding a protein for resistance to a particular drug or
antibiotic. The presence of this drug ensures that only the cells containing the plasmid
will divide. Thus, this antibiotic should be added to fresh culture medium prior to splitting
cells.
Trypsinize cells
4. Remove the cells to be split from the incubator. Tilt ask so that medium pools in
the corner of the ask away from the cell monolayer. Using a sterile Pasteur pipet,
vacuum aspirate the medium at the corner such that the pipet tip does not come into
contact with the cells.
5. Rinse cells with 5 ml sterile KPBS, being sure to wash entire surface in order to
remove all serum-containing medium. Aspirate the KPBS with a sterile Pasteur pipet
at the corner opposite the cells.
6. Repeat the wash described in steps 4 and 5.
7. Add 2 ml trypsin-EDTA and rock the ask back and forth, ensuring that all of the
cells are coated. Remove the trypsin-EDTA quickly (within 30 sec) using a sterile
Pasteur pipet.
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8. Tighten the cap of the ask and incubate in 37 C incubator for 2 to 5 min.
It is not necessary to place asks in the incubator after applying trypsin, but doing so
will speed up the process of detaching cells. Some cell lines are more sensitive to trypsin.
Overtrypsinizing cells can lead to a loss of cell-surface factors from which the cells cannot
recover. Some cells even require a rinsing step to remove any residual enzyme that is left
behind. To do this, resuspend the detached cells in KPBS, centrifuge several minutes at
800 g using a tabletop clinical centrifuge, and resuspend pellet in cell culture medium.
9. Tap ask to dislodge cells and check under microscope to make sure most cells are
detached and oating.
10. Using sterile technique, add an appropriate quantity of prewarmed DMEM/5% FBS
to the trypsinized ask, according to the desired split.
For example, if splitting 1:4, add 4 ml to the cells at this step.
Split cultures
11. Using a 5-ml pipet, triturate the cells 30 times by pipetting up and down, squirting
the medium against the bottom of the asks each time. Transfer the cells to the 75or 25-cm2 asks according to the desired split.
For the 1:4 example above, transfer 1 ml of the cell/medium mixture to the medium that
was transferred to the labeled ask in step 3.
12. Put the ask on its side and rock it gently to ll the bottom surface of the ask with
the cells.
13. Place the ask in the incubator and continue incubating. Loosen the cap of the ask
if using a CO2 incubator.
SUPPORT
PROTOCOL 2
FREEZING CELLS
In order to maintain proper quality control, it is recommended that cells not be passaged
more than 20 to 30 times. This ensures that they will always be at approximately the same
passage during infection experiments, to help maintain reproducibility and consistency. It
is therefore necessary to freeze the cell stocks in aliquots that can be rethawed as needed.
As a general rule of thumb, it is suggested that each time a new stock is thawed, it be
one of the older aliquots. Upon thawing, the cells should be expanded and then a portion
frozen, before two to three passages, for future use.
Materials
Phosphate-buffered saline with potassium (KPBS; see recipe), sterile
DMEM/5% NBCS medium (see recipe)
Dimethylsulfoxide (DMSO), sterile
70% ethanol
Conuent asks of Vero or HEp-2 cells (see Support Protocol 1)
Trypsin-EDTA (Invitrogen; also see recipe)
Liquid nitrogen
Tabletop clinical centrifuge
1.5-ml cryovials
Liquid nitrogen tank or freezer
Prepare CFM and rinse cells
1. Prewarm sterile KPBS in a 37 C water bath for 10 to 15 min.
HSV:
Propagation,
Quantication,
and Storage
2. Depending on the number of asks that are to be frozen, combine DMEM/5% NBCS
medium and DMSO as described in Table 14E.1.2 to prepare an appropriate amount
of cell freezing medium (CFM). Place CFM on ice.
14E.1.8
Current Protocols in Microbiology
Number of asks
2.7
300
3.6
400
4.5
500
5.4
600
3. Before use, wipe down all bottles (i.e., medium, KPBS, and trypsin-EDTA), aspirator,
pipetting devices, and biosafety hood with 70% ethanol.
4. Remove the cells that are going to be frozen from the 37 C incubator. Using a sterile
Pasteur pipet, aspirate the medium at the corner opposite the cell monolayer.
5. Rinse cell layer with 5 ml prewarmed sterile KPBS (step 1). Be sure to wash entire
surface in order to remove all serum-containing medium.
6. Using a sterile pipet, aspirate the KPBS with a sterile Pasteur pipet at the corner
opposite the cells
7. Repeat steps 5 and 6 for an additional wash.
Trypsinize cells
8. Add 2 ml trypsin-EDTA and rock the ask back and forth, ensuring that all of the
cells are coated.
9. Remove the trypsin-EDTA quickly (within 30 sec) using a sterile Pasteur pipet.
10. Tighten the cap of the ask and incubate in 37 C incubator for 2 to 5 min.
11. Tap the sides of the ask to dislodge cells.
12. Resuspend cells in 2 ml DMEM/5% NBCS and transfer to a 5-ml tube. From this
point forward, keep cells on ice.
Freeze cells
13. Centrifuge cells 3 to 4 min at 800 g, 4 C, using a tabletop clinical centrifuge,
and resuspend cell pellets in CFM at 1 107 cells/ml.
14. Distribute cells in CFM evenly between desired number of cryovials (750 l/
cryovial).
15. Transfer cells to a 80 C freezer as soon as possible and leave there overnight or up
to 2 weeks to allow cells to equilibrate, then transfer to 150 C in a liquid nitrogen
tank or liquid nitrogen freezer for long-term storage.
THAWING CELLS
Cells permissive for HSV are available from the ATCC and are provided as DMSOcontaining frozen stocks which must be properly thawed to ensure recovery of viable
cells. Choosing the type of cell in which to grow virus depends on the purpose of the
infection. Certain cells are highly permissive for virus replication and are therefore used to
prepare virus stocks. Others are more suited for the characterization of virus replication.
Table 14E.1.1 presents some examples of commonly used cell lines.
Note that the conditions outlined below are suitable for Vero and HEp-2 cell lines, which
are commonly used; however, media, culture vessel, optimal pH, and CO2 concentrations
may vary depending on the cell line being thawed.
SUPPORT
PROTOCOL 3
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Viruses
14E.1.9
Current Protocols in Microbiology
Materials
DMEM/5% FBS medium with antibiotics (see recipe)
Cryovial containing frozen cells (see Support Protocol 2, or purchase from ATCC),
maintained at 70 C or below
70% ethanol
75-cm2 ask (or alternative culture vessel)
Inverted tissue culture microscope
1. Prewarm DMEM/5% FBS medium at 37 C for 15 to 20 min.
2. Prepare a 75-cm2 ask containing 10 to 12 ml of the prewarmed culture medium
adjusted to optimal pH with 5% CO2 /95% air.
3. Remove vial containing cells from the freezer and immerse in a 37 C water bath.
Agitate so that cells thaw rapidly (within 1 min).
4. Wash outside of vial thoroughly with sufcient 70% ethanol to sterilize (5 ml).
5. In a sterile hood, transfer thawed cells to the ask from step 2. Pipet up and down
several times to ensure that all cells are distributed throughout the medium.
6. Place ask in 37 C incubator overnight.
7. On the following day, replace old medium (containing DMSO from the freezing
medium) with fresh prewarmed DMEM/5% FBS medium.
8. Monitor cell growth using inverted microscope. Passage cells (see Support Protocol
1) when they reach conuency (cell foci are touching each other).
BASIC
PROTOCOL 4
Materials
HSV:
Propagation,
Quantication,
and Storage
14E.1.10
Current Protocols in Microbiology
Animal DNA
Viruses
14E.1.11
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BASIC
PROTOCOL 5
Materials
Mild laboratory detergent
70% ethanol
Petroleum jelly
1.4 g/ml ammonium persulfate
Protein gel solutions A, B, and C (see recipe)
20% (w/v) SDS (APPENDIX 2A)
TEMED
30% acrylamide/bisacrylamide (see recipe)
1 protein running buffer (see recipe for 10)
Whole infected cell extract (see Basic Protocol 4)
Buffer A (see recipe)
4 disruption buffer (see recipe)
Gel-forming apparatus:
Glass plates for 20 20cm gel
2-mm spacers
Combs
Large binder clips
Syringes
Vertical electrophoresis apparatus, power supply, and cables
Bent needles for removing bubbles from electrophoresis chamber
Boiling water bath
Pour the gel
The gel must be made before the extract samples are prepared for loading.
1. Thoroughly wash glass gel plates with a mild detergent. Rinse, dry thoroughly, and
wipe down with 70% ethanol.
2. Using a syringe, outline the larger glass plate with a thin line of petroleum jelly along
both sides and the bottom.
There are numerous gel systems available. This method guarantees gels that never leak.
3. Place spacers on top of the petroleum on the glass and adjust into place. Add a thin
line of petroleum jelly on top of the spacers and carefully place the other glass on
top. Using large binder clips, clip both sides and the bottom to prevent leaks.
4. Stand plates up vertically. Using the appropriate-sized comb, mark on the plate at
1 cm from where the wells will end.
5. Prepare the separator gel in a 125-ml ask, according to the percentage of the gel
desired, typically (for 20 20 gel with 2-mm spacers):
HSV:
Propagation,
Quantication,
and Storage
4.8 ml H2 O
15 ml 1.4 g/ml ammonium persulfate
7.5 ml protein gel solution A
3.16 ml protein gel solution C
14E.1.12
Current Protocols in Microbiology
6. Upon mixing the above ingredients, immediately pour the gel, using a 10-ml pipet, up
to the line marked with the comb (see step 4; approximately the bottom three-quarters
of the gel).
The dimensions of the separator gel will be needed when detecting HSV proteins by
immunoblotting (see Basic Protocol 6).
7. Gently add a layer of water on top the separating gel using a Pasteur pipet and allow
the separator gel to polymerize.
8. When polymerized, remove the top layer of water using a syringe. Tilt the glass plates
until all of the water is removed.
9. Prepare the stacking gel (top 1/4 of the gel) in a 50-ml centrifuge tube, according to
the percentage of the gel desired, typically (for 20 20 gel with 2-mm spacers):
1.61 ml H2 O
3 ml 1.4 g/ml ammonium persulfate
750 l protein gel solution B
638 l 30% acrylamide/bisacrylamide
2 l TEMED.
10. Immediately pour the stacking gel using a 5-ml pipet. Put the comb into place and
wait for the stacking gel to polymerize completely.
11. Remove the clips and bottom spacer. Use a Kimwipe remove any excess petroleum
jelly.
Failure to remove excess petroleum jelly may cause the gel not to run properly.
12. Stand the gel vertically and align so smaller plate and comb face inward. Clip plates
onto a vertical electrophoresis apparatus.
13. Add 1 protein running buffer to the top and bottom chambers. Make sure that the
top does not leak.
14. Remove the bubbles between the two plates in the bottom chamber with a curved
(bent) needle on a syringe.
15. Mark the lanes on the glass plates.
16. Remove the comb and rinse the wells with buffer.
Samples can now be loaded onto the gel.
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14E.1.13
Current Protocols in Microbiology
Perform electrophoresis
20. Run the gel at a constant current of 70 mA.
Running a 2-mm 15% DATD-acrylamide gel at 70 mA typically takes 4 to 5 hr.
Materials
Gel containing separated HSV proteins (see Basic Protocol 5)
Transfer buffer with and without SDS (see recipes)
0.1% Ponceau S (optional; see recipe)
Phosphate-buffered saline (KPBS; see recipe)
5% milk/KPBS (see recipe)
Tris-buffered saline (TBS; see recipe)
Primary antibodies: HSV-specic antibodies are available from the
Rumbaugh-Goodwin Institute for Cancer Research (http://www.rgicr.org/)
Secondary antibody, alkaline phosphataseconjugated (Sigma or Fisher);
uorescently tagged antibodies may be obtained from Molecular Probes
1% BSA/KPBS (see recipe)
Tris-buffered saline with Tween 20 (TBST; see recipe)
AP buffer (see recipe)
15 mg/ml nitroblue tetrazolium chloride (NBT) in 70% dimethylformamide (DMF)
30 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in 100% DMF
Whatman no. 1 lter paper
0.45-m nitrocellulose transfer membrane
Plastic dish large enough to accommodate gel
Flat glass plate slightly larger than gel
Electroblotting apparatus: tank transfer system including transfer cassette and
power supply (e.g., Bio-Rad)
Prepare the transfer stack
1. Measure the dimensions of the separator gel. Cut two pieces of Whatman no. 1 lter
paper to a slightly larger size, i.e., allowing an extra centimeter on each side.
Avoid wasting nitrocellulose and lter paper by being accurate in gel dimensions.
HSV:
Propagation,
Quantication,
and Storage
3. Add transfer buffer without SDS to a plastic dish and place a at glass plate slightly
larger than the cut lter paper in the buffer.
4. Write top on one piece of lter paper. Wet the nitrocellulose and lter paper in
transfer buffer without SDS.
14E.1.14
Current Protocols in Microbiology
5. Place the bottom piece of lter paper on the glass plate, followed by the nitrocellulose.
Center and smooth to make sure there are no air bubbles trapped under the paper.
6. At this point, turn off the power supply to the protein gel. Discard the protein running
buffer in the top chamber. Unclip the gel and remove it from the chamber. Place the
gel at on a tabletop lined with paper towels. Slide out the side combs and gently
separate the two plates using a metal spatula.
7. Using a razor blade, carefully remove the stacking gel, slide it onto a paper towel,
and discard. Remove any gel below the blue buffer front line and slide it onto a paper
towel to discard. Cut a corner of the gel for orientation.
8. Roll the gel off the glass plates using a razor blade, allowing it to fall onto the nitrocellulose. Gently move the gel into place (congruent with nitrocellulose membrane).
9. Put the second, top piece of lter paper onto the gel to form a sandwich. Smooth
out any air bubbles. Remove the sandwich from the buffer.
11. Assemble and close the transfer cassette. Place cold transfer buffer with SDS into the
transfer apparatus rig. Insert the cassette into the rig nearest to the anode (red side),
oriented so that the top side of the cassette faces the black pole of the apparatus
(cathode).
12. Plug into power supply and run at 100 V in a cold room (4 C).
It will take 2 hr to completely transfer a 2-mm, 15% acrylamide gel.
13. Turn off the power. Take apart the cassette. Rinse cassette, sponges, and rig with tap
water to remove salts before storing.
Depending upon time available, the researcher may opt to air dry the nitrocellulose
membrane or go directly to next step and block in 5% milk. The membrane may also be
stained at this point using 0.1% Ponceau S (see recipe), which allows for visualization
of almost all the proteins on the membrane. This gives the researcher the ability to see
whether there are any air bubbles or if there was a problem with the transfer. This step is
not necessary, but it is advised.
15. Rinse the blot six times, each time with 10 ml TBS.
16. If it is desirable to cut the blot into sections (e.g., to stain with more than one antibody),
carefully cut the blot with a razor blade using the markers as a guide.
Loading prestained molecular weight markers onto the gel is advised if one desires to cut
the membrane.
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14E.1.15
Current Protocols in Microbiology
18. Add the appropriate primary antibody to each blot and incubate at 4 C.
Incubation with primary antibodies against viral proteins should take only 2 hr and may
be done at room temperature. However, detection of certain cellular proteins requires
overnight incubation at 4 C.
19. Remove the primary antibody. Rinse the blot three times, each time with 10 ml TBST
The antibody stock may be reused and should be stored at 4 C.
20. Dilute secondary antibody 1:1000 in 1% BSA/KPBS. Add the diluted secondary
antibody to the blot and incubate for exactly 1 hr at room temperature.
The secondary antibody must correlate with the primary. For example, if the primary
antibody is from mouse, the researcher must make sure to use an anti-mouse secondary
antibody. Antibodies should be diluted (e.g., 1:1000) in 1% BSA/KPBS. The authors
generally suggest secondary antibodies coupled to alkaline phosphatase.
21. Discard secondary antibody and rinse three times, each time for 15 min with 10 ml
TBST.
22. Rinse three times, each time for 5 min with 10 ml TBS.
23. Rinse once with 10 ml AP buffer for 1 to 2 min.
24. Prepare developer solution by diluting 15 mg/ml NBT and 30 mg/ml BCIP together
1:500 in AP buffer.
25. Add developer solution to the blot and wait for the bands to appear. Stop by rinsing
with water before the blot becomes over-developed.
BASIC
PROTOCOL 7
Materials
HSV:
Propagation,
Quantication,
and Storage
Ethanol
Cells of choice, growing in tissue culture
DMEM/5% FBS medium (see recipe)
Virus stock (see Basic Protocol 1)
199V medium (see recipe)
DMEM/5% NBCS medium (see recipe)
Phosphate-buffered saline (KPBS; see recipe)
2.5% paraformaldehyde (see recipe)
Acetone, 20 C
10 g/ml human immunoglobulin in 1% BSA/KPBS (see recipe for 1%
BSA/KPBS)
Primary antibody
1% BSA/KPBS (see recipe)
Fluorescently conjugated secondary antibody
ProLong Antifade Kit (Molecular Probes)
Clear nail polish
14E.1.16
Current Protocols in Microbiology
25-mm2 coverslips
6-well or 33-mm2 tissue culture dishes
Dark plastic box
Glass microscope slides
Additional reagents and equipment for splitting cells (see Support Protocol 1)
Prepare cover slips with cells
1. Dip a coverslip in ethanol and ame to sterilize. Place carefully in dish or well.
2. Seed an appropriate number of the cells of choice (see Support Protocol 1 for splitting
technique) in 2 ml DMEM/5% FBS onto the sterilized coverslips in the well/dish to
achieve conuency on the next day. Incubate cells overnight.
Infect cells
3. Optional: Perform a synchronized infection, by placing dishes on ice for 20 min
prior to infection and maintaining them on ice during virus absorption to ensure that
all cells are at the same point of the replication cycle throughout infection (Pomeranz
and Blaho, 1999).
This is especially useful when examining the kinetics of viral protein production.
4. Remove medium and infect cells by adding virus stock at an MOI of 15 in 199V
medium. Absorb virus 1 hr at 37 C (or on ice for synchronized infection).
Always keep the virus on ice and return the virus vials to 80 C as soon as virus has
been used.
5. Aspirate virus and add 2 ml DMEM/5% NBCS to each well/dish. Incubate 1 to 2 hr.
6. Remove cells from the incubator and aspirate off the medium from the edge of the
dish to ensure that the cells are not being removed from the coverslip.
The remaining steps need not be performed under sterile conditions.
7. Rinse cells twice, each time with 2 ml KPBS at room temperature, aspirating each
time.
Fix cells
8. Add 2 ml of 2.5% paraformaldehyde to each well and incubate at room temperature
for 20 min.
9. Aspirate the paraformaldehyde and add 1 ml acetone, 20 C, to each well. Place at
20 C for 3 to 5 min, then aspirate and rinse cells twice as in step 7.
Perform staining
10. Add 2 ml/well of 10 g/ml human immunoglobulin diluted in 1% BSA/KPBS and
block by incubating 1 hr to overnight at 4 C.
Treatment with this amount of human immunoglobulin was previously shown to be sufcient to neutralize Fc binding by the viral gE and gI proteins (Pomeranz and Blaho,
1999).
12. Remove dishes from the 4 C refrigerator and aspirate the blocking buffer. Rinse
twice with 2 ml KPBS at room temperature.
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13. Line the bottom of a dark box with Paralm. Place 50 l of primary antibody onto
the Paralm. Remove the coverslip containing the infected cells from the well/dish
and very carefully place it cell-side-down on the antibody solution in the dark box.
Incubate 1 hr at room temperature.
Pay special attention to where the cells are located relative to the coverslip. It is extremely
important to know the orientation of the coverslip in relation to being cell-side-up or
cell-side down.
14. Remove the coverslip from the box and place it cell-side up in a fresh well or dish.
Rinse twice with KPBS at room temperature, leaving the second rinse in the well or
dish.
15. Dilute the secondary antibody in 1% BSA/KPBS.
This dilution varies depending on the antibody. For example, FITC-coupled antibodies
usually use 1:300, while Texas Redcoupled antibodies use 1:150.
16. Remove the Paralm lining from the dark box and replace with fresh Paralm. Place
50 l of the diluted, uorescently-conjugated secondary antibody on the Paralm.
17. Remove the coverslip from the well and very carefully place it cell side-down on the
secondary antibody solution in the dark box. Incubate for exactly 45 min at room
temperature, making sure that the dark box is closed.
18. Remove the coverslip from the box and place it in a fresh well/dish, cell-side-up.
Rinse twice with 2 ml KPBS at room temperature, leaving the second rinse in the
well or dish.
20. Drop 5 l of the mounting solution onto a clean microscope slide. Remove the
coverslip from the well or dish and very carefully place it cell-side-down on the
mounting solution on the slide.
21. Seal the edges of the coverslip with clear nail polish and allow it to completely dry,
keeping the slides in the dark box. Place slides at 4 C overnight.
Slides will look the best when viewed the following day. Slides are typically viewed at
100 magnication with oil immersion using a uorescence microscope.
Acrylamide, 30%/bisacrylamide
Weigh out 145 g of acrylamide and 5 g bisacrylamide. Add 300 ml water and allow
these reagents to go into solution with stirring. Adjust volume to 500 ml with water.
Filter through a 0.45-m membrane. Store up to 1 year at 4 C in a glass bottle
wrapped with aluminum foil
HSV:
Propagation,
Quantication,
and Storage
CAUTION: A dust mask should be worn when weighing out powdered acrylamide. Predissolved liquid acrylamide may be substituted
14E.1.18
Current Protocols in Microbiology
AP buffer
10 ml 5 M NaCl
50 ml 1 M TrisCl, pH 9.5 (APPENDIX 2A)
2.5 ml 1 M MgCl2 (add 20.33 g of MgCl2 to 80 ml H2 O; stir and adjust volume
to 100 ml with H2 O; store indenitely at room temperature)
Adjust volume to 500 ml with H2 O
Store up to 1 month at room temperature
Bent Pasteur pipets (for sucking up plaques)
Apply heat near the tip of a Pasteur pipet over a low ame until it bends at a very
short 45 angle.
It is important that the tip of the pipet not be allowed to close during the heating.
BSA, 1% in KPBS
Dissolve 1 g BSA in 80 ml KPBS (see recipe). Adjust volume to 100 ml with KPBS.
Add 100 l of 20% (w/v) sodium azide for a nal concentration of 0.02%. Store up
to 3 months at 4 C.
Buffer A
5 ml 1 M TrisCl, pH 7.5 (APPENDIX 2A)
3 ml 5 M NaCl
1 ml 0.5 M EDTA, pH 8.0 (APPENDIX 2A)
4 ml 10% (v/v) Triton X-100 (Sigma)
H2 O to 100 ml
Store buffer with above ingredients up to 6 months at room temperature
Just prior to use, add the following protease inhibitors to an appropriate-sized aliquot
of Buffer A (e.g., 1 ml):
0.1 mM phenylmethylsulfonyluoride (PMSF; add from 0.1 M stock in methanol;
APPENDIX 2A or Sigma)
0.01 mM L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK;
add from 0.01 M stock in H2 O; Sigma)
0.01 mM L-1-chlor-3-(4-tosylamido)-4-phenyl-2-butanone (TPCK; add from
0.01 M stock in ethanol; Sigma)
Disruption buffer, 4
Combine the following in a 15-ml snap-cap tube:
4 ml 20% SDS (APPENDIX 2A)
2 ml of 1 M 2-mercaptoethanol
2 ml of 1 M TrisCl, pH 7.0 (APPENDIX 2A)
2 ml H2 O
2 mg of bromophenol blue (Sigma)
Store in aliquots up to 3 months at 20 C
DMEM/5% FBS or DMEM/5% NBCS medium
Add 6.25 ml of 10,000 U penicillin/10,000 g streptomycin (Pen/Strep), 25 ml fetal
bovine serum (FBS) or newborn calf serum (NBCS), and 50 l lter-sterilized 250
mg/ml Fungizone to 500 ml of Dulbeccos Modied Eagles Medium (DMEM).
In general, FBS is used to maintain cells to be passsaged while NBCS is used for cells to be
infected. While powdered DMEM may be used, premade liquid DMEM is preferred, since
it precludes the need for a large-scale lter-sterilization apparatus.
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Milk in KPBS, 5%
Dissolve 5 g nonfat dry milk in <100 ml KPBS (see recipe). Adjust volume to 100
ml with KPBS. Add 100 l of 20% (w/v) sodium azide. Prepare fresh.
Paraformaldehyde, 2.5%
Combine 3 ml of 10% (w/v) paraformaldehyde, methanol-free (store in dark at room
temperature) with 9 ml KPBS (see recipe). Prepare fresh.
Only make enough for the experiment to be performed.
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Propagation,
Quantication,
and Storage
14E.1.20
Current Protocols in Microbiology
199V medium
Combine the following in a sterile (autoclaved) 500-ml glass bottle:
430 ml sterile H2 O
50 ml 10 Hanks Balanced Salt Solution (HBSS; APPENDIX 2A or commercial supplier, e.g., Invitrogen)
6.25 ml 10,000 U penicillin/10,000 g streptomycin (Pen/Strep; Invitrogen)
5 ml 67 mM L-glutamine (0.5 g L-glutamine, e.g., Invitrogen, in 50 ml sterile H2 O)
Filter sterilize using 0.22-m lter
Add 9.35 ml 7.5% (w/v) NaHCO3 (7.5 g NaHCO3 in 80 ml sterile H2 O)
Stir and adjust volume to 100 ml
Filter sterilize in a biosafety hood using an 0.22-m lter
If color of the solution is not cherry red (pH 7.0), add sterile 10 M NaOH dropwise
to the bottle until the solution turns that color
Store up to 6 months at 4 C
Versene
Add 8.0 g NaCl, 0.2 g, KCl, 1.15 g Na2 HPO4 , and 0.2 g KH2 PO4 to 800 ml water. Stir
until all chemicals are in solution. Add 0.2 g disodium EDTA and adjust the volume
to 1 liter with water. Continue mixing until the EDTA is in solution. Autoclave,
cool, and store indenitely at 4 C.
COMMENTARY
Background Information
HSV-1 is the prototype of a family of highly
cytolytic viruses, the Herpesviridae, whose
lytic replication cycle ultimately leads to the
destruction of infected cells. HSV-1 is a neurotropic herpesvirus that causes a variety of
infections in humans ranging from benign le-
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HSV:
Propagation,
Quantication,
and Storage
Sterility
Standard sterile technique for cell culture
must be used when working with HSV. Microbial contamination of cells will cause signicantly reduced virus yields and may result
in contaminated virus stocks, which will then
be passed along to the next set of cells to be
used for infections. In the undesirable event
that the virus stock becomes infected with bacteria, yeast, or mold, the following may be performed to cure the contamination. Using the
spoiled virus sample, infect fresh cells to create
Anticipated Results
Infection of Vero or HEp-2 cells with HSV
will result in obvious CPE in less than a
24-hr period. When low-MOI infections are
performed (see Basic Protocols 1 and 2), obvious, well isolated infectious centers (plaques)
will be observed. When infections are performed at an MOI of 5 (see Basic Protocols 4 and 7), all cells in the monolayers
will display CPE. Representative results from
immunoblotting and indirect immunouorescence analyses of HSV-infected cells may be
found in Pomeranz and Blaho (1999, 2000).
14E.1.22
Current Protocols in Microbiology
Protocol
Operation
Time required
Basic Protocol 1
3 days
Basic Protocol 2
5 days
Basic Protocol 3
Plaque purication
1 week
Support Protocol 1
Splitting cells
45 min
Support Protocol 2
Freezing cells
45 min
Support Protocol 3
Thawing cells
15 min
Basic Protocol 4
3 days
Basic Protocol 5
Gel electrophoresis
2 hr
Basic Protocol 6
Immunoblotting
1 day
Basic Protocol 7
3 days
Time Considerations
One single-step replication cycle of HSV
takes between 12 to 18 hr for completion.
Therefore, all infections take a minimum of
one day to perform. In situations where multiple rounds of replication are required (see
Basic Protocols 4 and 7), it is necessary to
leave the infections for multiple days. The
immunoblotting and indirect immunouorescence procedures (see Basic Protocols 6 and
7) are multifaceted methods containing several subprotocols. Accordingly, these techniques require more than 1 day for completion. Convenient stopping points are noted in
each of these protocols. Time allotments for
all protocols in this unit are summarized in
Table 14E.1.3.
Literature Cited
Pomeranz, L.E. and Blaho, J.A. 1999. Modied
VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection.
J. Virol. 73:6769-6781.
Pomeranz, L.E. and Blaho, J.A. 2000. Assembly
of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22. J. Virol.
74:10041-10054.
Roizman, B. and Knipe, D.M. 2001. Herpes simplex
viruses and their replication. In Virology, 4th ed.
(D.M. Knipe and P.M. Howley, eds.) pp. 23992459. Lippincott-Raven, Philadelphia.
Key References
Internet Resources
http://www.stdgen.lanl.gov/stdgen
Web site maintained by the Los Alamos National
Laboratory Bioscience Division that includes compilation and analysis of molecular sequence information pertaining to sexually transmitted bacteria
and viruses. Dynamic graphics and extended analyses are available for all organisms. Annotation is
accomplished by a combination of automation and
hand review of each record.
http://darwin.bio.uci.edu/faculty/wagner
Home page of Dr. Edward Wagner, Professor of Microbiology and Molecular Genetics at the University of CaliforniaIrvine. It provides an introduction
to HSV. Here one can explore herpes virus research
with Dr. Wagner on the following topics: herpes
simplex virus (HSV); the HSV genome; HSV replication; use of DNA microarrays to analyze gene
expression in HSV-infected cells; microarray analysis of cellular transcript abundance as a function
of HSV infection; temporal patterns of HSV-2 transcripts; analysis of herpes simplex virus promoters;
structural properties of herpes simplex virus promoters; and latent infections by HSV.
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UNIT 14E.2
ABSTRACT
Epstein-Barr virus (EBV) was first reported as the etiological agent of Burkitts lymphoma
in 1964. Since then, EBV has also been associated with nasopharyngeal carcinoma, which
is highly prevalent in Southeast Asia, as well as infectious mononucleosis, complications
of AIDS, and transplant-related B cell lymphomas. This virus has further been linked with
T cell lymphomas and Hodgkins disease, establishing the concept of a wide spectrum
of EBV-associated malignant disorders. So far, there are a number of EBV-infected
cell lines established that can be induced for production of infectious viral progeny and
that facilitate the study of the mechanism of EBV-related infection, transformation, and
oncogenesis. This unit describes procedures for the preparation of EBV virion particles
and in vitro infection of cells with EBV. In addition, procedures for quantitation and
C 2007
storage of the virus are provided. Curr. Protoc. Microbiol. 6:14E.2.1-14E.2.21.
by John Wiley & Sons, Inc.
Keywords: Epstein Barr virus r induction r infection r quantitation r B lymphocyte
INTRODUCTION
This unit focuses on techniques for infection, propagation, quantitation, and long-term
storage of Epstein-Barr virus (EBV), as well as the immortalization of primary human
B lymphocytes in vitro by EBV. A series of procedures are detailed that will be valuable
to the novice as well as the more experienced virologist working with EBV. Numerous
details that have been incorporated into the protocols over time, which are now part of the
standard techniques used in working with EBV, are outlined. These techniques depend
on the healthy growth of cell cultures and the length of time that individual cultures are
kept growing or passaged for induction and harvesting of the virus. Most techniques
are provided in great detail so that they can be easily established elsewhere; varying
strategies are included, which can be of interest and can provide for some flexibility in
the labs that will use these protocols.
CAUTION: Epstein-Barr Virus is a Biosafety Level 2 or 2+ (BSL-2) pathogen. Follow
all appropriate guidelines for the use and handling of pathogenic microorganisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidified 37 C, 5% CO2
incubator unless otherwise specified.
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Current Protocols in Microbiology 14E.2.1-14E.2.21, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc14e02s6
C 2007 John Wiley & Sons, Inc.
Copyright
14E.2.1
Supplement 6
BASIC
PROTOCOL 1
EBV: Infection,
Propagation,
Quantitation and
Storage
Figure 14E.2.1 (A) Entrance to a Biosafety Level 3 (BSL-3) laboratory. (B) Laminar ow hood
with biosafety HEPA lter. (C) Ultracentrifuge with swinging-bucket rotor.
14E.2.2
Supplement 6
aspirated into the flask will be immediately lysed in hypochlorous acid (HClO). The
setup is illustrated in Figure 14E.2.1B.
Cells collected for induction should be resuspended in 10 ml complete medium and
supplemented with the final concentration of antibody mentioned above. The resuspended
cells should then be transferred to a culture flask and placed at 37 C in an incubator with
5% CO2 for induction. The lytic reactivation of the virus can take as long as 4 to 5 days,
but the induction of the lytic transactivator Zta can be seen within 24 to 48 hr post
induction. Virus can then be collected by harvesting the supernatant and concentrated by
high-speed centrifugation (Fig 14E.2.1C).
Materials
EBV-positive B cells (see Basic Protocol 7): Akata cells infected with GFP-tagged
EBV are recommended (Kanda et al., 2004; to obtain cells, contact Dr. Kanda at
tkanda@igm.hokudai.ac.jp)
Complete RPMI medium containing 10% FBS (see recipe)
Anti-IgG antibody
Centrifuge with swinging-bucket rotor
Additional reagents and equipment for harvesting EBV from cell supernatant
(Basic Protocol 5)
1. Suspend logarithmically growing EBV-infected Akata cells to a final concentration
of 510 106 cells/ml with fresh complete RPMI medium containing 10% FBS.
2. Add anti-IgG antibodies to a final concentration of 100 g/ml.
3. After 48 hr culture at 37 C, harvest cells by centrifuging for 5 min at 800 g,
room temperature. Retain the supernatant, which includes the virus (also see Basic
Protocol 5).
The Akata cells in the pellet are activated and produce viruses. They can be used to check
the expression of EBV early antigens by immunoblotting or immunofluorescence.
BASIC
PROTOCOL 2
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at 220 V and 975 F. Here, a specific protocol performed in the authors laboratory
will be described; however the initial amount of cells infected with EBV can be varied
depending on the amount of virus to be produced.
Materials
EBV-positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2
(generated in Dr. Erle Robertsons laboratory; erle@mail.med.upenn.edu)
Complete RPMI medium containing 10% FBS (see recipe) and serum-free RPMI
1640 medium (e.g., Invitrogen)
Phosphate-buffered saline (PBS; APPENDIX 2A)
1g/ml BZLF1 expression vector in TE buffer, pH 8.0 (APPENDIX 2A) or distilled H2 O
Centrifuge with swinging-bucket rotor
0.4-cm-gap electroporation cuvette
Gene pulser II system (Bio-Rad)
Additional reagents and equipment for harvesting EBV from cell supernatant
(Basic Protocol 5)
NOTE: B95.8, LCL1, and LCL2 are all EBV-positive B cells lines that can be used as a
resources for production of the virus, with no significant difference between them.
1. Collect 1.01.5 107 cells in exponential growth phase by centrifuging 5 min at
216 g, room temperature, in a swinging-bucket rotor.
2. Remove supernatant. Wash cells once by resuspending in PBS, then centrifuging
5 min at 216 g, room temperature, and removing the supernatant.
3. Gently resuspend the cells in 400 l serum-free RPMI 1640. Mix with 10 g of a
BZLF1 expression construct.
4. Transfer the cells to a 0.4-cm gap electroporation cuvette.
5. Electroporate the cells at 220 V and 975 F using a Bio-Rad gene pulser
(Fig. 14E.2.2).
EBV: Infection,
Propagation,
Quantitation and
Storage
14E.2.4
Supplement 6
Figure 14E.2.2
6. Transfer the cells into 10 ml complete RPMI medium containing 10% FBS. Incubate
the cells at 37 C in a 5% CO2 incubator for 3 to 5 days, allowing the production of
viral progeny.
7. Harvest cells by centrifuging for 5 min at 800 g, room temperature. Retain the
supernatant, which contains the virus. Also see Basic Protocol 5.
BASIC
PROTOCOL 3
TPA is a well-known activator of protein kinase C (PKC), which has been shown to
induce the reactivation of herpesviruses such as EBV and KSHV in certain latently
infected cells (Lazdins et al., 1987; Davies et al., 1991; Gradoville et al., 2002). In the
case of EBV, TPA activates viral lytic replication by inducing the expression of the major
viral intermediate-early gene BZLF1.
Materials
EBV-positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2
(generated in Dr. Erle Robertsons laboratory; erle@mail.med.upenn.edu)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Complete RPMI medium containing 10% FBS (see recipe)
2000 (40 g/g/ml) 12-O-tetradecanoyl-phorbol-13-acetate (TPA; Sigma),
sterilized by filtration through 0.22-m membrane
Centrifuge with swinging-bucket rotor
Additional reagents and equipment for harvesting EBV from cell supernatant
(Basic Protocol 5)
NOTE: B95.8, LCL1, and LCL2 are all EBV-positive B cells lines that can be used as a
resources for production of the virus, with no significant difference between them.
1. Harvest 1.0 106 EBV-positive cells by centrifuging 5 min at 216 g, room
temperature, in a swinging-bucket rotor.
2. Remove supernatant. Wash cells once by resuspending in PBS, then centrifuging
5 min at 216 g, room temperature, and removing the supernatant.
3. Resuspend the cells in 10 ml fresh complete RPMI medium containing 10% FBS.
Add 2000 (40 g/ml) TPA to a final concentration of 1 (20 ng/ml).
4. Incubate the cells at 37 C in a 5% CO2 incubator for 3 to 5 days, allowing the
production of viral progeny.
5. Harvest cells by centrifuging for 5 min at 800 g, room temperature. Retain the
supernatant, which contains the virus. Also see Basic Protocol 5.
BASIC
PROTOCOL 4
Materials
EBV-positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2
(generated in Dr. Erle Robertsons laboratory; erle@mail.med.upenn.edu)
Phosphate-buffered saline (PBS; APPENDIX 2A)
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BASIC
PROTOCOL 5
ISOLATING EBV
Harvesting of EBV from Cell Supernatant
The induction of EBV by the methods described in Basic Protocols 1 to 4 leads to lytic
replication to produce viral progeny. Eventually, the cells harboring EBV are lysed and
the infectious virions are released into the culture supernatant. A portion of the virion
pool, however, may also be trapped within cells that are not lysed, or may be associated
with cellular organelles. Usually, three cycles of freeze-thaw lead to further release of
virions from intact cells and organelles, and thus increase viral production (Lee et al.,
2001; Lan et al., 2005).
Materials
Cells induced for EBV replication (Basic Protocols 1 to 4)
Dry ice
Isopropanol
Centrifuge with swinging-bucket rotor
15-ml conical centrifuge tubes
Container suitable for resisting extremely cold temperatures
0.45-m syringe filter
1. Centrifuge induced cells 12 min at 300 g, room temperature.
2. Transfer most of the supernatant into a fresh 15-ml tube.
For instance, transfer 9 ml if the cell culture is 10 ml total.
EBV: Infection,
Propagation,
Quantitation and
Storage
3. Resuspend the cells in the remaining supernatant and perform three cycles of freezethaw in the following manner.
a. Add an appropriate amount of dry ice to a container capable of resisting extremely
cold temperature changes and mix with isopropanol at a 1:1 ratio.
14E.2.6
Supplement 6
Figure 14E.2.3 (A) Components used in this unit, including 0.45-m lter, different size syringes,
and collecting tube for ltering virus. (B) Filtering virus in a laminar-ow hood.
b. Incubate the tube containing cells in the dry ice/isopropanol mixture for 5 min.
c. Immediately transfer the tube to a 37 C water bath and incubate for 5 min or
until it is thawed.
d. Repeat the freeze-thaw cycle described in substeps b and c, above, three times.
e. Remove debris by centrifuging 10 min at 1000 g, room temperature. Recover
supernatant.
4. Combine the supernatant with the previous 9 ml (see step 2).
5. Filter the supernatant using a 0.45-m syringe filter (Fig. 14E.2.3).
BASIC
PROTOCOL 6
Materials
70% (v/v) ethanol
EBV-containing supernatant (Basic Protocol 5)
Phosphate-buffered saline (PBS; APPENDIX 2A)
10-ml ultracentrifuge tube
UV light source for sterilization
Ultracentrifuge with swinging-bucket rotor (Fig. 14E.2.1C)
Refrigerated centrifuge with fixed-angle rotor accommodating 1.7-ml
microcentrifuge tubes
1. Sterilize a 10-ml ultracentrifuge tube by overnight exposure to UV light.
2. Carefully wash out the ultracentrifuge rotor with 70% ethanol.
3. Transfer the supernatant into a 10-ml ultracentrifuge tube.
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Supplement 6
BASIC
PROTOCOL 7
Materials
Human B cell lines (suspension), human epithelial cell lines (adherent), or primary
human B cells (APPENDIX 4C)
Complete RPMI medium containing 10% FBS (see recipe)
Supernatant containing EBV infectious virions (Basic Protocol 5 or 6)
1 trypsin/EDTA solution (e.g., Invitrogen)
Complete DMEM medium containing 10% FBS (see recipe)
15-ml conical polypropylene centrifuge tubes
Centrifuge with swinging-bucket rotor
75-cm2 tissue culture flasks or 100-mm tissue culture plates
96-well tissue culture plate (optional; for producing lymphoblastoid cell line)
NOTE: Medium containing 10% FBS is not always necessary; lower serum concentrations have also been shown to work well for a number of cell lines.
2a. Wash the cells once by adding 10 ml PBS, centrifuging as in step 1, and removing
the supernatant.
3a. Resuspend pellet in 5 ml complete RPMI containing 10% FBS.
4a. Mix supernatant containing EBV virions with cells.
Although there is currently no method for titering EBV, based on the authors experience,
a quantity of virions collected from 1 107 induced EBV-infected cells should be used
to infect every 1 107 cells.
14E.2.8
Supplement 6
Figure 14E.2.4
system.
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7c. Plate the cells into a 96-well plate at 100 l for each well
The desired cell number is 20,000 per well.
8c. Incubate the cells at 37 C with 5% CO2 for 1 week, then add another 100 l fresh
medium to each well (for a total volume of 200 l).
9c. Every third day, aspirate out about half the medium from each well and replace
with fresh medium (Fig. 14E.2.4). Repeat this process for about 4 to 8 weeks
until lymphoblastoid cells are clearly visible by microscopy (see Fig. 14E.2.7; cells
typically look like a clump).
BASIC
PROTOCOL 8
Materials
EBV: Infection,
Propagation,
Quantitation and
Storage
14E.2.10
Supplement 6
Figure 14E.2.5
A9 cells (mouse B lymphocyte cell line used here as recipient cells; ATCC
#CRL-1811) grown as monolayer in 100-mm culture dish
47% (w/v) PEG (mol. wt. 8000)
Serum-free DMEM medium (e.g., Invitrogen)
Growth medium for A9 cells: complete DMEM medium containing 10% FBS (see
recipe)
1 trypsin/EDTA solution (e.g., Invitrogen)
EBV-A9 selection medium B (see recipe)
6-well tissue culture plates
15- and 50-ml centrifuge tubes
Centrifuge and plate carrier
8-, 5-, and 3-m pore-size Nucleopore polycarbonate filters (Whatman)
25-cm2 tissue culture flasks
100-mm tissue culture plates
Prepare EBV-infected human lymphoid cells
1. Wash exponentially growing EBV-infected and EBV-negative (control) human lymphoid cells once with serum-free RPMI 1640 medium. Dilute to 2.5 105 cells/ml
in the same medium.
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The EBV-negative cells cannot make microcells, which are associated with the presence
of the virus. The difference between the EBV-positive cells and EBV-negative controls will
become apparent at step 13.
Some cells, e.g., fibroblasts, are resistant to micronuclei induction by Colcemid treatment
(see below). In such a case, a different cell line should be used (Kelleher et al., 1998;
Kugoh et al., 1999).
16. Centrifuge 10 min at 400 g, room temperature, to pellet the purified microcells.
Resuspend pellet in 5 ml of serum-free DMEM medium containing 100 g/ml
phytohemagglutinin (PHA).
14E.2.12
Supplement 6
QUANTITATION OF EBV
Quantitation of EBV Virions by Quantitative PCR Using DNA
of Known Copy Number as Standard
BASIC
PROTOCOL 9
Epstein-Barr virus (EBV) virions can be quantified by determining the amount of DNA
encapsulated within the viral particles. Quantitation of virions based on the DNA content
is more accurate, compared to the conventional plaque assay; also, plaque assay has not
proven to be successful in quantitation of EBV virions. In order to isolate viral DNA,
virions are purified by ultracentrifugation as described previously. Since the centrifugation may also pellet proteins as well as other DNAs that may interfere with the exact
quantitation of the virus particles, these purified virions are subjected to further purification by density-gradient centrifugation. Density-gradient centrifugation is done either
on a linear sucrose or Nycodenz step gradient. Both gradients are equally good for the
purification of virions.
Materials
Concentrated EBV virion suspension (Basic Protocol 6)
30% and 60% (w/v) sucrose solutions
20% and 35% (w/v) Nycodenz (e.g., Sigma)
0.5 and 1 phosphate-buffered saline (PBS; see APPENDIX 2A for 1
concentration)
Lysis buffer (see recipe)
12:12:1 (v/v/v) phenol:chloroform:isoamyl alcohol
3 M sodium acetate, pH 5.2 (APPENDIX 2A)
100% ethanol, ice-cold
Primers for quantitative PCR (Table 14E.2.1)
DNA of known copy number as standard for quantitative PCR (Bookout et al.,
2006)
15-ml ultracentrifuge tubes
Gradient maker
Ultracentrifuge
18.5-G needles and syringes
MWCO 10,000 dialysis tubing, and dialysis clips
60 C water bath
Additional reagents and equipment for quantitative PCR (Bookout et al., 2006)
Purify virions
To purify concentrated virions on a linear sucrose gradient.
1a. Prepare a 10-ml linear 30% to 60% sucrose gradient in a 15-ml ultracentrifuge tube
using a gradient maker. Add 0.5 ml concentrated virion suspension to the top of the
gradient.
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Current Protocols in Microbiology
Supplement 6
2a. Centrifuge the tubes 2 hr at 30,000 g, 4 C, which separates the virions from
cell debris. Collect the band containing virions by puncturing the tube with 18.5-G
needle. Discard the remaining gradient solution as biohazard waste.
The band is generally located at the middle of the gradient and is clearly visible, resembling a white ring.
3a. Transfer the contents of the band into dialysis tubing and dialyze 3 to 6 hr against
4 liters of 0.5 PBS to remove sucrose. Pellet the purified virions by centrifugation
for 1 hr at 40,000 g, 4 C.
2b. Collect the band containing virions by puncturing the tube with 18.5-G needle.
Discard the remaining gradient solution as biohazard waste.
The band is generally located at the middle of the gradient and is clearly visible, resembling a white ring.
3b. Transfer the contents of the band into dialysis tubing and dialyze 3 to 6 hr against 4
liters of 0.5 PBS to remove the Nycodenz. Centrifuge 1 hr at 40,000 g, 4 C, to
pellet the virion particles. Discard supernatant as biohazard waste.
EBV: Infection,
Propagation,
Quantitation and
Storage
Regions
Bam C
GCAGGGCTCGCAAAGTATAG
TGCGGAAGTGACACCAAATA
Bam E
TACTGCCACCAGTACCACAACA
GGCCGACATTCTCCAAGATAA
EBNA2
CTCTGCCACCTGCAACACTA
ATTTGGGGTGCTTTGATGAG
LMP2
TGCAATTTGCCTAACATGGA
TGGACATGAAGAGCACGAAG
Bam W
CCAGACAGCAGCCAATTGTC
GGTAGAAGACCCCCTCTTAC
14E.2.14
Supplement 6
ALTERNATE
PROTOCOL
Alternatively, comparison of the Ct values (see Basic Protocol 9 and Bookout et al., 2006)
of viral DNA and a cell line carrying known copy number of the EBV genome may also
be used for the quantitation of virion. For example, Namalwa cells (request from Dr.
Erle S. Robertson; erle@mail.med.upenn.edu) carry two copies of EBV genome (Speck
et al., 2003), and thus can be used as a standard for quantitation of EBV virions. The
Ct value from episomal DNA of known number of Namalwa cells, multiplied by 2, will
represent the total EBV copies. Calculation based on the comparison of Ct values of
virion DNA with the Ct value of the control Namalwa EBV will give an approximate
number of virion particles (Fig. 14E.2.6).
Figure 14E.2.6 (A) Typical graph showing Ct value of DNA template used for amplication. Ct value for all four
samples is shown in their respective color. (B) Standard curve plotted based on the number of copies calculated
based on the Ct value.
Animal DNA
Viruses
14E.2.15
Current Protocols in Microbiology
Supplement 6
BASIC
PROTOCOL 10
Materials
EBV-infected or lymphoblastoid cells (see Basic Protocols 7 and 8)
Fetal bovine serum (FBS; APPENDIX 2A)
EBV-infected-cell freezing medium (see recipe)
Cryotubes
Liquid nitrogen or ultracold freezer at 140 C
1. Pellet 107 exponentially growing EBV-infected or lymphoblastoid cells and resuspend them in 1.5 ml FBS in a cryotube.
2. Add 0.5 ml of EBV-infected-cell freezing medium containing glucose and DMSO
to the cryotube. Mix by inverting a few times.
3. Place cryotube with cells in 80 C for 1 week, then transfer to liquid nitrogen or
ultracold freezer at 140 C.
BASIC
PROTOCOL 11
Materials
Frozen EBV-infected cells in cryotubes (Basic Protocol 10)
Growth medium appropriate for cells used (see Reagents and Solutions)
Circular floating tube rack (Bioexpress, http://www.bioexpress.com; cat. no.
R-8011-2)
15-ml conical polypropylene centrifuge tubes
Centrifuge
25-cm2 tissue culture flasks
1. Place the cryotubes in a bubble rack. Shake the rack to help thaw the cells, usually
for 1 to 2 min.
2. Transfer the sample to a 15-ml conical centrifuge tubes with 10 ml growth medium
using a 1-ml disposable pipet.
3. Centrifuge the cells for 5 min at 1200 g, room temperature. Aspirate the medium
to just above the cell pellet, wash the pellet again with 10 ml medium, then centrifuge
as before. Repeat the wash a total of three times.
4. Transfer the cell suspension to a 25-cm2 flask and incubate in a 37 C, 5% CO2
incubator.
It is recommended that 1.0 107 suspension cells be incubated in at least 3 ml complete
medium.
5. Increase the volume once cells clearly start to survive. Check daily for the first 5 days.
Supernatant containing EBV virus particles can be stored without adding DMSO. Unlike
the cells, supernatant containing the EBV virion does not require rapid thawing.
EBV: Infection,
Propagation,
Quantitation and
Storage
14E.2.16
Supplement 6
Lysis buffer
10 mM TrisCl, pH 8.5 (APPENDIX 2A)
1 mM EDTA, pH 8.0 (APPENDIX 2A)
1% (w/v) N-laurylsarcosine (Sarcosyl)
0.1 mg/ml proteinase K
Percoll microcell gradient mixture
46.7% (v/v) DMEM (e.g., Invitrogen)
10% (v/v) fetal bovine serum (FBS; APPENDIX 2A)
46.7% (v/v) Percoll (Sigma)
70 mM NaCl
Animal DNA
Viruses
continued
14E.2.17
Current Protocols in Microbiology
Supplement 6
24 mM HEPES pH 7.2
19 g/ml cytochalasin B
1 g/ml Colcemid
COMMENTARY
Background Information
Epstein-Barr virus (EBV) was the first
characterized human tumor virus (Epstein
et al., 1964). EBV is a ubiquitous human herpesvirus associated with numerous human
malignancies. EBV is predominantly associated with the infection of two target tissues in
vivo: B lymphocytes, where the infection is
predominantly latent, and oropharyngeal epithelial tissue, in which the infection is strictly
lytic, with the production of viral progeny
(Epstein et al., 1964; Chan et al., 2002; Chan
et al., 2004). Both target tissues are susceptible to EBV-associated malignant change, leading to tumors either of B cell origin, such as
Burkitts lymphoma (BL), Hodgkins disease,
and AIDS/transplantassociated immunoblastic lymphomas, or of epithelial cell origin, such
as nasopharyngeal carcinoma (NPC; Epstein
et al., 1964; Chan et al., 2002; Chan et al.,
2004; Hammerschmidt and Sugden, 2004).
EBV is a large, double-stranded DNA
virus that shares common structural features
with other herpesviruses. Following primary
lytic infection, this virus typically establishes
lifelong latent infection in the host (Miller,
1989). Once EBV establishes latency in cells,
it stays in the cells as a circular episome with
limited latent gene expressed (Miller, 1989).
To date, numerous cell lines have been generated which are stably infected with EBV. These
cell lines can be induced with either chemical
inducers or the EBV intermediate early gene
BZLF1 to produce viral progeny. They greatly
facilitate studies on EBV both in vitro and
in vivo.
EBV: Infection,
Propagation,
Quantitation and
Storage
A number of important variables can determine the extent to which the induction of
EBV will be successful. Careful monitoring of
the cell culture, the length of time in culture,
and the number of passages of the cultures
all play a significant role in the propagation
of EBV for storage. EBV has been successfully induced using Ig cross-linking (Basic
Protocol 1), expression of the intermediate
early transactivator BZFL1 from a heterologous promoter, or chemical inducers, which
include the phorbol ester TPA or a combination of TPA and sodium butyrate (SB). These
14E.2.18
Supplement 6
Figure 14E.2.7
EBV infection.
Lymphoblastoid cell line, (A) 4 weeks post EBV infection and (B) 8 weeks post
Anticipated Results
The protocols provided above to induce
EBV are all very straightforward. In general,
virus produced by induction of 1.0 107 EBVinfected cells using chemical inducers or Ig
cross-linking can yield more than 50% infection efficiency when used for infection of 1.0
107 permissive cells. Sometimes, induction
of EBV by transfecting EBV-infected cells
with a BZLF1 expression construct may not
be as efficient as use of chemical inducers or
Ig cross-linking, depending on the transfection efficiency. However BZLF1 transfection
can mimic natural viral reactivation. Retroviral
technique can greatly increase the transduction
of BZLF1.
To produce LCLs, 4 to 8 weeks are necessary (Fig. 14E.2.7). Usually, more than 50%
of wells will eventually form LCLs.
Time Considerations
As described in the protocol, induction of
EBV will take 3 to 5 days. Concentration, on
the other hand, will take 3 to 4 hr. Usually, a viral pellet is visible at the bottom of the tube after concentration of EBV by ultracentrifuging
the supernatant. Infection of cells also requires
3 to 4 hr, as described above, and the infection
efficiency varies depending on the permissiveness of the cell type and the amount of virus
added to the cells.
Literature Cited
Bookout, A.L., Cummins, C.L., Kramer, M.F.,
Pesola, J.M., and Mangelsdorf, D.J. 2006.
High-throughput real-time quantitative reversetranscription PCR. Curr. Protoc. Mol. Biol.
15.8.1-15.8.24.
Borza, C.M. and Hutt-Fletcher, L.M. 1998. EpsteinBarr virus recombinant lacking expression of
glycoprotein gp150 infects B cells normally but
is enhanced for infection of epithelial cells. J.
Virol. 72:7577-7582.
Cantaloube, J.F., Piechaczyk, M., Calender, A.,
Lenoir, G., Minty, A., Carriere, D., Fischer, E.,
and Poncelet, P. 1990. Stable expression and
function of EBV/C3d receptor following genomic transfection into murine fibroblast L cells.
Eur. J. Immunol. 20:409-416.
Carel, J.C., Frazier, B., Ley, T.J., and Holers, V.M.
1989. Analysis of epitope expression and the
functional repertoire of recombinant complement receptor 2 (CR2/CD21) in mouse and human cells. J. Immunol. 143:923-930.
Cen, H., Williams, P.A., McWilliams, H.P., Breinig,
M.C., Ho, M., and McKnight, J.L. 1993. Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody
response in solid organ transplant recipients
with posttransplant lymphoproliferative disorders. Blood 81:1393-1403.
Chan, A.T., Teo, P.M., and Johnson, P.J. 2002. Nasopharyngeal carcinoma. Ann. Oncol. 13:10071015.
Animal DNA
Viruses
14E.2.19
Current Protocols in Microbiology
Supplement 6
Chan, A.T., Teo, P.M., and Huang, D.P. 2004. Pathogenesis and treatment of nasopharyngeal carcinoma. Semin. Oncol. 31:794-801.
EBV: Infection,
Propagation,
Quantitation and
Storage
14E.2.20
Supplement 6
Animal DNA
Viruses
14E.2.21
Current Protocols in Microbiology
Supplement 6
UNIT 15A.1
ABSTRACT
Lymphocytic choriomeningitis virus (LCMV) is an enveloped, ambisense RNA virus
and the prototypic virus of the arenavirus group. It can cause viral meningitis and other
ailments in humans, but its natural host is the mouse. The LCMV/mouse model has
been useful for examining mechanisms of viral persistence and the basic concepts of
virus-induced immunity and immunopathology. This unit discusses strain differences
and biosafety containment issues for LCMV. Recommendations are made for techniques
for propagating LCMV to high titers to quantify it by plaque assay and PCR techniques
and to preserve its infectivity by appropriate storage. Curr. Protoc. Microbiol. 8:15A.1.1C 2008 by John Wiley & Sons, Inc.
15A.1.11.
Keywords: BHK cell
virus r PCR r plaque
INTRODUCTION
Lymphocytic choriomeningitis virus (LCMV) is the prototype virus of the arenavirus
group. It is an enveloped, ambisense RNA virus containing two RNA segments: L, which
encodes an RNA-dependent RNA polymerase and a zinc finger binding Z protein; and S,
which encodes a nucleoprotein (NP) and a glycoprotein precursor (GP0) that is cleaved
into two subunits, GP1 and GP2 (Welsh, 2000). LCMV causes a persistent infection in its
natural host, the mouse, but it is capable of infecting a wide range of animals, including
humans. LCMV is easy to isolate from the wild, and many strains have been isolated.
Most studies in scientific laboratories have focused on derivatives of three isolates originating in the early 1930s: the Armstrong strain, isolated from a monkey undergoing a
lymphocytic choriomeningitis (hence the name); the Traub strain, isolated from a laboratory colony of persistently infected mice; and the WE strain, isolated from a human
after exposure to persistently infected mice. The Armstrong strain is sometimes referred
to as neurotropic, whereas the Traub and WE strains are sometimes referred to as
viscerotropic. The neurotropic designation is a confusing misnomer because each
of these strains can grow well in the brain. However, high levels of viral replication
in the viscera seem to either distract or clonally exhaust T cells, preventing a strong
T celldependent meningitis and encephalitis from occurring. LCMV has been an important model for studying T celldependent pathology in the brain.
Many variants of these strains also exist. Notably, the clone 13 derivative of the Armstrong
strain and the docile derivative of the WE strain seem to replicate better in mice than
their respective parent strains and are more likely to cause the clonal exhaustion of T
cells by high antigen load (Moskophidis et al., 1993; Zajac et al., 1998). Escape variants
bearing mutations in Tcell or antibody epitopes have been generated (Lewicki et al.,
1995; Ciurea et al., 2000). Recently, reverse genetic techniques for LCMV have been
developed, making it possible to do sophisticated molecular studies and to generate
recombinants between LCMV and other viruses (Lee and de la Torre, 2002).
Current Protocols in Microbiology 15A.1.1-15A.1.11, February 2008
Published online February 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc15a01s8
C 2008 John Wiley & Sons, Inc.
Copyright
Animal RNA
Viruses
15A.1.1
Supplement 8
LCMV is considered an Old World arenavirus and may have originated in Africa. It
is closely related to Lassa virus, which causes severe and potentially lethal infections
of humans in West Africa. LCMV has disseminated throughout the world in its Mus
musculus host.
CAUTION: Prior to undertaking any experiments outlined in this unit, the researcher
should read and understand the information presented in the Safety Considerations
section.
CAUTION: Established strains of LCMV are considered Biosafety Level 2 (BSL-2)
pathogens. Follow all appropriate guidelines and regulations for the use and handling of
pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
CAUTION: New human or field isolates of LCMV are considered Biosafety Level 3
(BSL-3) pathogens. Follow all appropriate guidelines for the use and handling of
pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B)
for more information.
CAUTION: Follow all appropriate guidelines and regulations for the use and handling
of human-derived materials. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
SAFETY CONSIDERATIONS
LCMV can cause persistent or acute infections in animal colonies and is a threat to
rodents and primates in veterinary facilities. Therefore, it is best to keep LCMV-infected
animals well separated from other animals. Most unexpected infections in animal facilities with LCMV have come when the source of LCMV was unknown, e.g., from
an unknowingly contaminated cell line or animal that entered the facility. Usually an
LCMV-infected animal colony can be safely maintained with appropriate containment,
e.g., a biocontainment suite under negative pressure with a two-door autoclave, allowing
one to autoclave the bedding material on its way out of the isolated area. It is important
to have a facility with two or more exit doors so that animals can never escape.
LCMV is also a human pathogen, and in some areas 5% of the human population is
seropositive (Welsh, 2000). The agent can cause a variety of syndromes, from malaise to
meningitis or encephalitis. Death from LCMV infection is exceedingly rare, and patients
nearly always recover without sequellae. The recommended biosafety level for LCMV
strains has been ambiguously listed by the NIH/CDC as BSL-2 or BSL-3. At one time
the neurotropic strains were considered BSL-3, presumably because they were thought
to be a greater hazard to humans. That, however, was an unfortunate characterization
because the neurotropic strains are no more neurotropic to humans and certainly no more
infectious in humans than the viscerotropic strains. They simply cause meningitis and
encephalitis more easily in the mouse because they grow relatively poorly throughout
the host, and the T cells are more free to attack the brain. The neurotropic Armstrong
strain is now considered a BSL-2 agent, along with the viscerotropic agents.
LCMV:
Propagation,
Quantitation, and
Storage
There are no reliable quantitative data on the relative threats of the different strains to
humans. Anecdotally, the authors have heard of more infections in laboratory personnel
of strain WE origin than of strain Armstrong origin, although it is possible that the clone
13 variant of Armstrong may be more virulent, as it has caused laboratory infections. The
NIH/CDC manual Biosafety in Microbiological and Biomedical Laboratories (BMBL)
5th edition now recommends BSL-2 for most procedures with most strains, but it recommends BSL-3 practices for new human or field isolates or for procedures that would
result in high-titer aerosols.
15A.1.2
Supplement 8
PROPAGATION OF LCMV
The LCMV -dystroglycan receptor is a ubiquitous protein expressed in many cell types
and in different species, and the virus can consequently grow in a wide variety of cell
types from many species, including mouse, hamster, monkey, and human (Cao et al.,
1998). The best yields are from fibroblast or epithelial cell lines because it grows poorly
in lymphocytes. Very good yields, 23 108 plaque forming units (pfu) per ml, can be
obtained in cultures of baby hamster kidney cells, specifically BHK21, which do not shed
any endogenous retrovirus (like many mouse cell lines) that may contaminate the end
product. Good titers can be obtained at 48 hr by inoculating monolayers (or suspension
cultures, an option with BHK21/13s cells) with a multiplicity of infection (MOI) of 0.03
to 0.1 pfu/cell or at 72 hr with an MOI of 0.003 to 0.01 pfu/cell. The advantage of a
low-multiplicity infection is that it reduces the problem of viral interference and gives rise
to high-titer stocks. For optimal yields these cells should be in a highly active metabolic
state, and the monolayer should be 50% to 75% confluent at the time of infection.
BASIC
PROTOCOL 1
Materials
Baby hamster kidney (BHK) cells, lines 21 or 21/13s
BHK propagation medium
Lymphocytic choriomeningitis virus (LCMV; MOI of 0.03 to 0.1 pfu/cell for
harvesting after 48 hr or MOI of 0.003 to 0.01 pfu/cell for harvesting after 72 hr)
75-cm2 (T-75) or 150-cm2 (T-150) tissue culture flasks, or tissue culture roller
bottles
Plastic centrifuge tubes of appropriate size
Refrigerated centrifuge, 4 C
Procedure
1. Select an appropriate size plastic tissue culture vessel for propagation, depending on
the volume to be harvested.
These may be T75 or T150 flasks, or roller bottles for larger scale propagation.
2. Seed flasks with BHK cells in BHK propagation medium. Use 30 to 40 ml/T-75
flask, 60 ml/T-150 flask, or 150 ml/roller bottle and incubate.
BHK cells divide quickly and can undergo up to two divisions a day at 37 C, 5% CO2 .
The specific cell density to initiate the cultures should probably be empirically derived,
due to differences in the rates of divisions of different cell preparations.
3. When vessels are 50% confluent, decant the culture fluid and infect with virus in a
limited volume (3 ml/T-75, 6 ml/T-150, 25 ml/roller bottle). Occasionally tilt flasks
during the infection period and have roller bottle turning at about 1.5 rpm.
4. After 1 to 1.5 hr, add BHK propagation medium to previous levels and incubate 2 to
3 days (depending on MOI).
LCMV does not cause substantial lysis of cells visible as cytopathic effect in these cultures,
but there will likely be a reduction in cell density compared to uninfected control cultures.
Animal RNA
Viruses
15A.1.3
Current Protocols in Microbiology
Supplement 8
There may be many BHK cells in the culture fluid, and a second and similar centrifugation
might be required to clear the culture fluid.
6. Keeping the virus cold at all times after the harvest, dispense the culture fluid into
aliquots and store indefinitely at 70 C.
BASIC
PROTOCOL 2
Materials
Vero cells (African green monkey kidney cells; ATCC)
Eagles MEM with 10% (v/v) heat-inactivated (56 C for 30 min) fetal bovine
serum (FBS)
Lymphocytic choriomeningitis virus (LCMV) sample to be tested
Vero cell propagation medium (see recipe)
Phosphate-buffered saline (PBS; APPENDIX 2A)
2 LCMV plaque assay medium (see recipe)
1% (w/v) Seakem agarose-ME (Lonza), recently boiled (in a microwave)
1% (w/v) neutral red (aqueous solution); store up to several months at 4 C
6-well petri plates
96-well microtiter plates
Platform rocker
Prepare cells and virus
1. Seed 5 105 Vero cells onto six-well petri plates in 4 ml/well Eagles MEM with
10% FBS and incubate.
2. When the monolayers are 80% confluent in 1 to 2 days (depending on how fast the
cells are growing), decant the medium and replace with 1 ml fresh medium
3. Prepare a series of 10-fold dilutions of the test virus sample in Vero cell propagation
medium
For large scale titrations this can be done in 96-well microtiter plates by serially transferring 20 l of inoculum into wells containing 180 l medium, changing pipet tips with
each transfer.
Infect cells
4. When dilutions are complete, add 100 l to the Vero cell monolayers, starting with
the most dilute sample and using the same pipet for increasing concentrations.
5. Incubate plates 60 to 90 min, with gentle rocking every 20 to 30 min.
6. Optional: For best accuracy, after this adsorption/penetration period, remove the
medium and wash the monolayers with PBS.
LCMV:
Propagation,
Quantitation, and
Storage
This will synchronize the infection and will result in more homogenous plaque sizes.
However, this is an additional step that increases the possibility of contamination and
may not be necessary for most experiments.
15A.1.4
Supplement 8
Visualize plaques
7. Prepare an agarose overlay by combining equal volumes of 2 LCMV plaque assay
medium with recently boiled 1% agarose-ME solution in water that has cooled in a
42 C water bath.
This will be about 10 min after the boiling step. The agarose solution can also just be left
on the bench top and judged satisfactory to use by it not being uncomfortable to touch by
ones inner wrist.
8. To each monolayer add 4 ml agarose overlay. Allow medium to gel for 15 min.
9. Incubate the plates 4 days.
10. Stain the plates with 1.5 ml of a 1:10,000 dilution of neutral red (from a 1% aqueous
solution) made up in 1:1 2 LCMV plaque assay medium/1% agarose and incubate
overnight.
This is the same medium as that used for the overlay (step 7), but with neutral red added.
Neutral red is self-sterilizing, and a 1% solution can be made up in double-distilled water
and be satisfactorily kept in the refrigerator for several months.
If the neutral red starts crystallizing into clumps, avoid adding any clumps onto the plates
because they may kill the cells.
Plaques should be visible the next day.
Analyze results
11. To calculate a titer in plaque-forming units (pfu)/ml, select a petri well with a
sufficient number of plaques to give a reliable count and not too many plaques
(where they would be superimposed on each other).
For example, choose plates with 20 to 70 plaques.
BASIC
PROTOCOL 3
Animal RNA
Viruses
15A.1.5
Current Protocols in Microbiology
Supplement 8
the end of the reaction, the qPCR machine brings the mixture to a temperature that is
suitably below the expected Tm of the resultant product generated. The machine then
increases the temperature of the reaction by half-degree intervals with a fluorescence
capture after each half-degree increase. This will result in a melt curve that can then be
used to confirm the Tm of the resulting product. This final step helps to ensure that a
single expected product is generated during the reaction.
The other method used to quantify DNA product takes advantage of the 5 -exonuclease
activity of Taq polymerase and fluorescent resonant energy transfer (FRET). A singlestrand oligo is designed to anneal to a sequence within the product generated during
the PCR reaction. This oligo has been labeled with two fluorochromes, typically a
higher-energy fluorochrome designated the reporter at the 5 end, and a lower-energy
fluorochrome designated the quencher at the 3 end. The oligo is designed to have a
higher Tm than the primers, as the oligo must be 100% hybridized to the PCR product
for the assay to be accurate. While the fluorochromes are within close proximity, no
fluorescence is observed, as the fluorescence of the reporter is quenched by the 3
quencher; but as Taq DNA polymerase degrades the oligo, the reporter and quencher are
separated, and fluorescence emission is observed.
In this protocol two quantitative PCR reactions are performed, one using LCMV primers
and an LCMV-specific oligo, and one using beta-actin (normally produced by these
cells) and SYBR Green I. The beta-actin qRT-PCR is done to control for variations that
may occur during the RNA isolation or reverse transcription steps. Both reactions are
performed using the same cDNA sample. Quantification during the qRT-PCR reaction is
only possible with use of a dilution series of a standard to compare the relative amount
of product generated during the reaction. In other words, qRT-PCR cannot be used to
directly quantify RNA levels, but is only useful as a method of comparison by way of
quantification against a standard. In the following protocol, a qRT-PCR is described that
measures relative LCMV RNA levels using the oligo method for LCMV, as initially
reported by Roberts et al. (2004). This is then compared to a beta-actin qRT-PCR using
primers initially reported by Miller et al. (2004) using SYBR Green.
Materials
Test sample (cells infected with LCMV)
SuperScript First-Strand Synthesis for RT-PCR (Invitrogen)
Double-distilled (dd) H2 O
500 mM Tris buffer
5 g/l bovine serum albumin (BSA)
30 mM MgCl2
2.5 mM (each) dNTPs
SYBR Green I nucleic acid stain, 10,000 concentration (Molecular Probes)
10 M LCMV GP forward primer: 5 -TGC CTG ACC AAA TGG ATG ATT-3
10 M LCMV GP reverse primer: 5 -CTG CTG TGT TCC CGA AAC ACT-3
10 M beta-actin forward primer: 5 -CGA GGC CCA GAG CAA GAG AG-3
10 M beta-actin reverse primer: 5 -CGG TTG GCC TTA GGG TTC AG-3
1 M fluorescein (Bio-Rad)
10 M LCMV Taq Man MGB oligo: 6FAM-TTG CTG CAG AGC TT MGBNFQ
(Applied Biosystems)
5 U/l Taq DNA polymerase (Promega)
LCMV:
Propagation,
Quantitation, and
Storage
15A.1.6
Supplement 8
1. Extract RNA from the test sample using 2-ml Eppendorf Phase Lock Gel tubes
according to current manufacturers protocol.
2. Convert RNA to cDNA using the Invitrogen SuperScript First-Strand Synthesis
System for RT-PCR (also see UNITS 16D.3 & 16F.1).
3. On ice, prepare the following two PCR reaction mixes (one for LCMV and one for
beta-actin):
4. Place two 50-l aliquots (i.e., duplicates) of the reaction mixture into an iCycler iQ
PCR plate and seal with iCycler iQ Optical Tape.
5. Carry out the PCR reactions.
a. To measure beta-actin cDNA: Carry out the following program for beta-actin
real-time PCR protocol in an iCycler iQ real-time PCR detection system:
Initial step:
40 cycles:
Final step:
2 min 30 sec
30 sec
25 sec
25 sec
indefinite
95 C (denaturation)
95 C (denaturation)
62 C (annealing)
72 C (extension)
72 C (hold).
b. To measure LCMV GP cDNA: Carry out the following program for LCMV realtime PCR protocol in an iCycler iQ real-time PCR detection system:
Initial step:
40 cycles:
Final step:
2 min
15 sec
1 min
indefinite
50 C
95 C (denaturation)
60 C (annealing and extension)
60 C (hold).
Analyses of data obtained are provided in the Commentary section (e.g., see Critical
Parameters, qRT-PCR).
Animal RNA
Viruses
15A.1.7
Current Protocols in Microbiology
Supplement 8
BASIC
PROTOCOL 4
STORAGE OF LCMV
LCMV is a heat-labile, enveloped virus that needs to be kept cold, or it will rapidly lose
infectivity. Storage should be at 70 C, preferably in the presence of some protein, such
as 10% fetal bovine serum, which will enhance stability. Even with that, there will be
an 50% loss in titer with each freeze-thaw cycle. Virus purified away from protein
contaminants will be extremely unstable with a freeze-thaw, unless the purified virus is
at a sufficiently high protein concentration to stabilize itself. When thawing the virus, do
so quickly at 37 C and immediately put on ice.
LCMV:
Propagation,
Quantitation, and
Storage
Quantifying LCMV
Many techniques for quantifying RNA levels using PCR approaches are currently available, and two such workable techniques are
suggested in this unit. It is also possible to
use probe sets that allow quantification of two
cDNA products within the same tube (different fluorescent outputs, i.e., multiplexing), but
this protocol has not been tested within our
laboratory at the time of this writing. The
primers shown in this unit for the qRT-PCR
assay are designed to quantify LCMV GP1-
15A.1.8
Supplement 8
Critical Parameters
Autointerference
Of major importance in the generation
of high-titer virus stocks is avoidance of
the problem of autointerference (Welsh and
Oldstone, 1978). LCMV very rapidly generates defective interfering (DI) viruses, especially late in its infecting cycle, where the ratio of DI to standard viruses increases. These
DI viruses will greatly interfere (as much as
1000-fold) with the propagation of standard
virus if they are present in the inoculum.
To prevent the accumulation of DI virus in
seed stocks, these seed stocks should be initiated at a low multiplicity of infection and harvested just prior to the peak of standard virus
synthesis. Serial passage of virus should always be with a diluted inoculum, because otherwise the DI virus proportion will increase.
Similarly, for the propagation of high titer
virus for purification, the carefully prepared
seed stock should also be diluted, to ensure
that cells are not initially co-infected with DI
and standard virus. High-multiplicity infections (e.g., MOI = 1 to 10) rarely produce
high-titer stocks, unless the seed stock is virtually devoid of DI virus, and that is a rarity.
The presence of DI viruses in stocks can often be detected in plaque assays, where, at high
concentrations of virus the monolayers may
look normal, without detectable cytopathic effect. Under those conditions plaques will be
seen on monolayers receiving a more diluted
inoculum, where there is a lower likelihood
that a cell will be co-infected with a standard
and a DI virus. LCMV, under optimal conditions, should grow to titers of 12 108
pfu/ml.
High titers of virus can also be obtained
by freeze-thawing or sonically disrupting cells
because more than half of the infectivity in a
culture may be cell-associated, but this virus
will be heavily contaminated with cell debris
and may not be useful in some types of experiments.
Host cells
BHK cells. For optimal LCMV proliferation and plaque assays, cells must be in a
suitable and healthy condition. Our subjective
(though not quantitatively assessed) observation is that viral yields are poor if BHK cells
are heavily confluent prior to seeding vessels
with cells for the production of viral stocks,
and that seeding the vessels with cells previously growing in log phase is more suitable.
Also, waiting for BHK cell monolayers to be
confluent before infection seems to reduce viral yield. BHK cells metabolize media very
quickly, so limiting the amount of medium in
order to increase the viral concentration may
not be wise because it is likely to reduce the
numbers of healthy BHK cells and the subsequent production of virus.
Vero cells. For plaque assays it is important
to use Vero cells that have been carefully maintained in culture and have not been allowed
to overgrow vessels during confluence. Vero
cells sometimes alter their growth characteristics and become less useful in plaque assays
after continuous passage in culture. Avoid mycoplasma contamination (see APPENDIX 3B).
qRT-PCR
The RNA levels of each of the tissue samples in each of the qPCRs are quantified by
use of a standard. It is critical that quantification of RNA levels is performed at early cycles
during the PCR reaction. At earlier cycles, all
PCR reagents are in excess and the reaction occurs in an exponential manner, with a doubling
of PCR product occurring after each cycle. As
the reaction progresses, reagents become limiting, and different samples will generate different amounts of product at each cycle. The
amount of relative LCMV RNA calculated is
then divided by the amount of relative betaactin RNA calculated, and this number will be
a relative measure of how much LCMV RNA
is present in each sample.
Troubleshooting
Propagation of LCMV
BHK cells do not stick to roller bottles.
Slow down the RPM, especially during the initial seeding of the vessels. BHK cells slough
off from monolayers into culture fluid. This is
common for the BHK21/13s cell line, which
can be adapted to suspension culture. Usually
this does not pose any problems for generating high viral titers, but they should be cleared
from the culture fluid by centrifugation before
aliquoting virus.
Low viral titers. Viral titers of <3 107
pfu are suboptimal; this could be caused by not
harvesting the culture fluid at the peak of viral
production, which may need to be empirically
determined by titrating virus at different times
after infection. It should be noted that viral
titers sharply decline within 12 hr of the peak
titer, even though the cells remain with only
mild cytopathic effects, such as reduced cell
growth. Low viral titers can also be caused by
inoculating monolayers with too high a dose
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15A.1.9
Current Protocols in Microbiology
Supplement 8
of virus stock containing DI virus (see Critical Parameters, Autointerference). This can
be overcome by generating seed stocks from
very low dose inocula (MOI = 0.001 to 0.01
pfu/cell) and harvesting just before the peak in
titer, because thereafter, DI virus will accumulate. Dilutions of this stock could then be used
as inocula for the production of high-titer viral
stocks.
Quantitation of LCMV infectious units by
plaque assay
Plaques show abnormal morphology.
Sometimes LCMV plaques develop concentric rings with a bulls eye effect. These are
more readily apparent when incubator CO2
levels are 5% or higher and less apparent at
lower CO2 concentrations.
Plaques are hard to read. This can happen
when cell monolayers or media are suboptimal; sometimes plaques become clearer if the
stained plates are left in the incubator for an
extra day or two. The contrast in the plaque
assay can also be enhanced by adding a 0.5 ml
concentrated acetic acid on top of the gel in the
plaque assay plate. In 5 to 10 min the staining
will be briefly enhanced, and the plaques will
need to be counted immediately because the
cells will soon die thereafter.
Vero cells slough off monolayer. This can
be due to contamination with mycoplasma or
to a drift in the nature of Vero cells with continuous passage: use new or earlier passage Vero
cells. The effect could also be due to problems
with the medium or incubator. If the agarose
is repeatedly boiled and used, it will become
more concentrated due to evaporation of water, and the more concentrated agarose can be
toxic for cells.
qRT-PCR
It is suggested that all reagents purchased
for the qRT- PCR are of molecular biology
grade, i.e., DNase and RNase free. All molecular biology work should be done using sterile aerosol filter tips because small amounts
of a contaminating product are quickly amplified during a PCR. The MgCl2 concentration
within the PCR may need to be empirically adjusted. It is known that small variations within
the amount of MgCl2 may drastically alter the
efficiency of the PCR. The parameters for PCR
amplification listed (times and temperatures)
may be further optimized because these may
vary from machine to machine.
LCMV:
Propagation,
Quantitation, and
Storage
Time Considerations
Infection of cells with virus can be accomplished within 2 hr. Harvest of virus from culture fluid as well as dispensing into aliquots
can be done within 1 hr, including centrifugation steps. Infection of plaque assay plates,
incubation, and overlay with medium/agarose
can be completed within 2 hr. RNA isolation,
if done according to the manufacturers protocol, takes about 3 hr. Conversion to cDNA
takes 2 to 3 hr, and setup for the PCR takes
2 to 3 hr, with the actual reaction time varying depending on the setup of the machine
(typically 2 to 4 hr).
Literature Cited
Anticipated Results
15A.1.10
Supplement 8
H., and Zinkernagel, R.M. 2000. Viral persistence through selection of neutralizing antibodyescape variants. Proc. Natl. Acad. Sci. U.S.A.
97:2749-2754.
Lee, K.J. and de la Torre, J.C. 2002. Reverse
genetics of arenaviruses. Curr. Top. Microbiol.
Immunol. 262:175-193.
Lewicki, H.A., Tishon, A., Borrow, P., Evans, C.F.,
Gairin, J.E., Hahn, K.M., Jewell, D.A., Wilson,
I.A., and Oldstone, M.B. 1995. CTL escape viral
variants. 1. Generation and molecular characterization. Virology 210:29-40.
Miller, A.T., Wilcox, H.M., Lai, Z., and Berg, L.J.
2004. Signaling through Itk promotes T helper 2
differentiation via negative regulation of T-bet.
Immunity. 21:67-80.
Moskophidis, D., Lechner, F., Pircher, H., and
Zinkernagel, R.M. 1993. Virus persistence in
acutely infected immunocompetent mice by
exhaustion of antiviral cytotoxic effector cells.
Nature 362:758-761.
Animal RNA
Viruses
15A.1.11
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Supplement 8
UNIT 15B.1
Sindbis virus is able to infect a wide variety of vertebrate and invertebrate cells. In
nature, this virus demonstrates a complex life cycle, moving from the mosquito vector
into the mammalian host, which in turn reinfects the mosquito vector. In cell culture,
Sindbis virus produces lytic infections in mammalian cells while establishing persistent
infections in cultured mosquito cells, as in the infected mosquitoes. The mechanism
for the establishment of persistence in mosquito cells is not well understood, making
this virus system a good model for the study of viral persistence. The tool of reverse
genetics (in which an RNA genome is reverse-transcribed into cDNA) has become an
invaluable tool for the study of this virus by producing many types of mutations in the
viral cDNA. Careful genetic analysis of mutants produced by this or any other mutagenic
method should be done using both mammalian and insect cell systems to assure that
the installed mutation is not a host-range-specic mutation (Hernandez et al., 2003). In
general, both host systems can be used with relative ease. The specic choice of cell
line for the propagation of virus is determined by the type of study required. Mammalian
cells lend themselves to metabolic labeling with radiolabeled isotopes while mosquito
cells do not. Both host cell types can be used as indicator cells for virus titration by
plaque formation. Insect cells have been used for the production of host-range mutants
that do not grow abundantly in mammalian cells (Hernandez et al., 2003). These host
range mutants were titered on both mammalian (baby hamster kidney, BHK) and insect
(C7-10) cell lines to determine any change in infectivity of the mutant on one cell line
or another. Mammalian and mosquito cells are genetically and biochemically distinct.
These divergent hosts of Sindbis virus respond distinctly to drug treatments (ScheefersBorchel et al., 1981). In one particular study, the block to Sindbis virus production by
the drug chloroquine could only be demonstrated in mosquito cells (Hernandez et al.,
2001).
Cell propagation methods will be described for BHK (see Basic Protocol 3), chicken
embryo broblast (CEF; see Support Protocol 2), and mosquito cell cultures (see Support
Protocol 4). While many different vertebrate cell types are used for the propagation of
Sindbis virus, infection and transfection of baby hamster kidney (BHK) cells will be
described in detail (see Basic Protocol 1 and Alternate Protocol 1). Primary cultures of
chicken embryos (CEF) are also widely used for the cultivation of the virus and will also
be discussed. The BHK protocols are easily adaptable to the infection of other mammalian
cells. The most common insect cells in use include mosquito Aedes albopictus isolates
originating from the virus-free cell line originally prepared by Singh (Singh, 1967; Brown
et al., 1976). Cells provided by Sonya Buckley (Yale Arbovirus Research Unit) from the
original Singh line have been used in the authors laboratory for the clonal selection
of a subline U4.4. Additional clonal isolates derived from the Singh cultures include
the C6/36 and C7-10 cell lines. The C6/36 isolate was derived by Igarashi (1978) and
provided by Kenneth Ekels (Walter Reed Army Institute). C7-10 cells were derived
from LT-C-7 cells (Sarver and Stollar, 1977) and provided by Victor Stollar (Rutgers
Medical School). The appropriate mosquito cell line to use for specic applications is
determined by the investigator, because each of these cell lines responds differently to
infection by Sindbis virus. The U4.4 and C6/36 cultures do not display cytopathic effect,
while the C7-10 cultures will display signicant cytopathology prior to establishment of
a persistent infection. Both mammalian and insect cell cultures will produce wild-type
Sindbis virus titers in the range of 109 to 1010 pfu/ml. The cell lines maintained in this
Animal RNA
Viruses
Contributed by Raquel Hernandez, Christine Sinodis, and Dennis T. Brown
Current Protocols in Microbiology (2005) 15B.1.1-15B.1.34
C 2005 by John Wiley & Sons, Inc.
Copyright
15B.1.1
laboratory have not been deposited with the ATCC but are available from the authors upon
request (rhernan2@unity.ncsu.edu).
All methods employing mosquito cell propagation (see Support Protocol 4), infection (see
Basic Protocol 4 and Alternate Protocol 2), and transfection (see Alternate Protocol 3) are
identical for each of the different insect cell lines, except where noted. Virus freezing and
storage enables the production of large quantities of virus stock, which remains viable for
over 10 years when kept frozen. The stability of frozen virus facilitates the collection of
samples from multiple experiments, which can subsequently be evaluated for virus titer
by plaque assay. Plaque assays using mammalian (see Basic Protocol 3) or insect cell
monolayers (see Basic Protocol 5) are routinely used for quantitation of infectious Sindbis
virus and will be discussed in detail. Plaque purication of Sindbis virus is described in
Basic Protocol 6.
BASIC
PROTOCOL 1
Materials
Sindbis Virus:
Propagation,
Quantication,
and Storage
Baby hamster kidney (BHK) cells (ATCC #CCL10), growing in culture (see
Support Protocol 1)
1 complete EMEM medium (see recipe)
Sindbis virus (SVHR) stock, titered (see Basic Protocol 3)
1 PBS-D (see recipe for 10) containing 3% FBS (heat-inactivated at 56 C; see
APPENDIX 2A)
Glycerol, sterile
Liquid nitrogen
15B.1.2
Current Protocols in Microbiology
Platform rocker
25- or 75-cm2 culture asks (the authors have found that vented asks are less
susceptible to contamination with mold than the standard asks)
15- or 50-ml conical polypropylene centrifuge tubes, sterile
NOTE: It is standard practice to grow a stock of virus from which additional virus
stocks will be grown prior to any additional work with the virus. This practice avoids
the production of defective interfering particles which will accumulate upon successive
serial passage of high concentrations of virus. Generally, an MOI of 0.01 is required for
production of this stock virus, referred to as the stock stock, i.e., the primary source
from which stock virus is grown. This stock stock does not need to be gradient puried.
In the event that it is suspected that the virus source is contaminated by other virus strains
or by mutants, plaque purication may be required. Generally, infection at an MOI of
0.01, which is the method used to replenish the stock stock, is sufcient to suppress the
production of defective interfering particles. Stock virus may be produced from any cell
line of choice; stock virus grown in BHK cells yields a high titer and has the advantage
that the progress of infection can be followed by evaluating the cytopathic effect.
NOTE: Sindbis virus is very sticky and will bind to glass and plastic surfaces. The authors
have found that Corning polypropylene tubes (microcentrifuge tubes, self-standing, and
conical) bind less virus than other brands of plastic.
1. Subculture BHK cells in 1 complete EMEM medium the day prior to infection in
75- or 25-cm2 asks such that the monolayer will be 90% conuent at the time of
infection.
The subculture ratio will differ with individual cell lines, depending on the level of
metabolism. The subculture dilution should produce a 90% conuent monolayer within
24 hr. The approximate number of mammalian cells should be calculated as in step 2,
below.
Determination of the degree of conuence is highly subjective and the assessment will
differ from person to person. In general, the cells should cover 90% of the culture surface
area to be described as being 90% conuent.
2. Calculate the amount of virus needed for the desired multiplicity of infection (MOI)
according to the following formula.
no. cells MOI = pfu needed
This requires knowledge of the number of cells in the culture vessel to be infected. For
example, when calculating the MOI for BHK cells in a 90% conuent 75-cm2 ask, the
number of cells is 2 107 cells. A 90% conuent 25-cm2 ask would contain 6
106 cells. An experiment requiring the infection of a 25-cm2 ask at an MOI of 50 would
proceed as follows. The number of cells (6 106 ) is multiplied by the desired MOI (50),
which equals 3 108 total pfu. This amount of virus should be diluted in the smallest
volume that will cover the cells, so as not to dilute the virus excessively. If asks are used,
a rule of thumb is 1 ml/75 cm2 of surface area.
An MOI of 50 to 100 is used for experiments that require the infection of all the cells in
the culture, or for a single growth-cycle experiment.
If suspension cells are used, follow the above guidelines as to the number of cells to be
infected and the MOI to be used. Centrifuge the cells out of the culture medium at low
speed (200 g ) and proceed with infection in a Corning conical tube as described below.
3. Remove the appropriate amount of virus from 80 C freezer and thaw the vial on
ice. Once virus has thawed, dilute to the desired concentration in 1 PBS-D/3%
FBS. Add appropriate amount of virus suspension to the monolayer.
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Current Protocols in Microbiology
4. Place ask on rocking platform for 1 hr at room temperature, with cap closed tightly.
Caps on asks should be tightened to help retain the CO2 dissolved in the remaining
EMEM medium and maintain neutral pH. Alternatively, the PBS-D may be additionally
buffered by the addition of 10 to 20 mM HEPES, pH 7.2 (see recipe for 1 M stock solution).
5. Remove inoculum and add 5 to 7 ml fresh 1 complete EMEM medium for a 75-cm2
monolayer or 3 ml of the same medium for a 25-cm2 monolayer. Resume incubation
with the caps loosened to allow for CO2 exchange.
If more concentrated virus is desired, add 3 ml medium to a 75-cm2 monolayer.
Suspension cells should be removed from the tube and placed in a new ask, again
following the cell concentration guidelines described above.
6. Examine monolayers periodically for cytopathic effect (CPE), which generally becomes evident between 18 and 24 hr post-infection.
Cell lines will differ in the time period in which CPE is demonstrated. Some indicators
of CPE in BHK cells include the cells becoming long and thin, the cytoplasm assuming a foamy appearance, aggregations of cell nuclei, and, in advanced stages, cell
lysis.
7. When CPE is observed, transfer medium from asks to 15- or 50-ml conical
polypropylene centrifuge tubes, pooling like samples if desired.
8. Centrifuge 10 min at 1000 g to remove cell debris. Decant virus-containing supernatant into a new tube.
A separate, dedicated centrifuge should be reserved for infected cells and viruses.
If purication on tartrate gradient is to be performed (see Basic Protocol 2), use
the supernatant from this step as the starting material for that protocol (do not add
glycerol).
BASIC
PROTOCOL 2
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.4
Current Protocols in Microbiology
Materials
Virus supernatant (see Basic Protocol 1, step 8)
15% and 35% (w/v) potassium tartrate in PBS-D (see recipe for PBS-D), 4 C
1 PBS-D (see recipe for 10), 4 C
Beckman UltraClear centrifuge tubes (25 89mm)
Beckman ultracentrifuge with SW28 rotor and buckets (or equivalent), prechilled
to 4 C
Ring stands
Small tube clamps
Hand-held low-intensity (e.g., 20-W) lamp
30- to 100-ml gradient former (e.g., Bio-Rad model 385)
1. Measure the volume of viral supernatant and determine the number of gradients that
will be needed.
Because manipulations will be done at the benchtop, the samples from this step forward
should not be considered sterile for critical applications. If a sterile puried preparation
is required, samples should be lter sterilized (see step 8).
The SW28 rotor used to centrifuge the gradient will only hold six gradients; fewer are
possible but more tubes will require extra runs.
2. For each gradient to be run, carefully layer 12 ml of 15% potassium tartrate onto a
layer of 6 ml of 35% potassium tartrate (see Fig. 15B.1.1) in a Beckman UltraClear
centrifuge tube. Place the tubes into the SW28 rotor buckets and weigh them upright
(the authors use a small beaker cushioned with tissue for this purpose). Add 20 ml
(typically) of virus. Add 1 PBS-D dropwise to balance the tubes.
Figure 15B.1.1 Schematic of the step gradient used for virus purication. The 35% potassium
tartrate step is shown at the bottom of the tube. 15% potassium tartrate is overlaid onto this step
to form the second layer. The culture supernatant containing the virus forms the nal layer on the
gradient. After the centrifugation, a virus band (shown as light gray) will be visible at the interface
between the 15% and 35% steps.
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Take special care to weigh the buckets and tubes, and load the centrifuge rotor according
to the manufacturers tolerances and specications as found in the manual. Failure to do
so can result in severe damage to the rotor and the instrument.
A visible layer should be seen between the 35% tartrate, the 15% tartrate and the virus
supernatant layers. Take care that the tube is full but not overowing.
The rotor, buckets, and solutions should be prechilled to 4 C. If an odd number of gradients
is required, prepare a blank gradient and overlay with buffer. Use this tube to balance
the rotor.
Sindbis Virus:
Propagation,
Quantication,
and Storage
Figure 15B.1.2 Schematic of a linear gradient. A linear gradient is used as the second purication
step. As with the step gradient, 35% potassium tartrate will be at the bottom of the tube and in
the rst chamber of the gradient former. The gradient former will mix the 35% and 15% solutions
together to form a linear gradient. The diluted virus band from the rst step gradient (Fig. 15B.1.1)
is pipetted onto the top of the linear gradient.
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Current Protocols in Microbiology
7. Using a gradient former, prepare a continuous gradient from 15% to 35% potassium
tartrate at a volume not less than three-quarters the virus solution to be added. Layer
the diluted virus over the continuous gradient (see Fig. 15B.1.2).
Read the manufacturers manual for instructions on use of the gradient former. As with the
step gradient described above, the tube should be full without overowing after addition
of the diluted virus sample.
ALTERNATE
PROTOCOL 1
Successful transfection of cells with infectious RNA requires the investigator to handle
the cells and transcripts using RNase-free sterile technique. For a detailed description of
the synthesis of infectious transcripts from Sindbis virus cDNA using SP6 polymerase,
refer to Rice et al. (1987) and Liu et al. (1996). After the cells are trypsinized and pelleted,
they are washed with RNase-free buffer to remove contaminating RNase found naturally
in all cells. For a description of the preparations necessary for solutions and supplies
(glassware and plasticware) to be used for RNase-free protocols, see APPENDIX 2A in this
manual, Maniatis et al. (1982), and Gilman (2002).
The following protocol has been modied from Lilijestrom and Garoff (1991).
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidied 37 C, 5% CO2
incubator unless otherwise specied.
Materials
Baby hamster kidney (BHK) cells (ATCC #CCL-10), growing in culture (see
Support Protocol 1)
1 complete EMEM medium (see recipe) without gentamicin
1 PBS-D (see recipe for 10; use RNase-free PBS-D as indicated in protocol)
0.25% trypsin (see recipe)
Versene solution (see recipe)
RNA transcript(s) from cDNA(s) of interest (see Support Protocol 3)
75- and 25-cm2 tissue culture asks
Siliconized, low-retention RNase-free microcentrifuge tubes (Fisherbrand cat. no.
02-681-331)
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Current Protocols in Microbiology
2. The next day, decant medium from asks and wash the monolayer once with 5 ml of
1 PBS-D.
3. Mix 25 ml of thawed 0.25% trypsin and 75 ml of versene solution, and prewarm
to 37 C. Add this to the monolayer (2 ml for 25-cm2 ask; 5 ml for 75-cm2 ask).
Incubate at room temperature until cells begin to detach from ask, then resuspend
cells and disrupt cell clumps by pipetting up and down with a 5- to 10-ml pipet. Add
an equal volume of 1 complete EMEM medium without gentamicin to stop the
reaction.
Mix the trypsin and versene solutions just prior to the trypsinization step. Be careful not
to leave cells in the trypsin longer than it takes for the cells to begin to detach from the
asks. Cell trypsinized for too long do not recover well from electroporation. Pipet the
cells up and down with a small-bore pipet (5 to 10 ml) to break cell clumps. The FBS in
the completed medium will neutralize the trypsin.
4. Centrifuge cell suspension 5 min at 500 g, room temperature, in a tabletop centrifuge, and discard supernatant. Combine all pellets into one tube, if multiple tubes
have been used.
6. Dilute a small portion of the cell pellet 1:10 or 1:50 in 1 PBS-D and count on a
hemacytometer (Strober, 1997). Keep remaining cells on ice.
7. Adjust the volume of the cell suspension so that the concentration is 1 to 2
107 cells/ml.
Sindbis Virus:
Propagation,
Quantication,
and Storage
8. For every RNA sample to be transfected, place a 400-l aliquot of the cell suspension
into an siliconized, low-retention RNase-free microcentrifuge tube.
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Current Protocols in Microbiology
9. Add RNA to the cells (in a volume of 20 l containing not more than 10 g total
RNA) and transfer the mixture into a 2-cm gap-length electroporation cuvette.
10. Pulse the cell/RNA mixture once at 1.5 kV, 25 F, and innite resistance.
The time constant after pulsing one time should be 0.7 sec; if the time constant is low,
e.g., 0.2 sec, repeat the pulse.
BASIC
PROTOCOL 3
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidied 37 C, 5% CO2
incubator unless otherwise specied.
Animal RNA
Viruses
15B.1.9
Current Protocols in Microbiology
Materials
Baby hamster kidney (BHK) cells (ATCC #CCL-10), growing in culture (see
Support Protocol 1)
1 and 2 complete EMEM medium (see recipe)
SVHR diluent (see recipe) or TM diluent for BHK cell assay (see recipe)
Virus stock to be titered (e.g., Basic Protocol 1, Basic Protocol 2, or Alternate
Protocol)
2% agarose (see recipe)
2 PBS-D (see recipe for 10)
2% neutral red stock solution (see recipe)
20 mM HEPES, pH 7.2 to 7.4 (see recipe; for less stable virus)
25-cm2 tissue culture asks (the authors have found that vented asks are les
susceptible to contamination with mold than the standard asks)
Dilution tubes: 13 100mm borosilicate glass test tubes
Dilution tube rack
Platform rocker
Light box
Prepare cells and virus dilutions
1. Determine the number of asks required, with one ask per dilution of virus, plus
a few control asks. The day before the plaque assay, split BHK cells into 25-cm2
asks containing 6 106 cell/ask, using 1 complete EMEM medium such that
they will be 80% to 90% conuent on the next day.
If the cells are ready in the morning and the assay is to be done in the afternoon, the
cells can be stored at room temperature with the caps closed tightly, or moved to a 28 C
incubator to slow growth.
2. Remove the virus to be titered from the freezer and thaw on ice.
3. Fill the required number of dilution tubes with 900 l SVHR diluent (for wild-type
virus) or TM diluent (for unstable virus).
4. Prepare serial dilutions of the virus by adding 100 l virus to the rst tube containing
900 l of diluent (101 ), vortexing that dilution at maximum speed, removing 100 l,
adding it to the next dilution tube (102 ), and continuing this process until the desired
dilutions have been made. Change pipet tips between dilutions.
Dilutions should be made immediately prior to use. Virus and dilutions should be kept on
ice at all times.
Infect cells
5. Pour growth medium out of asks (step 1) and infect each ask with 200 l of the
appropriate virus dilution.
Do not let monolayers dry out while adding virusi.e., do not pour medium off of too
many asks at one time.
6. Tighten the caps on the asks and place them on a platform rocker at room temperature
for 1 hr.
The caps on the ask should be tightened while they are rocking, since they are not in the
appropriate CO2 environment.
Sindbis Virus:
Propagation,
Quantication,
and Storage
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Current Protocols in Microbiology
Melt the agarose by autoclaving for 20 min or by microwaving for 1 min (for sterile
agarose stock, the medium must be autoclaved; microwaved agarose is not sterile). Allow
the agarose to cool but remain liquid by incubating in a 60 C water bath until needed.
Once the 60 C agarose is combined with the 37 C 2 medium, the temperature of the
mixture will be 40 C, and it will remain liquid for 5 min.
The agarose should be hot enough that it will not solidify too quickly, but allowed to cool
enough to allow one to touch the bottle before mixing with the medium (60 C to 70 C).
Bottles of medium at the correct temperature should not feel excessively hot to the touch.
Do not try to move asks before the overlay has solidied; the cell monolayer will tear.
Not all preparations of agarose are tolerated by cells in culture. In general, agarose
formulated for gel electrophoresis or chromatography is not suitable for tissue culture.
Use Sigma agarose as described in Reagents and Solutions.
8. Incubate asks for 2 days, periodically holding the asks up to the light to see if
plaques are visible.
The plaques will appear more opaque than the rest of the monolayer. Visible plaques are
an indication that the assay has worked up to this step.
9. For the stain overlay, prepare a 1:1 mixture of 2% agarose and 2 PBS-D (or a
1:1 mixture of 2% agarose and 2 EMEM/20 mM HEPES, pH 7.2 to 7.4, for less
stable virus). Add 2 ml of 2% neutral red stain stock solution per 100 ml for a nal
concentration of 0.06 % neutral red.
Final concentrations will vary with different neutral red preparations.
10. Remove the medium from each ask and add 5 ml of the staining mixture prepared
in step 9 to the monolayer. Cover the asks to protect cells from light and incubate
4 hr at 37 C.
Cells become light sensitive after they take up neutral red.
Figure 15B.1.3 Plaque assay asks. Note the appearance of SVHR plaques at 107 (ask 3),
108 (ask 2), and 109 dilutions (ask 1). Most plaques are large; however, medium-sized plaques
are also visible. Sindbis virus forms symmetrical plaques. At the lower dilutions, (107 ), plaques
are seen overlapping. Some monolayers, such as ask 2, may have monolayers that display faint
less well dened plaques.
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Viruses
15B.1.11
Current Protocols in Microbiology
Table 15B.1.1 Sample Data for Plaque Assay (also see Fig. 15B.1.3)
Flask 1
Flask 2
Flask 3
Dilution
109
108
107
No. of plaques
>50
dilutions made (e.g., 1 plaque on 106 ask, 10 plaques on 105 ask, and 100 plaques
on 104 ).
12. Count plaques using a light box. Calculate titer using the following equation, and
Table 15B.1.1 and Figure 15B.1.3 as examples, assuming that the 1 108 dilution
ask has six plaques.
6 plaques/(0.2 ml/1 108 dilution) = 3.0 109 pfu/ml.
Ideally the number of plaques in each dilution would differ by a factor of 10, but in practice
this number will vary. It is best to pick a dilution with an intermediate number of plaques,
e.g., 10 to 100, to calculate the virus titer. The titers are most reliable when more than
one dilution closely follows the dilution trend. Unusually high- or low-titer data should
be discarded.
SUPPORT
PROTOCOL 1
Materials
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.12
Current Protocols in Microbiology
1. Wash a conuent BHK cell monolayer once with 1 PBS-D, using 5 ml for a 25-cm2
ask or 15 ml for a 75-cm2 ask.
The authors have found that vented asks are les susceptible to contamination with mold
than the standard asks.
5a. For preparation of a single 25-cm2 ask: Pipet a 1-ml aliquot of the cell suspension
into 10 ml of 1 complete EMEM in the new 25-cm2 ask.
Up to three asks can be prepared from one initial 25-cm2 ask.
5b. For preparation of a large number of 25-cm2 asks: In a sterile bottle, pipet 10 ml of
1 complete EMEM for each ask to be prepared. Add 1 ml cell suspension to this
medium per each ask to be prepared. Aliquot 10 ml cell/medium mixture to each
new 25-cm2 asks, swirling continuously.
One 75-cm2 ask can be subcultured into nine 25-cm2 asks.
5c. For preparation of a single 75-cm2 ask: Resuspend cells from a single 25-cm2 ask
in 1 ml medium and transfer to a 75-cm2 ask containing 30 ml EMEM. Alternatively,
subculture a 75-cm2 ask into three 75-cm2 asks or nine 25-cm2 asks.
The optimum split ratio remains 1:3.
6. Carefully place asks in incubator with loosened caps and incubate 24 hr or until
conuent.
Care should be taken when placing the asks in the incubator so as not to disturb the cell
suspension.
SUPPORT
PROTOCOL 2
The use of CEF cultures for the cultivation of many animal viruses is still the gold
standard for propagation of Alphaviruses and is described here in detail. The high titers
of Sindbis virus obtained from BHK cells, however, have made the use of CEF cultures
less frequent.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidied 37 C, 5% CO2
incubator unless otherwise specied.
Animal RNA
Viruses
15B.1.13
Current Protocols in Microbiology
Materials
Fertile chicken eggs, 10 days old (Spafas;
http://www.criver.com/RM/spf/egg chickens.html)
70% ethanol
1 phosphate-buffered saline (PBS) with calcium and magnesium (see recipe)
0.25% trypsin
Versene solution (see recipe)
1 complete EMEM (see recipe)
Strong light source (75 to 100 W)
Small sterile beaker
Sharp scissors and forceps, sterile
50-ml conical tubes
37 C water bath
75-cm2 tissue culture asks
Additional reagents and equipment for counting cells (Strober, 1997), subculturing
BHK cells (see Support Protocol 1), and freezing cells (see Support Protocol 5)
Remove embryo from egg
1. Candle a 10-day-old egg by holding the egg in front of a bright light. Mark the
position of the air sac with a pencil.
Purchase the eggs so that they arrive at the 10-day-old stage. The embryo is seen as a
dark mass on the yolk surface. Discard eggs without embryos or with dead embryos.
2. Wash the egg with 70% ethanol and place it pointed-end-down in a small sterile
beaker.
3. Cut a large circular hole at the blunt end of the egg with a pair of sharp sterile scissors.
Remove the shell above the air sac with sterile scissors or forceps, taking care not to
touch the underlying membrane.
It is helpful to carefully poke the eggshell, thereby creating a small hole in the egg from
which to start cutting. Small scissors with thin blades are best for this purpose. Try to cut
the shell away as a large circular piece that can easily be pulled away. Avoid creating
shards of eggshell, which can fall into the egg and contaminate the embryo.
10. Mix 25 ml of thawed 0.25% trypsin and 75 ml of versene solution and prewarm to
37 C. Remove buffer from the macerated embryo using a pipet and add 20 ml of this
prewarmed solution. Shake tube gently at 37 C for 15 to 20 min.
15B.1.14
Current Protocols in Microbiology
11. Allow the large pieces to settle to the bottom of the tube. Using a pipet, transfer
the supernatant, containing the cell suspension, to a tube containing 25 ml of 1
complete EMEM.
The serum in the medium will inactivate the trypsin.
12. Centrifuge 10 min at 500 g, room temperature, and remove the supernatant. Resuspend cell pellet in 20 ml 1 complete EMEM.
The pellet will be loose, so care must be taken when removing the supernatant.
13. Take an aliquot of cell suspension and dilute 1:5 or 1:10 in PBS containing calcium
and magnesium. Count cells using a hemacytometer (Strober, 1997).
There should be 106 to 107 cells/ml.
A single embryo will yield 1.0 108 viable cells, enough, conservatively, to inoculate ten
75-cm2 asks. The number of embryos needed must be determined based on this number.
Culture CEF
14. Prepare each 75-cm2 ask required by adding 1 106 cells and 30 ml 1 complete
EMEM. Begin incubation.
Do not seed asks with less that 106 cells, the monolayers will not grow.
The authors have found that vented asks are less susceptible to contamination with mold
than the standard asks.
15. Subculture at a ratio of 1:3 up to three passages using the same technique described
for BHK cells (see Support Protocol 1).
The cells will remain viable for more than three passages. However, bear in mind that
there is variability in the cells from embryo to embryo. There are also changes in the cell
characteristics with increasing passage number.
16. Freeze cells in cryotubes at 106 cells/tube in 1 ml of freezing medium (see Support
Protocol 5).
BASIC
PROTOCOL 4
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
Materials
Mosquito cells (e.g., U4.4 cells; laboratory isolate; contact authors at
rhernan2@unity.ncsu.edu), growing in tissue culture in 75-cm2 asks
1 PBS-D (see recipe)
Serum-free M & M or EMEM medium (e.g., Invitrogen; also see recipes)
Sindbis virus (SVHR) stock, titered (see Basic Protocol 3 or 5)
Fresh 1 complete Mitsuhashi and Maramorosch (M & M) medium (see recipe) or
1 complete EMEM medium (see recipe)
Animal RNA
Viruses
15B.1.15
Current Protocols in Microbiology
Glycerol
Neutral-pH buffer (PBS-D or culture medium buffered to pH 7.2 to 7.4 using 10 to
20 mM HEPES)
28 C humidied 5% CO2 incubator (CO2 required only for use with EMEM)
75-cm2 tissue culture ask
Additional reagents and equipment for counting cells (Storber, 1999) and infecting
cell cultures with Sindbis virus and harvesting and storing the virus (see Basic
Protocol 1)
Prepare cells
1. For every 75-cm2 ask to be infected, transfer the contents of approximately one
and one-half 75-cm2 asks to a centrifuge tube. Centrifuge 5 min at 200 to 500 g,
room temperature. Remove the supernatant.
Mosquito cells grow as semisuspension cultures and are easily removed from the ask by
tapping the ask against a hard surface, or alternatively by pipetting up and down with a
5- to 10-ml pipet.
3. Resuspend the nal cell pellet in 1 ml of serum-free M & M or EMEM medium for
every ask to be infected. Count cells using a hemacytometer (Strober, 1997).
4. Pipet an aliquot of cell suspension containing 6 107 cells into each 75-cm2 ask
to be infected, with enough serum-free M & M or EMEM medium to cover the
monolayer (10 ml).
5. Incubate the ask of cells at 28 C for 1 hr, or until cells are well attached to the
surface of the ask.
CO2 is required only for use with EMEM.
Infect cells
6. Once cells are attached, remove medium and add the desired amount of virus to the
monolayer (see Basic Protocol 1, steps 2 to 3). Close caps tightly.
7. Place ask on a platform rocker and rock slowly at room temperature for 1 hr.
Slow rocking is needed to keep a minimum number of cells from lifting from the ask.
Most of the cells should remain attached to the ask after rocking. If cells have lifted from
the monolayer they should be removed with the inoculum.
Sindbis Virus:
Propagation,
Quantication,
and Storage
Virus may be harvested at between 24 and 72 hr post-infection (hpi) depending on the multiplicity of infection (MOI) used. Cytopathic effect (CPE) will not be evident in mosquito
cells, and therefore virus will be harvested based on time post-infection rather than CPE.
MOIs above 10 require 24 hr of growth. For an MOI of 1, asks would be allowed to
incubate 48 hr. To amplify virus from plaque (6000 pfu for SVHR; see Basic Protocol 6),
72 hr of incubation would be allowed.
15B.1.16
Current Protocols in Microbiology
11b. For preservation of highest infectivity level and virus particle/pfu ratio: Store virus
up to 5 days at 4 C in neutral-pH buffer.
This method preserves the highest level of infectivity. It is used for virus to be analyzed in
structural studies (Phinney et al., 2000; Phinney and Brown, 2000) and for the calculation
of particle-to-pfu ratios (Hernandez et al., 2003). Although titer is retained, this method
is wasteful in that unused virus is discarded.
ALTERNATE
PROTOCOL 2
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
2. Count cells (Strober, 1997) and calculate the amount of virus needed for the desired
multiplicity (MOI).
A 1:3 split of a conuent monolayer should give 6 107 cells per 75-cm2 ask in 2 days.
3. Pool all cells to be infected and centrifuge 5 min at 500 g, room temperature.
Remove supernatant.
Animal RNA
Viruses
15B.1.17
Current Protocols in Microbiology
Materials
Mosquito cells (e.g., U4.4 cells; laboratory isolate; contact authors at
rhernan2@unity.ncsu.edu), growing in tissue culture in 75-cm2 asks
1 PBS-D (see recipe for 10)
HBS buffer (see recipe), RNase-free, ice-cold
RNA transcript(s) from cDNA(s) of interest (see Support Protocol 3)
1 complete Mitsuhashi and Maramorosch (M & M) medium (see recipe)
1 M MOPS buffer (see recipe; for use with mosquito cell cultures and mutant
viruses requiring additional buffering)
50-ml conical polypropylene centrifuge tubes
Aerosol-barrier pipet tips, RNase free
RNase-free microcentrifuge tubes
Electroporation cuvettes 2-cm gap width
Electroporator
Additional reagents and equipment for counting cells (Strober, 1997) and
harvesting virus (see Basic Protocol 1)
Prepare cells
1. For every RNA sample to be transfected, transfer the contents of two 75-cm2 asks
of U4.4 cells (or other mosquito cell cultures) to a 50-ml conical polypropylene
centrifuge tube at 40 ml per tube (do not ll beyond 40-ml mark, or cells will become
contaminated). Hit side of ask sharply several times to loosen cells stuck to surface.
Centrifuge 5 min at 500 g, room temperature. Remove supernatants.
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.18
Current Protocols in Microbiology
2. Pool all cells, centrifuge 5 min at 500 g, room temperature, remove supernatant,
then add 5 ml of 1 PBS-D, centrifuge again as before, and remove the supernatant.
From this point it is important to use only RNase-free pipets, solutions, and aresol-barrier
pipet tips.
4. Resuspend cell pellet in 0.5 ml cold HBS buffer per RNA sample to be transfected.
5. Dilute a small aliquot of the cell pellet 1:10 or 1:50 in HBS buffer and count cells in
a hemacytometer (Strober, 1997). Keep remaining cells on ice.
6. Adjust the volume of the cell suspension so that the concentration is 5 107
cells/ml.
Transfect cells
7. For each RNA sample to be transfected, prepare an RNase-free microcentrifuge tube
containing 400 l cell suspension.
8. Add RNA to the cells and transfer the mixture into a 2-cm gap-width electroporation
cuvette.
9. Pulse the cell/RNA mixture once in an electroporate set at 2.0 kV, 25 F, and innite
resistance
The time constant after pulsing one time should be 0.7 sec.
10. Let the mixture sit in the cuvette 10 min at room temperature.
11. Transfer the mixture to a 25-cm2 ask containing 10 ml complete 1 complete
M & M medium. For unstable mutants, add 1 M MOPS buffer to a nal concentration
of 10 mM in the medium.
12. Incubate asks 48 hr in a humided 28 C, 5% CO2 incubator.
CO2 is required only for use with EMEM.
SUPPORT
PROTOCOL 3
Materials
RNase-free H2 O (Gilman, 2002; aslso see APPENDIX 2A)
Linearized cDNA of interest: do not treat with RNase
10 transcription buffer (see recipe)
dNTPs: ATP, UTP, GTP, and CTP (APPENDIX 2A)
m7 GpppG (New England Biolabs)
RNase inhibitor (Ambion)
SP6 RNA polymerase (New England Biolabs)
Animal RNA
Viruses
15B.1.19
Current Protocols in Microbiology
1 g linearized cDNA
1 transcription buffer
1 mM each ATP, UTP, and CTP
0.5 mM GTP
2 mM m7 GpppG
10 U 2 RNAse inhibitor
20 U SP6 polymerase
Incubate reactions at 40 C for 1 hr.
Also see Struhl (1999).
2. Check that the transcription reaction has worked by running 2 l of the reaction on
a 1% agarose gel for 1 hr at 80 mA. Stain with ethidium bromide (see Fig. 15B.1.4).
CAUTION: Ethidium bromide is a carcinogen and should be handled with gloves. See
manufacturers enclosed MSDS sheet and UNIT 1A.3 for appropriate handling conditions.
Agarose gel electrophoresis and ethidium bromide staining are detailed in Maniatis (1982)
and Voytas (1991).
Sindbis Virus:
Propagation,
Quantication,
and Storage
Figure 15B.1.4 1% agarose gel of transcripts from the transcription reaction. Lane 1, mass
ladder; 200 ng are present in the upper band. Lane 2, kb ladder; Sindbis virus cDNA (top bands
in lanes 3 to 6) runs just slightly above the uppermost 10 kb band. Lanes 3 to 6, transcription
reactions run prior to DNAse digestion. Note the nice tight bands of RNA above a smear of product.
This quality and quantity 500 g per 20-l reaction of RNA will produce a good transfection. The
smaller transcript in lane 4 is used as a positive control for the transcription reaction. The resulting
RNA is noninfectious and serves as a negative control for the cell transfection (see Alternate
Protocols 1 and 3).
15B.1.20
Current Protocols in Microbiology
3. After evaluating the reaction, add 4 U of RNase-free DNase and incubate reaction
15 min at 37 C.
It is imperative to remove the DNA. Excess nucleic acid will interfere with the transfection
and too much will kill the cells.
BASIC
PROTOCOL 5
Materials
C7-10 mosquito cells (laboratory isolate; contact authors at
rhernan2@unity.ncsu.edu) growing in 75-cm2 tissue culture asks
1 and 2 serum-free EMEM medium (see recipe)
1 and 2 serum-free MEM medium with Hanks salts (see recipe)
TM diluent for C7-10 plaque assay (see recipe)
SVHR sample to be titered (any cell source)
2 complete EMEM (see recipe)
2 complete MEM with Hanks salts (see recipe)
2% agarose (see recipe)
1 PBS-D (see recipe) containing 20 mM MOPS (prepare using 1 M MOPS
buffer; see recipe)
2% neutral red stock solution (see recipe)
25-cm2 tissue culture asks
Humidied 28 C 5% CO2 incubator (CO2 required only for use with EMEM)
Dilution tubes: 13 100mm borosilicate glass test tubes
Dilution tube rack
Platform rocker
Additional materials for counting cells (Strober, 1997)
Prepare cells
1. For every two 25-cm2 asks to be used in the plaque assay, transfer the contents of
one 75-cm2 ask of C7-10 mosquito cells to a 50-ml conical polypropylene centrifuge tube at 40 ml per tube (do not ll beyond 40-ml mark, or cells will become
contaminated). Hit side of ask sharply several times to loosen cells stuck to surface.
Centrifuge 5 min at 500 g, room temperature. Remove supernatants.
Animal RNA
Viruses
15B.1.21
Current Protocols in Microbiology
Infect cells
7. Pour off medium from asks and add 200 l of the appropriate virus dilution.
8. Tighten the caps on the asks and place them on a platform rocker at room temperature
for 1 hr with very slow rocking to avoid disrupting the fragile cell layer.
9. Prepare a 1:1 mixture of 37 C 2 complete EMEM medium and 37 C 2 complete
MEM medium with Hanks salts, then, in turn, prepare a 1:1 mixture of this combined
2 medium and 2% agarose. Remove inoculum from asks and overlay the cells in
each of the asks with 7 ml of this mixture.
Melt the agarose by autoclaving for 20 min or by microwaving for 1 min (for sterile
agarose stock, the medium must be autoclaved; microwaved agarose is not sterile). Allow
the agarose to cool but remain liquid by incubating in a 60 C water bath until needed.
Once the 60 C agarose is combined with the 37 C 2 medium, the temperature of the
mixture will be 40 C, and it will remain liquid for 5 min.
To this point, the C7-10 plaque assay protocol will take 3 hr depending on the number of
virus samples to be titered. This technique takes about twice as long as the plaque assay
on BHK cells.
Assay plaques
11. Prepare a 1:1 mixture of 2% agarose and 2 PBS-D/20 mM MOPS. Add 2 to 3 ml
of 2% neutral red stock solution per 100 ml of 1 agarose to a nal concentration of
0.04% to 0.06%.
12. Remove the medium from each ask and add 5 ml of the staining mixture prepared
in step 11 to the cells. Cover the asks to protect cells from light. Return asks to
humidied 28 C, 5% CO2 incubator overnight.
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.22
Current Protocols in Microbiology
14. Calculate virus titers as described above (see Basic Protocol 3, step 12).
SUPPORT
PROTOCOL 4
Materials
Mosquito cells growing in semisuspension
Medium: 1 complete Mitsuhashi and Maramorosch (M & M) medium (see
recipe) or 1 complete EMEM medium (see recipe)
Tissue culture asks
Humidied 28 C, 5% CO2 incubator (CO2 required only for use with EMEM)
1. Maintain mosquito cell lines in semisuspension in tissue culture asks at 28 C in
a 5% CO2 humidied incubator. Subculture every other day as described in the
following steps.
Conuent monolayers contain 6 107 cells per 75-cm2 ask. These cultures may be
subcultured every other day. Older or heavier cultures remain viable and are subcultured
after counting cells by adding 2 107 cells to each 75-cm2 ask.
Semisuspension cells adhere loosely to the substrate initially, and then begin to oat as
they age.
2. Hit the side sharply on any soft surface several times to loosen any cells that are stuck
to the surface of the ask. If the cells are aggregated into clumps, try to break up
the clumps by pipetting up and down several times using a 5- to 10-ml pipet before
transferring the cells to a new ask.
3. Transfer one-third of the volume of the cell suspension to each of three asks. To
each of these asks, add fresh medium to increase the volume to the original volume.
Animal RNA
Viruses
15B.1.23
Current Protocols in Microbiology
SUPPORT
PROTOCOL 5
Materials
BHK cells (ATCC #CCL-10)
0.25% trypsin (see recipe)
Versene solution (see recipe)
1 complete EMEM medium
Freezing medium (see recipe) made with EMEM for BHK cells or with either M &
M or EMEM for mosquito cells, depending on optimal growth medium
Mosquito cells: U4.4 and C7-10 (contact authors at rhernan2@unity.ncsu.edu) and
C6/36 cells (ATCC #CRL-1660)
Cryotubes
Nalgene Cryo 1 C freezing container
Liquid nitrogen freezer
For BHK cells
1a. Decant medium from a culture ask that is in log phase and not more than 90%
conuent.
The size of the ask or the quantity of cells will depend on the number of vials to be stored.
For example, a 75-cm2 ask of cells containing 2 107 cells will yield three 1-ml vials.
It is crucial that the cells to be frozen not be conuent but actively growing in log phase
for the cells to remain viable after the freezing procedure.
2a. Mix equal volumes of 0.25% trypsin and versene solution and prewarm to 37 C.
Wash cells once with PBS-D, then add a large enough volume of the trypsin/versene
solution to cover the monolayer. Allow digestion to proceed at room temperature
until the cells begin to detach from the ask.
3a. Stop the trypsinization reaction by adding an equal volume of 1 complete EMEM.
4a. Centrifuge 5 min at 500 g, room temperature, and decant off the trypsin solution.
5a. Resuspend cells in 1 ml of freezing medium per 6 106 cells.
6a. Aliquot cells into cryotubes and freeze slowly to 80 C at a rate of 1 C/min using a
Nalgene Cryo 1 C freezing container.
7a. After a minimum of 12 hr but not more than 24 hr, transfer the vials to a liquid
nitrogen freezer.
Failure to transfer the cells at the appropriate time will kill them.
Sindbis Virus:
Propagation,
Quantication,
and Storage
8a. To thaw cells, remove the appropriate vial from the liquid nitrogen freezer. Place in
a 37 C water bath until the medium is thawed. Pipet cell solution from vial into a
25-cm2 ask and slowly add 10 ml of prewarmed 1 complete EMEM dropwise to
the cells.
15B.1.24
Current Protocols in Microbiology
CAUTION: Gloves and goggles should be worn when removing vials from the liquid
nitrogen.
If there is a hissing sound emanating from the vial, N2 has leaked into the vial and the
cells will be contaminated. Discard a hissing vial. Likewise, discard cells that appear
foamy, as they contain dissolved N2 .
It is critical to add the warmed completed medium dropwise and slowly to allow the cells
sufcient time to equilibrate from the 5% DMSO/5% glycerol medium, which is not of the
same osmolarity. Cells that have been equilibrated poorly will appear blebbed and will
eventually lyse. These cells should be discarded.
9a. After the cells have attached to the ask (1 to 2 hr), discard medium in the asks, which
contains dead cells, DMSO, and glycerol that could retard growth of the monolayer.
Replace with 10 ml fresh 37 C, 1 complete EMEM medium and replace in the
incubator.
3b. Divide cells into 1-ml aliquots in cryotubes and freeze as described in steps 6a and
7a.
4b. Thaw and replate cells as described in steps 8a and 9a.
Mosquito cells are much less adherent than BHK; therefore, proceed as in step 9a, but
exercise additional care not to disturb the monolayer.
BASIC
PROTOCOL 6
Materials
Virus stock to be puried (see above protocols)
Virus diluent: 1 PBS-D (for wild-type SVHR; see recipe) or TM diluent for BHK
or C7-10 plaque assay as appropriate (for mutants; see recipes)
Soldering iron
Sterile Pasteur pipets and tubes
Additional reagents and equipment for plaque assay (see Basic Protocol 3 for BHK
cells or Basic Protocol 5 for mosquito cells)
NOTE: All solutions and equipment that comes into contact with living cells must be
sterile and aseptic technique must be used.
Animal RNA
Viruses
15B.1.25
Current Protocols in Microbiology
1. Prepare a plaque assay as in Basic Protocol 3 (for BHK cells) or Basic Protocol
5 (for mosquito cells), infecting with dilutions that will give 10 to 20 plaques in a
single ask.
Multiple asks will ensure that sufcient plaques are produced.
2. Choose plaques that are well formed and sufciently separated from one another to
ensure that only a single plaque is isolated. Circle the plaques to be picked.
3. If using asks, burn through the top of the ask with the hot soldering iron just above
the plaque to be picked.
4. Wet a sterile Pasteur pipet by pipetting up and down in the virus diluent solution.
This will reduce the tendency of the agarose to stick to the pipet.
5. Aspirate the chosen plaque with the Pasteur pipet, taking care to include all the
agarose above the plaque.
6. Pipet the agarose into 1 ml PBS-D or TM diluent (for unstable mutants) in a sterile
tube.
7. Elute overnight at 4 C for (for unstable mutants) or for 2 to 3 days at 4 C (for
wild-type SVHR).
8. Remove all pieces of agarose prior to using or storing the plaque-puried virus.
9. Optional: To ensure purity, repeat the above steps with the plaque-puried virus.
Agarose, 2%
Dilute agarose type I (Sigma, cat. no. A-6013) to 2% with tissue culture-grade
water. Autoclave in 50- to 100-ml aliquots to sterilize.
Before use, microwave 1 min to liquefy. Keep molten at 60 C until ready to dilute
with 2 complete EMEM (see recipe).
Store at room temperature up to 1 year.
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.26
Current Protocols in Microbiology
IMPORTANT NOTE: In this unit, complete media with supplements are not true percent
solutions. The percentage indicated represents the percentage of the original volume of
medium. This also holds for any other supplements added to the medium. For example, if
the initial volume of the medium is 400 ml, 10% FBS is 40 ml. Addition of other supplements,
such as 5% TPB would be 20 ml TPB, bringing the total volume to 460 ml.
If problems arise with the cell cultures, growth of the virus, or plaque formation, discard
old solutions and make fresh solutions.
Gentamicin is added to medium for BHK cells only. C6/36 and C7-10 cells do not require
gentamicin for general maintenance.
15B.1.27
Current Protocols in Microbiology
L-Glutamine,
100x
Weigh out 2.92 g L-glutamine and dissolve in a total volume of 100 ml 1 PBS-D
(see recipe for 10). Filter sterilize and divide into 50-ml aliquots in 100-ml bottles.
Store frozen up to 6 months; once thawed, discard after 2 weeks.
Glutamine is essential and very labile. It is thus added to already complete media as a
precaution.
HBS buffer
4.76 g HEPES
8.0 g NaCl
0.4 g KCl
0.1 g Na2 HPO4
1.1 g D-glucose
Tissue-culture-grade H2 O to 1 liter
Store up to 1 year at room temperature
HEPES (pH 7.2 to 7.4), 1 M
Weigh out 238.3 g HEPES and dissolve in a total volume of 1 liter tissue-culture
grade water. Adjust pH to 7.2 to 7.4 with NaOH. Autoclave 30 min to sterilize.
Store up to 1 year at room temperature.
MEM with Hanks salts, serum-free, 2x
MEM supplemented with Hanks salts, with glutamine and without sodium bicarbonate is purchased in powder form from Invitrogen. Prepare this medium as
described in the manufacturers instructions, using half the recommended amount
of water for the 2 solution of MEM. Filter sterilize. Store up to 6 months at 4 C
with containers sealed using Paralm.
Mitsuhashi and Maramorosch (M & M) medium, 1x serum-free and complete
Incomplete, serum-free medium:
0.2 g CaCl2 2H2 O
0.2 g KCl
0.1 g MgCl2 6H2 O
7.0 g NaCl
0.12 g NaHCO3 (dissolve separately and then add to mixture)
0.2 g NaH2 PO4 H2 O
5.0 g Yeastolate
4.0 g D-glucose
6.5 g lactalbumin hydrolysate
Tissue-culture-grade water up to 1 liter
Filter sterilize
Store incomplete medium up to 1 year at 4 C
Complete medium: Add 20% (v/v) heat inactivated fetal bovine serum (see note below on calculating percentages) and an additional 0.15% NaHCO3 . Store complete
medium 3 to 4 weeks at 4 C.
IMPORTANT NOTE: In this unit, complete media with supplements are not true percent
solutions. The percentage indicated represents the percentage of the original volume of
medium. This also holds for any other supplements added to the medium. For example, if the
initial volume of the medium is 400 ml, 10% FBS corresponds to 40 ml. Other supplements,
such as 5% TPB, corresponding to 20 ml TPB, bring the total volume to 460 ml.
Sindbis Virus:
Propagation,
Quantication,
and Storage
continued
15B.1.28
Current Protocols in Microbiology
The bicarbonate added to the incomplete, serum-free medium is used as a carbon source,
while the bicarbonate added to the complete medium is used to adjust the pH. M & M
medium is not buffered by bicarbonate and will not be affected by a CO2 environment, nor
is a CO2 incubator required. For additional buffering (e.g., if needed for mutant viruses),
add 20 mM MOPS, pH 7.2 to 7.4.
This medium has a high propensity for contamination with mold. Care should be taken to
keep the incubators clean. If problems arise with the cell cultures, growth of the virus, or
plaque formation, discard old solutions and make fresh ones.
PBS-D, 10x
2.0 g KCl
2.0 g KH2 PO4
80.0 g NaCl
11.3 g Na2 HPO4 or 21.6 g Na2 HPO4 7H2 O
Tissue-culture-grade H2 O to 1 liter
Autoclave 20 min
Dilute to 1 with sterile tissue-culture-grade water
Store up to 1 year at room temperature
Also see annotations to the recipe for PBS with calcium and magnesium; check that the pH
of the 1 solution is 7.4.
Animal RNA
Viruses
15B.1.29
Current Protocols in Microbiology
IMPORTANT NOTE: Do not adjust the pH of the 10 solutions 1 and 2, because this will
result in the wrong pH for the 1 solution. It is critical that all solutions that Sindbis virus
comes in contact with are neutral pH. Acidic pH quickly inactivates the virus.
Sindbis Virus:
Propagation,
Quantication,
and Storage
Versene solution
500 ml 1 PBS-D (see recipe for 10)
5 ml 0.1 M EDTA (see recipe)
1.5 ml 0.5% phenol red (see recipe)
Adjust pH to 7.4 with NaOH
Filter sterilize (if sterile stock solutions were not used)
Store up to 6 months at room temperature
15B.1.30
Current Protocols in Microbiology
COMMENTARY
Background Information
Alphaviruses are arthropod-borne viruses
that are transmitted to mammals when the
infected insect takes a blood meal. The Alphaviruses are found throughout the world and
cause many illnesses of clinical signicance.
While Sindbis virus primarily infects birds
and mammals, humans generally represent
a dead-end host, responding to infection
with limited viremia. Among the members
of the Alphavirus genus, Chickungunya and
ONyong-nyong viruses (Africa) produce
rash, arthralgia, and febrile illness in humans.
In the Western hemisphere, the Eastern (EEE),
Western (WEE), and Venezuelan encephalitis
viruses (VEE) are causes of encephalitis in
humans, which can be fatal. Encephalitis, or
inammation of the brain, occurs when the
viral infection crosses the blood-brain barrier
and infects the central nervous system (CNS).
VEE is a signicant veterinary and public
health problem in Central and South America
(Strauss and Strauss, 2002).
Sindbis virus, the prototype of the Alphaviruses, is an easily propagated virus which
has displayed the ability to infect all cell types
tested, whether of vertebrate or invertebrate
origin. Most of the difculties encountered
with the propagation of Sindbis result from
problems related to the cell cultures chosen for
its propagation or titration. The virus plaque
assay is the gold standard for the determination of virus infectivity. Not all viruses produce
a cytopathic effect, making it possible to perform a plaque assay for infectivity. However,
Sindbis virus is particularly amenable to this
method and produces large to pinpoint plaques,
depending on the virus strain or mutant. Although this assay requires several days before
the asks can be evaluated, the titers are reproducible. If care is not taken, however, the
error for the plaque assay can be quite large,
up to 0.5 logs. Repeating the assay three to
four times will minimize the amount of error,
and the accuracy can be improved to within a
2-fold error. Specically, infectivity measured
in pfu/ml is used to establish the relative infectivity of wild-type and mutant virus as a ratio of
particle/pfu. This measurement is required in
specic experiments evaluating infectivity of
mutant viruses (TM viruses; Hernandez et al.,
2003). It is particularly important when working with these or other mutants to monitor the
titers of the different viruses, because revertants can arise or contamination of the sample
with wild-type virus can occur. Infectious virus
Critical Parameters
Cell cultures
Of highest importance when growing Sindbis is the health of the cell cultures used. If the
BHK or mosquito cultures are growing well
and in log phase, virus should grow within
12 to 24 hr, depending on the MOI and the
cell line used. It is also of paramount importance that good sterile technique be followed
to avoid contamination of the cultures with microorganisms or cross-contamination between
mutant and wild-type virus.
For best results, virus should be thawed on
ice and all solutions used for dilution of the
virus and the plaque assays should be kept cold
and on ice. Care should be taken to avoid uctuations in the pH of the asks during infection
by closing the caps tightly and monitoring the
pH of the medium. Sindbis virus and many of
the mutants can be stored at 80 C for many
years. It is a good practice, however, to also
Animal RNA
Viruses
15B.1.31
Current Protocols in Microbiology
Table 15B.1.2 Common Problems Associated with the Sindbis Virus Plaque Assay, Their Possible Causes, and Solutions
Problem
Possible cause
Solution
No plaques
visible
Infection failed
Sindbis Virus:
Propagation,
Quantication,
and Storage
Serum
When purchasing new lots of serum, the authors routinely test a new lot, selected as that
which most closely matches the serum specications and chemical analysis of the serum
in current use (matched lot), for the ability
to maintain growth of U4.4 and BHK cells.
This is done by subculturing cells into medium
containing the test lot. Plaque formation of
SVHR stock on BHK cells is also tested. The
titer of the SVHR stock virus tested on BHK
cells grown in the new serum should match
the previous titer. The serum supplier should
have the lot analysis for any lot of serum.
This test should be performed prior to purchase
of large lots. The serum should be purchased
heat-inactivated, or heat inactivated after receipt by heating the thawed serum to 56 C for
30 min. The inactivation of complement is important for the propagation of Alphaviruses,
because all cattle contain antibodies directed
against mosquitoes and many Alphaviruses.
All mosquito cell lines are handled in the same
manner. Each of the mosquito cultures, U4.4,
C6/36, and C7-10 which originally were grown
in Mitsuhashi and Maramorosch media (M &
M), have also been adapted in the authors laboratory to grow in the same EMEM medium
described for BHK cells.
Troubleshooting
Failure of the plaque assay may be due to
many reasons. The most frequently encountered problems are listed in Table 15B.1.2.
Anticipated Results
It may require a few trials to master the
plaque assay. However, the protocols described
above should generate plaques, especially if
SVHR is being employed. The sensitivity of
the plaque assay for SVHR is not due to the
procedure itself, but rather to the low particle/pfu ratio of the virus, 5 particles/pfu.
Reproducible titers therefore result in good accuracy. The elution of a single plaque, in general, does not generate a large yield of virus,
1000 pfu/plaque; however, this is sufcient
for a second round of plaque purication or for
amplication of the plaque by infection of the
chosen cell line.
Once proper conditions are established,
mosquito cell culture is relatively simple and
can produce 8-fold more cells/ml than a
BHK culture of comparable size. An additional
advantage to working with this cell line is the
long viability of these cells. Mosquito U4.4
cells can be kept at 28 C for several months,
and viable cells can be rescued for subculture. Differences in the levels of virus produced from each of the mosquito cell cultures
have been observed, with C7-10 producing the
15B.1.32
Current Protocols in Microbiology
Time Considerations
Splitting mosquito cell cultures should only
take 15 to 30 min, depending on the number
of asks being split. In general, all infection
protocols require 1.5 to 2 hr to complete,
including rocking time. This also is the case
for the transfections, which require 1.5 to 3 hr
to complete, depending upon the number of
samples.
The C7-10 plaque assay protocol will take
3 hr depending on the number of virus sample to be titered.
Acknowledgement
The authors wish to acknowledge
Dr. Andrew Liss, Section of Molecular Genetics Microbiology and Immunology, University
of Texas at Austin, for the helpful hints and
details outlined in the CEF protocol (Support
Protocol 1).
Literature Cited
Brown, D.T., Smith, J.F., Gliedman, J.B., Riedel,
B., Filtzer, D., and Renz, D. 1976. Morphogenesis of Sindbis virus in cultured mosquito cells.
In Invertebrate Tissue Culture, Applications in
Medicine, Biology, and Agriculture (E. Kurstak
and K. Maramorosch, eds.) pp. 35-48. Academic
Press, New York.
Gilman, M. 2002. Preparation of cytoplasmic RNA
from tissue culture cells. In Current Protocols
in Molecular Biology (F.M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J.G. Seidman, J.A.
Smith, and K. Struhl, eds.) pp. 4.1.1-4.1.5. John
Wiley & Sons, Hoboken, N.J.
Hernandez, R., Lee, H., Nelson, C., and Brown,
D.T. 2000. A single deletion in the membraneproximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly. J.
Virol. 74:4220-4228.
Hernandez, R., Luo, T., and Brown, D.T. 2001. Exposure to low pH is not required for penetration of mosquito cells by Sindbis virus. J. Virol.
75:2010-2013.
Hernandez, R., Sinodis, C., Horton, M., Ferreira,
D., Yang, C., and Brown, D.T. 2003. Deletions
Animal RNA
Viruses
15B.1.33
Current Protocols in Microbiology
Sindbis Virus:
Propagation,
Quantication,
and Storage
15B.1.34
Current Protocols in Microbiology
UNIT 15F.2
Newcastle disease virus (NDV) is an avian pathogen. Some strains of the virus are important agricultural pathogens that cause often fatal systemic, respiratory, or neurological
disease in poultry. NDV is also a prototype paramyxovirus that has been utilized in laboratories to elucidate many fundamental principles of infection by this family of viruses.
Preparation and titration of purified virus stocks are basic procedures required in most
studies of this important pathogen.
Although NDV is found only in birds in nature, NDV will infect a wide variety of cell types
derived from certain mammals as well. Stocks of the virus can be prepared by growth
in either embryonated chicken eggs or cells grown in tissue culture. Preparation of virus
stocks by growth in embryonated eggs is an old and reliable method of producing stocks
of many different types of viruses and is, by far, the superior method for producing high
titer NDV stocks. This method has the added advantage of producing stocks of infectious
virus regardless of the virus strain. The same cannot be said of stocks produced in tissue
culture cells; while stocks of virulent NDV strains prepared in tissue culture cells are
infectious, stocks of avirulent NDV prepared in tissue culture cells are not infectious.
Both methods of NDV propagation are described in this unit.
Virus stocks grown in eggs are prepared by inoculating embryonated eggs with a zero
stock (low titer stock) of virus, and then harvesting and purifying progeny virus from
the infected eggs. Alternatively, virus can be purified from supernatants of infected
tissue culture cells. The procedure for preparation of the zero stocks is described in
Basic Protocol 2, followed by procedures for growing virus in embryonated eggs (Basic
Protocol 3), as well as the purification of egg-derived virus (Basic Protocol 4). Growth
and purification of virus derived from infection of tissue culture cells are described in
the Alternate Protocol.
The titration of NDV (determination of virus concentration in a preparation) can be
measured in several ways, but the two most frequently used are described in this unit.
Infectivity of the stock (the number of infectious virus particles) in plaque-forming units
(pfu)/ml is routinely determined by a plaque assay as described in Basic Protocol 1. A
relative measure of the concentration of total virus particles is routinely accomplished
by determining the hemagglutination titer and is described in Basic Protocol 5.
CAUTION: Virulent Newcastle disease viruses are Biosafety Level 3 (BSL-3) pathogens.
Follow all appropriate guidelines for the use and handling of pathogenic microorganisms.
See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. Perform
all work in a Class II biosafety cabinet (BSC).
CAUTION: Avirulent Newcastle disease viruses are Biosafety Level 2 (BSL-2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more
information. Perform all work in a Class II BSC.
CAUTION: Decontaminate all surfaces that have come in contact with virus using 70%
ethanol, 1% sodium dodecyl sulfate, or Vesphene. Autoclave all waste products and
autoclavable items. Nonautoclavable liquids may be brought to concentrations of 70%
ethanol, 1% sodium dodecyl sulfate, or 0.78% Vesphene for decontamination. Viruses
Animal RNA
Viruses
Contributed by Lori W. McGinnes, Homer Pantua, Julie Reitter, and Trudy G. Morrison
Current Protocols in Microbiology (2006) 15F.2.1-15F.2.18
C 2006 by John Wiley & Sons, Inc.
Copyright
15F.2.1
Supplement 1
removed from the BSC for centrifugation steps must be in sealed tubes, and the outsides of
the tubes must be cleaned with 70% ethanol. Centrifugation of BSL-3 virus preparations
must be performed in a BSL-3 laboratory.
IMPORTANT NOTE: Virulent or velogenic NDV has been classified as a Select Agent by
the United States Government. Refer to the CDC Select Agent Program for more information (http://www.cdc.gov/od/sap). Also refer to UNIT 1A.1 and other pertinent resources
(see APPENDIX 1B).
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aeseptic technique should be used accordingly.
BASIC
PROTOCOL 1
Materials
Avian tissue culture cells (e.g., ELL-O chicken cells; ATCC #UMNSAH/DF-1)
Virus stock (see Table 15F.2.1; Southeast Poultry Research Laboratory, Athens,
GA)
Tissue culture medium (e.g., DMEM with supplements; see recipe)
NDV overlay medium with or without trypsin, 46 C (see recipes)
Methanol (optional)
Giemsa stain (Sigma; optional)
35- or 60-mm tissue culture plates
Pipets
46 C water bath
NOTE: All culture incubations are performed in a humidified 37.5 C, 5% CO2 incubator
unless otherwise specified.
1. Prepare confluent monolayers of tissue culture cells by adding 7.5 105 cells or 3
105 cells per 60-mm or 35-mm tissue culture plates, respectively. Incubate 16 to
20 hr at 37 C or until cells are confluent.
These cell numbers can be added in any volume under the standard 5 ml for 60-mm and
2 ml for 35-mm plates. The cell numbers added is the important thing. Medium is then
added to bring the volumes up to the standard 5 ml or 2 ml. The surface area of the plates
is more important than the volume of the media overlaying the monolayer.
4. Add 0.2 ml virus dilution to the plate of tissue culture cells. Spread the inoculum by
rocking the plate.
Each dilution is usually plated in duplicate.
15F.2.2
Supplement 1
5. Incubate tissue culture cell monolayer with added virus for 45 min at 37.5 C.
During this time the virus is adsorbed onto the cells.
This step may also be performed at room temperature or at 4 C. Temperatures lower than
15 C to 20 C arrest infection at attachment. Attachment occurs from 4 C to 20 C, but
infection does not proceed until the cells are placed at the higher temperatures.
6. Cover cell monolayer with NDV overlay medium by adding down the side of the
plate 5 ml for a 60-mm plate or 2 ml for a 35-mm plate.
Use NDV overlay agar without trypsin for virulent strains of NDV. Use NDV overlay agar
with trypsin added for avirulent strains.
The original virus inoculum may or may not be removed with a pipet before adding the
agar overlay. The virus inoculum may dilute the overlay agar, resulting in failure of the
agar to solidify.
7. Allow plates to sit undisturbed at room temperature until the agar solidifies
(30 min).
8. Incubate plates until plaques appear.
The time required for plaques to appear depends upon the NDV strain used. Plaques of
virulent NDV can appear from 36 to 48 hr while plaques of avirulent NDV may require
40 hr or longer. Plaques can disappear after 48 to 72 hr.
9. Count plaques.
Plaques often appear as clear areas in the monolayer when holding the monolayer up to
a light. Alternatively, they may appear as whiter or opalescent areas in the monolayer.
Titer = number of plaques 5 dilution factor.
BASIC
PROTOCOL 2
Materials
Viral plaque (Basic Protocol 1)
Hanks balanced salt solution (HBSS; APPENDIX 2A or Gibco 24020; contains
calcium, magnesium, and phenol red)
Pasteur pipets
1.5-ml microcentrifuge tubes (polypropylene)
Microcentrifuge (Eppendorf or comparable)
Animal RNA
Viruses
15F.2.3
Current Protocols in Microbiology
Supplement 1
Figure 15F.2.1 Plates containing Newcastle disease virus plaques formed on monolayers of
avian cells (right) or COS-7 cells (left). The agar overlay has been removed and the cells on the
monolayers xed with methanol and stained with Giemsa stain. Plaques are the circular areas
with no stained cells.
2. Place the agar plug in 0.2 ml HBSS in a sterile 1.5-ml microcentrifuge tube, mix,
and incubate 30 min on ice.
3. Remove the agar by centrifuging 5 min at 3000 g, 4 C.
4. Plate several dilutions of the supernatant obtained from the single plaque as in Basic
Protocol 1 in order to obtain plates with individual, isolated single plaques.
5. Remove a single plaque and collect virus as described in steps 1 to 3.
7. Grow the virus (see Basic Protocol 3) and collect the allantoic fluid in a 15-ml
polypropylene centrifuge tube as for large virus preparations (see Basic Protocol 4,
steps 1 and 2).
15F.2.4
Supplement 1
9. Determine the virus titer of the supernatant (zero stock), using a plaque assay (Basic
Protocol 1).
10. Dispense aliquots of the zero stock and freeze up to 5 years at 80 C.
Aliquots of 0.5 ml are recommended.
BASIC
PROTOCOL 3
Materials
<10-day-old special pathogen free (SPF) eggs (e.g., Charles River/SPAFAS)
Zero stock (Basic Protocol 2)
Hanks balanced salt solution (HBSS; APPENDIX 2A or Gibco 24020; contains
calcium, magnesium, and phenol red) containing 2% (v/v) heat-inactivated FBS
(APPENDIX 2A)
70% ethanol
Humidified egg incubator (e.g., Lyon Electric)
Egg candler (e.g., Lyon Electric)
16-G, 10-mm needle
1-ml tuberculin syringe with an attached 26-G, 10-mm needle (e.g., Becton
Dickinson)
Nail polish or wax
1. Incubate eggs immediately upon arrival at 37.5 C in a humidified egg incubator.
Order eggs 2 weeks before the actual starting date and specify that eggs are to be <10 days
of age, preferably 8 days. If the eggs arrive cold, warm to room temperature prior to
placing them at 37.5 C.
2. Candle eggs at 10 days of age. Hold each egg up to a light source (egg candler) to
visualize the location of the embryo, membranes, and blood vessels. Discard eggs
with dead embryos.
Eggs with dead embryos will not have clear blood vessels and will have the embryo at
the bottom of the egg.
3. Turn the egg so the embryo is in the back and the yolk is in front. Mark with a pencil,
on the side of the egg opposite the embryo, a line where the membrane meets the air
sac. Also place a dot 5 mm above that line between blood vessels. The dot marks
the injection point (see Fig. 15F.2.2).
4. Dilute the zero stock in HBSS containing 2% heat-inactivated FBS so that there are
103 pfu in 0.1 ml.
Note that in Basic Protocol 2, step 6 for preparing a zero stock, virus recovered from a
single plaque is injected into a single egg without dilution. In contrast, diluted virus is
used in this protocol.
Animal RNA
Viruses
15F.2.5
Supplement 1
6. Using a 16-G, 10-mm needle, puncture each egg at the pencil dot.
Be careful to keep holes small.
7. Fill a 1-mm tuberculin syringe with attached 26-G, 10-mm needle with the diluted
zero stock and inject 0.1 ml of the virus dilution per egg.
Make sure needle is inserted such that it will enter the membrane below the line where
the membrane meets the air sac but not deep enough to penetrate the embryo.
Be careful not to push air into the eggs, particularly with the last 0.1 ml in the syringe.
Figure 15F.2.2 Injection of a 10-day-old chick embryo. (A) Cross section of a 10-day-old chick embryo with the major
structural components labeled. (B) Intact 10-day-old egg viewed with an egg candler. The top of the egg shows the position
of the pencil marks that locate the membrane at the bottom of the air sac and the position at which the injection needle
should be inserted. The diagram of the needle indicates the site of injection of virus. The needle is at a shallow angle to
avoid the embryo and is aimed away from the yolk sac. (C) Intact egg turned with the embryo in back and the yolk sac in
front.
NDV:
Propagation,
Quantication,
and Storage
15F.2.6
Supplement 1
11. As soon as the embryos are dead, place eggs at 4 C for at least 2 hr before collecting
allantoic fluid.
Incubation at 4 C terminates infection.
BASIC
PROTOCOL 4
The allantoic fluid must be removed from each infected egg. The allantoic fluid contains a high titer of virus; however, virus must be purified from the contaminating egg
components for most applications. Purification is accomplished by first concentrating the
virus by centrifugation. The resuspended virus is then sedimented through a 20% sucrose
solution onto a 65% sucrose pad by centrifugation, a step that further purifies the virus
from contaminating egg material and concentrates the virus. The virus obtained at the
20% to 65% sucrose interface is satisfactory for most applications. If necessary, further
purification can be attained by equilibrium centrifugation in a sucrose gradient.
Materials
Infected eggs (Basic Protocol 3)
70% ethanol
Hanks balanced salt solution (HBSS; APPENDIX 2A or Gibco 24020; contains
calcium, magnesium, and phenol red)
70% ethanol/1% (w/v) SDS
HBSS (APPENDIX 2A or Gibco 24020) containing 2% heat-inactivated FBS (APPENDIX
2A)
20%, 25%, 35%, 45%, 55%, and 65% (w/v) sucrose in standard buffer (see below
for standard buffer)
Standard buffer: 0.1 M NaCl/0.01 M TrisCl, pH 7.4 (APPENDIX 2A)/2 mM EDTA
Forceps
10-ml syringe with a 16-G needle
Microspoon-end spatula (e.g., VWR)
250-ml bottles for GSA rotor (or comparable)
Sorvall centrifuge (or comparable) with GSA rotor
Beckman Type 19 bottles
Deldrin cap assemblies (or comparable): sterilize by rinsing with 70% ethanol and
exposing to UV light for 20 hr
Beckman ultracentrifuge with type 19 rotor
10-ml glass Dounce homogenizer with a tight-fitting pestle
30-ml Sorvall centrifuge tube
Sorval SS-34 rotor
Beckman SW28.1 rotor with large buckets and small buckets
17- and 30-ml Beckman ultracentrifuge tubes (or comparable)
Ring stand with clamps
10-ml syringe with a 21-G needle
1- or 2-ml cryovials
80 C freezer
NOTE: Precool all centrifuges and rotors and chill all solutions and glassware used for
purification of virus.
NOTE: The following steps are for ten dozen infected eggs.
Animal RNA
Viruses
15F.2.7
Current Protocols in Microbiology
Supplement 1
2. Open the wide end of the eggs above the membrane using sterile forceps. Clear away
all shell fragments.
3. Break the milky-white air sac membrane covering the allantoic sac. Pull it to the
edges of the egg. Remove the fluid underneath the membrane using a 10-ml syringe
(16-G needle) and a microspoon-end spatula to push the membranes clear of needle
bore.
A single egg should yield between 3 and 10 ml fluid. The allantoic sac membrane, a clear
membrane under the air sac membrane, can also be broken to gain access to more fluid.
Care should be taken, however, not to break the yolk sac. If the yolk sack is broken, do
not remove more fluid from the egg.
After removal of fluid, eggs are autoclaved.
4. Place the harvested fluid directly into chilled, sterile 250-ml centrifuge bottles on
ice.
5. Centrifuge the pooled fluid 10 min at 4000 g (5000 rpm), 4 C in a precooled
Sorvall centrifuge with GSA rotor.
This will remove debris and erythrocytes.
Concentrate virus
6. Decant the supernatant into chilled, sterile Beckman Type 19 bottles.
7. Add HBSS to completely fill all bottles.
HBSS should be precooled to 4 C.
These bottles have a tendency to collapse if not completely filled.
Purify virus
11. Resuspend the pellet in 5 ml HBSS containing 2% heat-inactivated FBS using
10 strokes in a Dounce homogenizer.
The pellet is very sticky, thus, a dounce homogenizer is useful for resuspending the
particulate material.
12. Pour the resuspended material into a 30-ml Sorvall centrifuge tube.
13. Centrifuge 10 min at 6000 g (7000 rpm), 4 C using a SS-34 rotor.
This step removes large particulate matter. The virus is in the supernatant.
The pellet should be washed (steps 14 to 15) to release residual virus from the pellet.
14. Pour the supernatant into another 30-ml Sorvall centrifuge tube and keep on ice.
NDV:
Propagation,
Quantication,
and Storage
15F.2.8
Supplement 1
Figure 15F.2.3 Preparation of sucrose gradients used for virus concentration and purication
of Newcastle disease virus. (A) Construction of the discontinuous gradient described in Basic
Protocol 4, step 18. Resuspended virus is placed on top of the 20% sucrose layer. The position
of virus is indicated after centrifugation. (B) Construction of the continuous gradient described in
Basic Protocol 4, steps 22 to 24, used to further purify the virus preparation. The virus obtained
from A is diluted in standard buffer and then placed on top of the sucrose gradient. The position
of the virus is shown after centrifugation.
15. Add 5 ml HBSS containing 2% FBS to the pellet derived in step 13 and resuspend
with a Dounce homogenizer to release any residual virus.
16. Pour the resuspended pellet into a 30-ml Sorvall centrifuge tube and centrifuge 10 min
at 6000 g (7000 rpm), 4 C using an SS-34 rotor.
17. Combine the supernatants from steps 13 and 16 (10 ml) and centrifuge 10 min at
17,000 g (12,000 rpm), 4 C using a SS-34 rotor using a SS-34 rotor.
This step further clarifies the virus suspension. Virus remains in the supernatant.
20. Clamp the tube firmly on a ring stand in a Class II BSC. Collect the virus by
puncturing the bottom of the tube with a 21-G needle. Let the 65% sucrose drain
out. Collect the virus layer into a sterile tube as it drips from the bottom of the tube.
Animal RNA
Viruses
15F.2.9
Current Protocols in Microbiology
Supplement 1
This working stock is pure enough for most applications such as infections of cell
monolayers.
After harvesting the virus, autoclave the centrifuge tube and remaining sucrose.
21. Dilute the stock of virus by adding an equal volume of HBSS containing 2% FBS.
Dispense aliquots into cryovials and store up to 10 years at 80 C or proceed with
further purification.
Virus is stable through multiple freeze-thaw cycles.
26. Clamp the tube onto a ring stand. Carefully insert a 10-ml syringe with a 21-G needle
into the side of the tube just below the virus band. Remove the virus band by gently
pulling on the plunger of the syringe, sweeping the needle back and forth to remove
the entire virus band.
The virus can be entirely removed in 2 to 3 ml.
If necessary, this stock of virus may be concentrated in a discontinuous sucrose gradient
as described in steps 18 to 20.
Materials
Avian tissue culture cells (e.g., ELL-O chicken cells; ATCC #UMNSAH/DF-1)
Tissue culture medium (e.g., DMEM with supplements; see recipe)
Virus zero stock (Basic Protocol 2), titered
NDV:
Propagation,
Quantication,
and Storage
15F.2.10
Supplement 1
2. Infect cells with virus at a multiplicity of infection (MOI) of 10, i.e., add 10 plaque
forming units (pfu) per cell. Calculate the required volume of virus stock as follows:
no. cells to be infected 10/titer of virus stock (pfu/ml) = amount (ml) virus
stock to use.
The MOI may vary from 10 to 25. There are usually 8 106 cells in a confluent 100-mm
plate.
3. Dilute virus stock into tissue culture medium such that the appropriate number of
pfu are in 0.5 ml for infection of a 100-mm plate of cells.
4. Remove medium from the monolayer.
5. Add the 0.5 ml diluted virus to the monolayer and spread the inoculum over the
entire monolayer by rocking the plate.
6. Incubate tissue culture cell monolayer with added virus 45 min at 37.5 C.
During this time the virus is adsorbed onto the cells.
7. Remove the medium, wash the monolayers two times with 10 ml tissue culture
medium, and then add tissue culture medium to the cells.
This step, which removes unbound virus, is optional unless it is important to remove
residual virus in the inoculum.
11. Purify the virus by centrifugation through a 20% sucrose gradient onto a 65% sucrose
pad as described in Basic Protocol 4, steps 18 to 21.
BASIC
PROTOCOL 5
Animal RNA
Viruses
15F.2.11
Current Protocols in Microbiology
Supplement 1
Materials
Red blood cells (avian alsever or guinea pig alsever; BioLink)
PBS, ice cold (APPENDIX 2A)
1.5 ml microcentrifuge tubes
Microcentrifuge
Hemacytometer
Microscope
Microtiter plates (96-well, round bottom)
NOTE: Maintain all solutions on ice.
NDV:
Propagation,
Quantication,
and Storage
Figure 15F.2.4 A microtiter plate representative of a hemagglutination assay (HA) for two different preparations of virus (in duplicate). Rows A and B contain dilutions of stock 1, and rows C and
D, contain dilutions of stock 2. Row E wells show red blood cells (RBCs) without added virus, and
no hemagglutination is visible. Virus in each stock was diluted as indicated at the bottom of the
plate. Stock 1 hemagglutinated RBCs at dilutions up to 1:256 (RBCs formed a lattice), but not at
further dilutions; thus, the HA titer was 256. Stock 2 hemagglutinated RBCs up to dilutions of 1:32
but not at higher dilutions; thus, the HA titer was 32.
15F.2.12
Supplement 1
9. Into microtiter plate wells, dispense 10 l of each virus dilution and 10 l of PBS
with no virus for a negative control.
If possible, a positive control with a known amount of virus should also be included in
the assay. Assays should be performed in duplicate.
11. Incubate 1 hr at 4 C.
12. Score wells as negative hemagglutination by the appearance of RBC buttons.
The HA titer is the reciprocal of the last dilution of virus that results in hemagglutination
(lattice formation). The RBCs will settle out to form a button where there is insufficient
virus to agglutinate the RBCs (See Figure 15F.2.4).
15F.2.13
Current Protocols in Microbiology
Supplement 1
Add 55 ml of 1.8% (w/v) agar (prepare by heating in sterile water and cooling to
46 C)
Maintain medium up to 1 hr at 46 C
Additions to the media will depend upon the requirements of the cells used. For example,
some cells require added amino acids (essential amino acids).
This overlay is used for deriving plaques from virulent NDV strains.
COMMENTARY
Background Information
NDV:
Propagation,
Quantication,
and Storage
NDV is a member of the family Paramyxoviridae, which are enveloped, negativestranded, RNA viruses. The NDV genome encodes six genes, in the 3 to 5 gene order:
nucleocapsid protein (NP), phosphoprotein
(P), matrix protein (M), fusion glycoprotein
(F), hemagglutinin-neuraminidase glycoprotein (HN), and L protein (reviewed in Nagai et
al., 1989; Lamb and Kolakofsky, 2001; Morrison, 2003). The NP, P, and L proteins are
associated with the genome in the core of
the virus particle. This core is surrounded by
a membrane derived from host cell plasma
membranes. The M protein lines the inner surface of the host-derived membrane, and F and
HN are transmembrane glycoproteins, which
form the surface spike structures of virions.
The HN protein is the virus attachment protein
and binds to sialic acidcontaining receptors.
The F protein is directly responsible for the
fusion of the viral membrane and the cellular membrane during virus entry (reviewed in
Nagai et al., 1989; Lamb and Kolakofsky,
2001; Morrison, 2003).
In nature, many different strains or variants of NDV exist (see Table 15F.2.1 for a list
of the more commonly used strains). These
strains can be classified into two categories,
virulent and avirulent, reflecting their ability
to form plaques on monolayers of tissue culture cells and their ability to spread in an infected animal. This classification is based on
the sequence at the cleavage site of the F protein (Nagai and Klenk, 1977; Toyoda et al.,
1987; Glickman et al., 1988). The F protein
is synthesized as a precursor, F0 , which must
be proteolytically cleaved into disulfide-linked
heterodimer, F1 and F2 , to function as a fusion
protein (Nagai et al., 1976; Garten et al., 1980;
Rott and Klenk, 1988). F protein in virus particles must be cleaved for virus particles to
be infectious. The F protein found in virulent
viruses has a furin recognition sequence at the
cleavage site (Toyoda et al., 1987; Glickman
et al., 1988). This sequence (RXKR or RXRR)
is recognized and cleaved by a ubiquitous set
of host cell proteases located in the trans-Golgi
membranes of most cell types (reviewed in
Gotoh et al., 1992; Nagai, 1993). Thus,
15F.2.14
Supplement 1
virulent NDV readily forms plaques on monolayers of tissue culture cells and can spread in
many organs of birds. In contrast, the cleavage site of the F protein encoded by avirulent
viruses lacks a furin recognition sequence but
rather has single basic residues at the cleavage site (Nagai, 1993). Cleavage of these F
proteins is carried out by trypsin-like host proteases, but these proteases are extracellular and
are located only in respiratory and gastrointestinal tracts of birds or in the chorioallantoic
membranes of embryonated chicken eggs (Gotoh et al., 1990; Nagai, 1993). Avirulent strains
do not form plaques on monolayers of cells
grown in tissue culture, nor do they spread in
most tissues of birds. Virions released from infected tissue culture cells contain an uncleaved
F protein and are, therefore, noninfectious. Infectious stocks of these viruses can be prepared in embryonated eggs since the chorioallantoic membranes contain an enzyme (Factor
X) that will cleave the fusion protein of avirulent viruses. Avirulent NDV will form plaques
on monolayers of tissue culture grown cells if
an exogenous protease such as trypsin is added
to monolayers in the overlay media.
Historically, NDV strains have also been
classified into three categories depending upon
the time required for the virus to kill chicken
NDV straina
Egg virulencec
B1 Hitchner
avirulent
lentogenic
La Sota
avirulent
lentogenic
D26/76
avirulent
lentogenic
Queensland
avirulent
lentogenic
Ulster
avirulent
lentogenic
Roakin
virulent
mesogenic
virulent
mesogenic
Beaudette C
virulent
velogenic
Italien
virulent
velogenic
Texas G. B.
virulent
velogenic
Hertz
virulent
velogenic
Australia-Victoria
virulent
velogenic
Miyadera
virulent
velogenic
Animal RNA
Viruses
15F.2.15
Current Protocols in Microbiology
Supplement 1
Table 15F.2.2 Troubleshooting Guide for Newcastle Disease Virus Growth, Purication, and
Assay Procedures
Problem
Possible cause
Solution
Plaque assay
Contamination
Lumpy agar
See below
Nonpermissive cells
No virus in inoculum
NDV strain not lethal to embryos Harvest allantoic fluid and titer
within 70 hr
by plaque assay or HA
Purification of Virus
Very little fluid in eggs
NDV:
Propagation,
Quantication,
and Storage
15F.2.16
Supplement 1
Table 15F.2.2 Troubleshooting Guide for Newcastle Disease Virus Growth, Purication, and
Assay Procedures, continued
Problem
Possible cause
Solution
Harvested fluid is
yellow
Repellet supernatants
RBCs expired
Remake PBS
Defective RBCs
Hemagglutination Assay
No RBC in pellet after
washes
No HA activity
All dilutions
hemagglutinate
Results inconclusive
Improper mixing
Anticipated Results
Plaque assays
Plaques will appear as clear or opalescent
areas within the monolayers. These are areas
of cell death that result from the spreading infection. Plaques may be directly seen through
the agar overlay or after removing the agar
overlay and staining the cell monolayers with
Giemsa as shown in Figure 15F.2.1.
To determine the titer of the virus stock,
count plaques on plates where the number
of plaques are easy to see and easy to count
(30 to 100). The titer of the stock (pfu/ml)
is equal to the number of plaques multiplied
by the dilution factor multiplied by five (assuming an inoculum of 0.2 ml). For example,
if one counted 30 plaques on the 107 dilution plate, the titer is 30 107 5 = 1.5
109 pfu/ml.
Plaque assay
Plaques derived from virulent strains of
NDV appear at 36 to 40 hr. Plaques derived from avirulent NDV in the presence
of trypsin may take from 40 to 72 hr to
appear.
Hemagglutinin assay
Expect 46 103 pfu/HA unit.
Time Considerations
Animal RNA
Viruses
15F.2.17
Current Protocols in Microbiology
Supplement 1
Literature Cited
Alexander, D.J. 1997. Newcastle disease and other
avian paramyxoviruses. In Diseases of Poultry
(B.W. Cainek, H.J. Barnes, C.W. Beard, L.R.
McDougald, and L.J. Saif, eds.) pp. 541-570.
Iowa State University Press, Ames, Iowa.
Beard, C.W. and Hanson, R.P. 1984. Newcastle disease. In Disease of Poultry (M.S. Hofstad, ed.)
pp. 452-470. Iowa State University Press, Ames,
Iowa.
Garten, W., Berk, W., Nagai, R., Rott, R., and Klenk,
H.D. 1980. Mutational changes of the protease
susceptibility of glycoprotein F of Newcastle
disease virus: Effects on pathogenicity. J. Gen.
Virol. 50:135-147.
Glickman, R.L., Syddall, R.J., Sheehan, R.M., and
Bratt, M.A. 1988. Quantitative basic residue requirements in the cleavage activation site of
the fusion glycoprotein as a determinant of
virulence of Newcastle disease virus. J. Virol.
62:354-356.
Gotoh, B., Ogasawara, T., Toyoda, T., Inocencio,
N.M., Hamaguchi, M., and Nagai, Y. 1990. An
endoprotease homologous to the blood clotting
factor X as a determinant of viral tropism in
chick embryo. EMBO J. 9:4189-4195.
Gotoh, B., Ohnishi, Y., Inocencio, N.M., Esaki,
E., Nakayama, K., Barr, P.J., Thomas, G., and
Nagai, Y. 1992. Mammalian subtilisin-related
proteinases in cleavage activation of the
paramyxovirus fusion glycoprotein: Superiority of furin/PACE to PC2 or PC1/PC3. J. Virol.
66:6391-6397.
NDV:
Propagation,
Quantication,
and Storage
15F.2.18
Supplement 1
UNIT 15G.1
This unit covers several techniques for propagating, quantifying, and storing human influenza A viruses from existing stocks (see Basic Protocols 1 and 2) or from primary
clinical specimens (see Alternate Protocols 2 and 4). Virus isolation is a highly sensitive
and useful technique for the identification of viral infections. An important advantage
of virus isolation is the amplification of the virus from the original specimen, making it
available for further antigenic and genetic characterization. Influenza viruses are quantified either by a unit of hemagglutination, which is not a measure of an absolute amount
of virus but is an operational unit dependent on the method used for the hemagglutination assay titration (see Basic Protocol 3), or by determining infectious units using
the 50% tissue culture infectious dose assay (see Basic Protocol 4), 50% egg infectious
dose assay (see Basic Protocol 5), or plaque assay (see Basic Protocol 6). After isolating
and quantifying human influenza, the product must be properly stored to maintain virus
viability.
CAUTION: The protocols presented in this unit are for use with contemporary human influenza virus subtypes, which must be handled under Biosafety Level 2 (BSL-2)
conditions. For biosafety levels recommended for noncontemporary or nonhuman influenza viruses, refer to the influenza agent summary statement in the Biosafety in
Microbiological and Biomedical Laboratories manual published by the Centers for Disease Control and Prevention and National Institutes of Health (see interim guidelines at
http://www.cdc.gov/flu/pdf/h2n2bsl3.pdf). All work with infectious influenza virus should
be conducted in a class II biological safety cabinet. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for additional information.
IMPORTANT NOTE: Use of trade names and commercial sources is for identification
only and does not imply endorsement by the Public Health Service or by the U.S.
Department of Health and Human Services.
BASIC
PROTOCOL 1
Madin Darby canine kidney (MDCK) cells (ATCC# CCL-34) are the preferred host
for the isolation and characterization of influenza A and B viruses, but not influenza
C viruses due to the incompatibility of sialic acid moieties on the cell surface with the
viral receptor specificity. Influenza viruses are also able to replicate in a few primary,
diploid, and continuous cell cultures. The propagation of influenza viruses in primary
monkey kidney (PMK) cells (Viro-Med Laboratories or BioWhittaker) and R-mix fresh
cells (Diagnostic Hybrids) is presented in Alternate Protocol 1. For preparation of a
virus stock from a primary clinical specimen, refer to Alternate Protocol 2. The protocol
described below is for the propagation of existing human influenza A and B virus stocks
in MDCK cell culture.
Materials
Influenza virus stock
Madin Darby canine kidney (MDCK) cells confluent in a 75-cm2 flask (see
Support Protocol 1)
Phosphate-buffered saline (PBS) containing potassium (APPENDIX 2A)
cDMEM/7.5% BSA (see recipe)
Influenza virus growth medium (see recipe)
Contributed by Kristy J. Szretter, Amanda L. Balish, and Jacqueline M. Katz
Current Protocols in Microbiology (2006) 15G.1.1-15G.1.22
C 2006 by John Wiley & Sons, Inc.
Copyright
Animal RNA
Viruses
15G.1.1
Supplement 3
2. Remove the MDCK growth medium using a sterile 25-ml pipet and wash the
MDCK monolayer two times with 10 ml of room temperature PBS and once with
cDMEM/7.5% BSA, removing washes with sterile 10-ml pipets.
This cell line requires the cells to be confluent, i.e., completely covering the surface of the
flask, before the virus is inoculated. If the monolayer is overgrown (i.e., cells overlapping),
it is less sensitive to virus infection.
Fetal bovine serum (FBS) inhibits viral entry and must be removed for efficient infection
of cells.
4. Inoculate flask with 1 ml virus and rotate to cover monolayer with inoculum. Take
care not to add medium directly onto the monolayer as this may disrupt the cells.
5. Incubate inoculated flasks for a minimum of 30 min or up to 1 hr at 37 C.
Incubation for >1 hr may cause the MDCK cell monolayer to dry out.
Inuenza:
Propagation,
Quantication,
and Storage
15G.1.2
Supplement 3
(TPCK)-trypsin for virus growth, but do require the addition of 0.5% FBS to medium
instead of BSA. Limitations of PMK cells include lower viral titers, contamination by
adventitious simian agents such as foamy viruses, and batch-to-batch variability.
ALTERNATE
PROTOCOL 2
Specimens for virus isolation should be kept at 4 C immediately after collection and
inoculated into susceptible cell cultures as soon as possible. If the specimen cannot
be processed within 48 to 72 hr, the specimen should be frozen up to 1 year at or
below 70 C. Primary influenza specimens require the addition of antibiotics prior to
inoculating into cell culture (see Support Protocol 3). Due to the unknown amount of
influenza virus in the clinical specimen, it may require multiple passages in cell culture
for the virus to grow. Typically, two passages may follow the initial inoculation before a
specimen is considered to be negative by virus isolation.
2. Remove the MDCK growth medium using a sterile 25-ml pipet and wash the MDCK
monolayer two times with 10 ml of room temperature PBS and one time with
cDMEM/7.5% BSA, removing washes with sterile 10-ml pipets.
Fetal bovine serum (FBS) inhibits viral entry and must be removed for efficient infection
of cells.
4. Inoculate flask with 200 l virus and rotate to cover monolayer with inoculum. Take
care not to add medium directly onto the monolayer as this may disrupt the cells.
5. Incubate inoculated flasks for 30 min to 1 hr at 37 C.
After 1 hr the monolayer will begin to dry out.
8. Harvest cell culture supernatant into 50-ml tubes, when at least 75% of the cell
monolayer is exhibiting CPE.
Typical CPE by influenza viruses include rounding up of infected cells and detachment
from culture flask. If no CPE is present, perform a hemagglutination assay (see Basic
Protocol 3). If HA titer is <8 HAU, then 600 l of undiluted supernatant should be
passaged into a new MDCK flask. Primary clinical specimens should be passaged up to
three times. If no HA titer is detected after three passages, the virus should be considered
unrecoverable (i.e., influenza virus was not recovered from the specimen by this method).
Animal RNA
Viruses
15G.1.3
Current Protocols in Microbiology
Supplement 3
2. Inoculate each shell vial monolayer with 0.2 ml primary clinical specimen.
This volume is sufficient to cover the monolayer in a shell vial. Do not pipet inoculum
directly onto the monolayer as this may disrupt the cells.
3. Centrifuge shell vials for 30 to 60 min at 700 g, at room temperature (24 to 27 C).
Centrifugation of shell vials allows for more efficient absorption of virus into the cell
monolayer.
5. Observe the shell vials during the 72-hr incubation and harvest supernatant into
15-ml tubes at the first signs of cytopathic effect (CPE). Dispose of tissue culture
materials in an appropriate biological waste container.
Inuenza:
Propagation,
Quantication,
and Storage
Typical CPE by influenza viruses include rounding up of infected cells and detachment
from culture flask.
15G.1.4
Supplement 3
BASIC
PROTOCOL 2
In the past, influenza viruses were often isolated in embryonated eggs; however, today, the
majority of laboratories use cell culture due to availability and ease of isolation of some
contemporary human influenza strains in mammalian cells versus embryonated eggs.
Nevertheless, many laboratory-adapted viruses and all vaccine strains are egg-grown and
may be propagated readily in 10- to 11-day-old embryonated chicken eggs.
Materials
Influenza virus stock
Egg diluent: antibiotic-supplemented KPBS or tryptose phosphate broth (see
recipes)
10- to 11-day-old embryonated chicken eggs (see Support Protocol 2)
70% ethanol
Glue (Elmers), nonsterile
22-G, 1 1 /2 -in. and 18-G, 1 /2 -in. needles
1-ml syringe
33 to 35 C incubator
Forceps, sterile
10-ml pipets
50-ml plastic concical tubes
2-ml cryovials, sterile (Nunc)
Additional reagents and equipment for performing a hemagglutination test (see
Basic Protocol 3)
NOTE: All equipment and solutions coming into contact with eggs must be sterile and
proper sterile technique should be used accordingly.
Inoculate eggs
1. Dilute influenza virus stock in egg diluent.
The concentration of the seed stock will determine how dilute the inoculum should be.
Typically, a 1:100 to 1:1000 dilution of virus will yield optimal virus growth (104 to 106
50% egg infectious dose (EID50 ) or 0.01 to 0.1 HAU of virus).
Either KPBS or tryptose phosphate brothbased diluent can be used in eggs.
2. Arrange three eggs with the airsac up. Spray tops of eggs with 70% ethanol.
Three to five eggs are recommended to produce sufficient volume of allantoic fluid for
future use. Also, eggs may die during the incubation time due to bacterial contamination,
virulence of inoculated virus, etc., therefore, the more eggs inoculated, the better the
chance to obtain a viable stock of virus. One egg produces 3 to 10 ml allantoic fluid and
0.1 to 1 ml amniotic fluid.
Allow the alcohol to evaporate before proceeding to next step.
3. Punch a small hole in the shell over the air sac using an 18-G, 1 /2 -in. needle and
dispose of needle in an appropriate sharps container.
4. Aspirate 0.6 ml diluted virus sample into a 1-ml syringe with a 22-G, 11 /2 -in. needle.
Animal RNA
Viruses
15G.1.5
Current Protocols in Microbiology
Supplement 3
Figure 15G.1.1
5. Insert the needle at a 45 angle into the allantoic cavity and inoculate 0.2 ml influenza
virus dilution; repeat with the other two eggs (Fig. 15G.1.1).
6. Discard syringe and needle into a sharps safety container.
7. Seal the hole punched in the eggshell with a drop of glue.
Care should be taken not to contaminate glue in bottle with virus.
10. With sterile forceps, break the shell over the air sac and push aside the allantoic
membrane with the forceps, taking care not to break the yolk.
11. Using a 10-ml pipet, aspirate the allantoic fluid and place in a labeled 50-ml plastic
conical tube. Dispose of eggs in an appropriate biological waste container.
12. Centrifuge tubes 5 min at 500 g, 4 C, to pellet any blood cells and tissue fragments.
Transfer clarified fluid into fresh 50-ml tube. Keep on ice (4 C).
Inuenza:
Propagation,
Quantication,
and Storage
15G.1.6
Supplement 3
ALTERNATE
PROTOCOL 4
With original clinical material, the amniotic sac is inoculated at the same time as the
allantoic cavity. Virus inoculated into the amniotic sac that surrounds the embryo can
replicate directly in the embryonic tissue and the resulting virus is released into the
amniotic fluid. However, the amniotic sac will yield only a small volume of fluid and
thus subsequent passage into the allantoic cavity may be required to produce sufficient
volume for testing.
2. Inoculate three eggs per specimen; label eggs to clearly identify original sample.
3. Punch a small hole in the shell over the air sac using an 18-G, 1 /2 -in. needle and
dispose of needle in appropriate sharps container.
4. Aspirate 0.6 ml clinical specimen into a 1-m syringe with a 22-G, 11 /2 -in. needle.
5. Using an egg candler apparatus, hold the egg up to light source and locate the embryo.
6. Insert the needle into the amniotic sac and inoculate 0.1 ml primary clinical specimen
into the sac. Withdraw the needle about 1 /2 -in. and inoculate 0.2 ml primary clinical
specimen into the allantoic cavity (Fig. 15G.1.1).
7. Repeat with remaining clinical specimen and eggs.
8. Discard syringe and needle into a sharps safety container.
9. Seal the holes punched in the egg shells with a drop of glue.
Care should be taken not to contaminate glue in bottle with virus.
11. Harvest the allantoic fluid as described in Basic Protocol 2, steps 9 through 11. Keep
tubes on ice (4 C).
12. Invert the egg so that the embryo and amniotic sac hang down and are clearly visible
and separate from the egg yolk. Using a 1-ml syringe and 22-G, 1 1 /2 -in. needle,
pierce the air sac and remove as much amniotic fluid as possible from around the
embryo. Dispose of eggs in an appropriate biological waste container.
Keep allantoic and amniotic samples separate as the amniotic fluid may contain more
virus than the allantoic fluid. The volume of allantoic fluid recovered may vary from 5 to
10 ml, while the volume of the amniotic fluid recovered may vary from 0.1 ml to 1 ml.
The amniotic cavity provides a richer source of susceptible cells for virus replication and
is used to help viruses from primary specimens grow more efficiently.
13. Centrifuge tubes 5 min at 500 g, 4 C, to pellet any blood cells and tissue fragments.
Transfer clarified fluid into fresh tube. Keep tubes on ice (4 C).
Animal RNA
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15G.1.7
Current Protocols in Microbiology
Supplement 3
14. Perform a hemagglutination test on both the allantoic and amniotic fluids. (see Basic
Protocol 3).
If hemagglutination test is negative or titer is <8 HAU, passage the specimen two more
times before reporting inability to recover virus from the specimen. If HA test results in
titers >8 HAU, specimens can be dispensed into aliquots and stored.
15. Dispense allantoic and amniotic fluid into 2-ml aliquots in 2-ml sterile cryovials and
store <1 year at 70 to 80 C or at 135 to 150 C for long-term storage. (see
Basic Protocol 7).
BASIC
PROTOCOL 3
Materials
Phosphate-buffered saline (PBS) containing potassium (APPENDIX 2A)
Influenza virus stock (e.g., allantoic or amniotic fluid)
Standardized turkey red blood cells (see Support Protocol 4)
96-well V-bottom microtiter plate (Nunc)
NOTE: Keep virus stock on ice (4 C) during the HA test to maintain virus infectivity.
1. Pipet 50 l PBS into wells 2 through 12 across a 96-well V-bottom plate
(Fig. 15G.1.2).
2. Pipet 100 l influenza virus stock into the first column of the 96-well plate.
3. Perform a two-fold dilution series across the 96-well plate by transferring 50 l
between wells, disposing of the final 50 l from the last well.
4. Add 50 l standardized turkey red blood cells to all wells. Tap the plate gently to
mix.
Approximately 5 ml standardized turkey red blood cells will be needed per 96-well plate
used in this assay.
Inuenza:
Propagation,
Quantication,
and Storage
15G.1.8
Supplement 3
Figure 15G.1.2
Figure 15G.1.3 Reading a hemagglutination titration assay plate. Positive samples will look pink
as the red blood cells are held in solution by the virus. Negative samples will look clear with a red
dot, since the red blood cells settle to the bottom of the V-bottom plate.
6. Observe endpoint of agglutination and record titer per 50 l of sample (Fig. 15G.1.3).
Red blood cells will settle to the bottom of the V-bottom well in negative samples, while
red blood cells will agglutinate in positive samples. The endpoint should be read as the
last well showing complete agglutination.
7. Store virus stocks with desired HAU titers (see Alternate Protocol 4, step 14). Dispose
of materials in an appropriate biological waste container.
BASIC
PROTOCOL 4
The 50% tissue culture infectious dose (TCID50 ) assay is a method to measure the amount
of infectious virus in a sample by determining the highest dilution of the sample that
can infect 50% of cells in culture. In this procedure, the virus sample is diluted across a
96-well tissue culture plate containing MDCK cells. The titration should be performed
in quadruplicate.
Materials
MDCK cells in 96-well tissue culture plate (see Support Protocol 1)
Influenza virus growth medium (see recipe)
Influenza virus stock
96-well tissue culture plate (Costar)
Inverted microscope
Animal RNA
Viruses
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Current Protocols in Microbiology
Supplement 3
NOTE: All equipment and solutions coming into contact with cells must be sterile and
proper sterile technique should be used accordingly.
NOTE: All culture incubations are performed in a 37 C, 5% CO2 humidified incubator.
1. Remove MDCK growth medium from plate and wash cells by adding 350 l influenza
virus growth medium to all wells. Aspirate the medium and repeat washing step.
Care should be taken not to disrupt the cell monolayer.
2. Add 100 l influenza virus growth medium to all wells, except the first column, of
a 96-well tissue culture plate.
3. Thaw influenza virus stock and dilute 1:100 in influenza virus growth medium.
4. Add 146 l diluted virus to first column of 96-well tissue culture plate and perform a
1 / log dilution series by transferring 46 l between wells, disposing of final 46 l
2
10
after the eleventh column. Do not add virus to the final column as this is the cell
control for the assay.
5. Incubate plates 2 hr at 37 C.
6. Remove inoculum and gently wash once with 250 l influenza virus growth medium.
7. Add 200 l influenza virus growth medium to all wells and incubate up to 72 hr at
37 C, observing for endpoints in cytopathic effect (CPE).
Typical CPE by influenza viruses include rounding up of infected cells and detachment
from culture flask.
9. Record endpoint of CPE and determine TCID50 titer per 100 l using the ReedMuench method (see Support Protocol 5).
BASIC
PROTOCOL 5
Materials
Egg diluent (see recipe)
Influenza virus stock
10- to 11-day-old embryonated chicken eggs (see Support Protocol 2)
Standardized turkey red blood cells (see Support Protocol 4)
1.5-ml microcentrifuge tubes, sterile
Forceps, sterile
96-well V-bottom plate (Nunc)
Additional reagents and equipment for inoculating eggs (see Basic Protocol 2)
Inuenza:
Propagation,
Quantication,
and Storage
NOTE: All equipment and solutions coming into contact with embryonated eggs must
be sterile and proper sterile technique should be used accordingly.
15G.1.10
Supplement 3
1. Label ten 1.5-ml microcentrifuge tubes 1 through 10 and fill each with 450 l egg
diluent.
2. Pipet 50 l influenza virus stock into 450 l egg diluent in the first tube and perform
a ten-fold dilution down to 1010 by transferring 50 l between tubes.
Changing tips between tubes will decrease the chance of sample carryover and will result
in a more accurate titration of the virus.
3. Inoculate three eggs with 100 l per dilution as described in Basic Protocol 2, steps 1
to 8. Incubate eggs 48 hr at 35 C (optimal for influenza A) or 72 hr at 33 C (optimal
for influenza B).
If the type of virus is unknown, incubate eggs at 35 C.
7. Add 50 l standardized turkey red blood cells to all wells and incubate 30 min at
room temperature (24 to 27 C).
Approximately 5 ml standardized turkey red blood cells will be needed per 96-well plate
used in this assay.
9. Determine the EID50 titer per 100 l by the Reed-Muench method (see Support
Protocol 5).
BASIC
PROTOCOL 6
Materials
2 plaque assay medium (see recipe)
1.6% (w/v) agarose solution
Influenza virus stock
Madin-Darby canine kidney (MDCK) cells confluent in 6-well tissue culture plates
(see Support Protocol 1)
Plaque assay wash medium (see recipe)
2 mg/ml TPCK-trypsin working stock (see recipe)
70% ethanol
0.3% crystal violet solution
37 and 56 C water baths
Forceps, sterile
Inverted microscope
Current Protocols in Microbiology
Animal RNA
Viruses
15G.1.11
Supplement 3
NOTE: All equipment and solutions coming into contact with cells must be sterile and
proper sterile technique should be used accordingly.
1. Before beginning the plaque assay, warm 2 plaque assay medium in a 37 C water
bath and place 1.6% agarose solution in a 56 C water bath.
The 2 plaque assay medium should be warm enough so as not to solidify the 1.6%
agarose solution when mixed together; also it should not be too hot, which will kill
the MDCK cell monolayer when the media solution is added. The 1.6% agarose solution should be kept at 56 C to keep the solution from solidifying before use. When
both solutions are mixed together the temperature of the resulting solution should be
suitable for immediate addition to the MDCK cell monolayer while maintaining cell
viability.
3. Remove the MDCK growth medium from the 6-well tissue culture plates and
wash the MDCK monolayer three times with room temperature plaque assay wash
medium.
For wash steps, either a sterile 10-ml pipet or 1000-l pipettor may be used to remove
and add the medium. Do not pipet medium directly onto the monolayer as this may disrupt
the cells.
Fetal bovine serum (FBS) inhibits viral entry and must be removed for efficient infection
of cells.
4. Perform a ten-fold dilution series starting at 101 and diluting virus samples down to
1010 in plaque assay wash medium. Change disposable pipet or pipet tips between
tubes to decrease the chance of sample carryover.
5. Inoculate 6-well tissue culture wells in duplicate with 100 l diluted virus sample
and rotate tissue culture plate to cover monolayer with inoculum. Take care not to
add medium directly onto the monolayer as this may disrupt the cells.
6. Incubate inoculated plates for a minimum of 30 min or up to 1 hr at 37 C.
Incubation for >1 hr may cause the MDCK cell monolayer to dry out and result in reduced
cell viability.
7. Wash wells two times with room temperature plaque assay wash medium.
For wash steps, either a sterile 10-ml pipet or 1000-l pipettor may be used to remove
and add the medium. Do not pipet medium directly onto the monolayer as this may disrupt
the cells.
Inoculum does not need to be removed before washing MDCK cell monolayer.
9. Mix 1:1 2 plaque assay medium with 1.6% agarose solution and immediately
add 2 ml of agarose medium solution to each inoculated well. Let solidify at room
temperature (24 to 27 C).
For one 6-well tissue culture plate, mix together 7 ml of 2 plaque assay medium with
7 ml of 1.6% agarose solution.
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15G.1.12
Supplement 3
10. Incubate 6-well tissue culture plates at 37 C. Use an inverted microscope to observe
the MDCK monolayer for plaque formation daily.
Plaques will appear as small clear areas in the monolayer.
Current Protocols in Microbiology
11. After 72 hr, carefully remove the agar plug from each well using sterile forceps and
disposing of the agar in a biological waste container. Take care to avoid scratching
the MDCK cell monolayer or letting the agar plugs rotate during removal, as this
will make it difficult to accurately count plaques.
12. Pipet 2 ml of 70% ethanol into each well and incubate 20 min at room temperature
(24 to 27 C) to fix the MDCK cell monolayer.
Do not pipet 70% ethanol directly onto the monolayer as this may disrupt the cells.
13. Remove ethanol and add 1 ml crystal violet solution to each well. Incubate 10 min
at room temperature (24 to 27 C) to stain MDCK cell monolayer.
14. Remove crystal violet solution and wash wells with water to rinse away excess stain
solution.
15. Let plates dry overnight at room temperature (24 to 27 C) before counting plaques.
16. Count plaques in each well and determine the plaque forming units (pfu) per milliliter
using the following formula: pfu/ml = (no. of plaques dilution factor 10).
Plaques suitable for counting will appear as discrete clear circular zones on the background of the purple stained MDCK cell monolayer. At low dilution of virus, the monolayer
may be completely destroyed, resulting in no or minimal purple staining in the wells. The
pfu/ml calculation should be based on the dilution of virus sample that gives >10 plaques
per well but is still countable (i.e., <150 plaques).
BASIC
PROTOCOL 7
ALTERNATE
PROTOCOL 5
SUPPORT
PROTOCOL 1
Animal RNA
Viruses
15G.1.13
Current Protocols in Microbiology
Supplement 3
Materials
Madin-Darby canine kidney (MDCK) cells (ATCC# CCL-34) grown confluent in a
75-cm2 flask (see Support Protocol 1)
Trypsin/EDTA (GIBCO)
Heat-inactivated fetal bovine serum (FBS; APPENDIX 2A)
MDCK growth medium (see recipe)
75- or 25-cm2 tissue culture flasks; or 6- or 96-well tissue culture plates (Corning)
Additional reagents and equipment for counting cells using a hemacytometer
(APPENDIX 4A)
NOTE: All equipment and solutions coming into contact with cells must be sterile and
proper sterile technique should be used.
NOTE: All culture incubations are performed in a humidified 37 C, 5% CO2 incubator.
1. Decant growth medium from a confluent 75-cm2 flask of MDCK cells, and add 5 ml
trypsin/EDTA prewarmed to 37 C.
2. Gently rock the flask to distribute the trypsin/EDTA over the entire MDCK cell
monolayer. Remove the trypsin/EDTA with a pipet.
3. Repeat one time with an additional 5 ml of trypsin/EDTA.
4. Add 1 ml prewarmed trypsin/EDTA, ensuring that entire cell monolayer is bathed
in trypsin/EDTA and incubate flask at 37 C until all cells detach from the plastic
surface (5 to 10 min).
Gentle shaking or tapping of the flask may help cells detach.
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and Storage
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Figure 15G.1.4 Viable and nonviable chicken eggs as apparent from candling. This black and
white facsimile of the gure is intended only as a placeholder; for full-color version of gure go to
http://www.currentprotocols.com
In a darkened room, hold the airsac end of the egg to the candler light source and
gently rotate it while observing the morphology of the chick embryo. A healthy embryo
has a well-defined airsac, free-flowing membranes, uniform vein development and
reacts to the light of the candler. It is very important to screen out broken, unhealthy,
very porous, or dead embryos, as these eggs will not support efficient influenza virus
growth. Figure 15G.1.4 depicts viable and non-viable chicken eggs as viewed by a
KUHL candler. Alternatively, refer to the instruction manual of the egg candler for more
instructions.
NOTE: Egg incubations are performed in a 34 C, 50% humidity incubator with rotation
for eggs <9 days old. Cell culture incubators can be used for eggs >9 days old, but the
CO2 must be turned off or the embryos will suffocate.
SUPPORT
PROTOCOL 3
Materials
Specimen in collection vials (e.g., from nasal, throat, or combined nasal/throat
swabs; or nasopharyngeal, nasal, or throat aspirates or washings)
10 mg/ml gentamicin (GIBCO)
2- to 4-mm glass beads (VWR)
Polypropylene microcentrifuge tubes, sterile
Cryovials (Nunc)
NOTE: Clarified supernatants can be used to directly inoculate cell culture flasks or eggs.
Animal RNA
Viruses
15G.1.15
Current Protocols in Microbiology
Supplement 3
Materials
Whole blood mixed 1:1 in Alsevers solution (APPENDIX 2A)
Phosphate-buffered saline (PBS) containing potassium (APPENDIX 2A)
Cotton gauze, sterile
50-ml conical centrifuge tube
Table 15G.1.1 Properties of RBC from Different Species
Chicken
Turkey
Guinea pig
Human type O
0.5%
0.5%
0.75%
0.75%
Incubation time at 25 C
30 min
30 min
1 hr
1 hr
Button
Button
Halo
Halo
Concentration
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SUPPORT
PROTOCOL 5
The Reed-Muench method (Reed and Muench, 1938) can be utilized to determine the
dilution of virus sample required to yield a 50% positive result. The advantage to using
this method rather than a 100% endpoint is that the 50% endpoint is less affected by
variations within the titration experiment. It is important to note that the unit of infectivity
measured by this endpoint method may require more than one infectious particle. With
the Reed-Muench method, it will be necessary to use a large number of small groups
of test samples at different dilutions (see Basic Protocols 4 and 5 for set up of titration
experiments). At the end of the titration experiment, determine the number of positive
and negative samples at each dilution. Using these numbers, a percentage of infected
samples can be calculated for each dilution. The dilution that would infect 50% of test
samples is estimated using the following formulas:
proportional distance formula = [(% positive value >50%) 50%]/
[(% positive value >50%) (% positive value <50%)]
Knowing the proportional distance between dilutions, the 50%-endpoint can be calculated
using the exact dilutions used:
log infectious dose 50 = (log dilution >50%) + (proportional distance
log dilution factor)
The reciprocal of this number is used to express the titer in infectious units per unit
volume.
An example an ID50 calculation using the Reed-Muench method is shown in
Figure 15G.1.5.
Animal RNA
Viruses
15G.1.17
Current Protocols in Microbiology
Supplement 3
Figure 15G.1.5
15G.1.18
Supplement 3
Inuenza:
Propagation,
Quantication,
and Storage
stock, 2 mg/ml
10 ml DMEM
20 mg trypsin, TPCK-treated, type XIII from bovine pancreas (Sigma)
Filter sterilize for up to 0.2-m membrane
Dispense into aliquots
Store aliquots at 70 C until reagents expire
Dispose of unused thawed TPCK-trypsin, do not refreeze, as the TPCK-trypsin is not stable
after thawing.
Animal RNA
Viruses
15G.1.19
Current Protocols in Microbiology
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Inuenza:
Propagation,
Quantication,
and Storage
15G.1.20
Supplement 3
and can potentially result in an influenza pandemic if able to spread efficiently (Wright and
Webster, 2001).
Human influenza A viruses are transmitted
directly between individuals via aerosolized
droplets generated by coughing and sneezing
or indirectly through contact with fomites on
contaminated surfaces (Alford et al., 1966;
Lidwell, 1974; Bean et al., 1982). Infection
begins in the nasal and tracheal passageways,
and rapidly spreads throughout the upper and
lower respiratory tract. Apical shedding of
virus from respiratory epithelial cells generally limits viral infection to the respiratory tract. Clinical symptoms of an acute influenza A virus infection can range from mild
to severe and typically include fever, cough,
headache, malaise, and anorexia. Each year in
the United States, 36,000 people, on average,
die from complications of influenza virus infection, with 90% of these deaths being in the
elderly population aged >65 years (Thompson
et al., 2003).
Virus isolation is a highly sensitive and useful technique for the diagnosis of influenza
virus infection when used with clinical specimens of good quality. In fact, isolation of a
virus in cell culture along with subsequent
identification by immunologic or genetic techniques or by electron microscopy are standard
methods for virus diagnosis. One important
advantage of virus isolation is that this method
amplifies the virus from the original specimen
and makes it available for further antigenic
and genetic characterization, and also for drugsusceptibility testing if required. The propagation or use of laboratory-adapted strains or
influenza strains of animal origin in laboratories also attempting isolation of viruses from
human clinical specimens for diagnostic or research purposes is not recommended, since
there is a risk of contaminating the diagnostic
specimens with laboratory or animal strains
with high growth phenotypes.
In the past, human influenza viruses often
were isolated in embryonated eggs; however,
today the majority of laboratories use cell culture. Since vaccine candidate viruses must be
isolated in eggs, the trend to use cell cultures
has decreased the availability of suitable vaccine viruses. For this reason, laboratories that
have the capability to isolate influenza in eggs
are encouraged to continue. However, some
strains of influenza, and in particular, recent
human field isolates, are difficult to isolate and
grow in eggs, even after amniotic inoculation.
For this and other reasons, MDCK cells are
the preferred cell line for isolation of human
influenza viruses from clinical specimens.
a conformational change essential for membrane fusion in the endosome and the release
of the nucleic acid allowing for replication to
proceed. Although MDCK cells lack such an
endogenous protease, addition of exogenous
trypsin to the medium at a typical concentration of 1 to 2 g/ml is sufficient for proteolytic cleavage of the HA and the generation of
infectious progeny. The trypsin used to propagate the virus is heat stable L-1-tosylamide2-phenylethyl chloromethyl ketone (TPCK)treated trypsin. This should not be confused
with the trypsin-EDTA solution used to remove cell monolayer from flasks during cell
passage, which is not suitable as a source of
trypsin for virus propagation. Each new working stock of TPCK-trypsin should be titered
for optimal activity and lack of toxicity to the
cell monolayer. The optimal concentration of
trypsin will produce maximal amounts of virus
in infected culture supernatants but will not
result in destruction of uninfected monolayers
over the period of incubation. If the trypsin
concentration is too high, cells will round
up and detach from the plastic, i.e., present
with morphology similar to the cytopathic effect observed in virus-infected cultures. Fetal
bovine serum reduces the virus infectivity and
may also inhibit the activity of trypsin. Therefore, removal of all traces of serum contained
in growth medium by gently rinsing monolayers multiple times is an important prerequisite
for virus inoculation.
Ten- to eleven-day-old embryonated eggs
are optimal for growth of influenza A and B
viruses. It is generally convenient to purchase
freshly fertilized eggs from a supplier and to
incubate them in the laboratory for the required
time. The use of an egg incubator that automatically turns the eggs several times a day,
and maintains constant humidity and temperature is optimal, but a humidified CO2 incubator
that has the CO2 turned off to avoid asphyxiating the embryos may be used successfully
if eggs are turned manually several times a
day. From 4 to 5 days post-fertilization, the
embryos should be clearly visible. A low percentage of eggs should be expected to be nonviable and should not be used for inoculation.
The percent of nonviable eggs may increase in
the summer months in warmer climates. Viability can be determined prior to inoculation
by candling eggs.
The methods described here provide a
source of a quantified infectious influenza
virus stock for use in many laboratory procedures. However, if large quantities of viral
antigens are required, e.g., for use in ELISA or
Anticipated Results
The extent of influenza virus growth in
either MDCK cells or embryonated eggs
is strain-dependent. Human viruses isolated
from primary clinical specimens may grow
poorly in either eggs or tissue culture (titers
of 4 to 16 HAU per 50 l and titers of 107
ID50 /ml or 106 pfu/ml) until the virus has
been adapted to grow in vitro. In contrast,
many laboratory strains that have been passaged numerous times in eggs may achieve
titers of 256 to >1024 HAU per 50 l or 108
to 109 ID50 /ml or 107 to 108 pfu/ml. For antigenic characterization, hemagglutination units
should be 8 HAU. In tissue culture, virus release into the supernatant may be readily monitored at regular intervals by removing a small
amount to detect rising hemagglutination
activity.
Adaptation of human influenza viruses
to grow in eggs may select for variants
that possess amino acid substitutions in the
HA molecule that can also affect biological
Animal RNA
Viruses
15G.1.21
Current Protocols in Microbiology
Supplement 3
properties (Katz et al., 1990). The acquisition of amino acid substitutions in the HA in
viruses repeatedly passaged in MDCK cells
has also been reported (Mochalova et al. 2003).
The potential for such changes should be considered when repeatedly passaging influenza
viruses in eggs or tissue culture and may be
identified by sequence analysis.
Time Considerations
Human influenza A viruses must be incubated for 48 hr, whereas influenza B viruses
require a 72-hr incubation period. Since clinical material should be blind-passaged at least
twice, it may take up to 14 days before the
results of virus diagnosis can be stated to be
virus not recovered. Optimal infectivity of
a virus stock will be achieved when the virus
is kept on ice/4 C during harvest, clarification of fluid and estimation of virus HAU, and
then frozen immediately at or below 70 C.
Viruses may be stored for several years at
70 C, but prolonged storage at this temperature will cause a reduction in virus viability. Incubation times for the hemagglutination
assay must be followed to produce accurate
results; 30 min for avian RBC and 1 hr for
mammalian RBC at room temperature is recommended. The plaque assay requires 4 days;
a half day to set up, a 72-hr incubation, and a
half day to process and read plates. During the
plaque assay setup, work quickly after mixing
the warm 2 plaque assay medium with the
1.6% agarose solution, to ensure that the media mixture does not solidify prior to applying
to 6-well tissue culture plates, but is not too
hot for the MDCK cell monolayer. The optimal temperature of the overlay media should
be in the range of 37 C to 42 C.
Literature Cited
Alford, R.H., Kasel, J.A., Gerone, P.J., and Knight,
V. 1966. Human influenza resulting from aerosol
inhalation. Proc. Soc. Exp. Biol. Med. 122:800804.
Arora, D.J., Tremplay, P., Bourgault, R., and
Boileau, S. 1985. Concentration and purification of influenza virus from allantoic fluid. Anal.
Biochem. 144:189-192.
Azzi, A., Bartolomei-Corsi, O., Zakrzewska, K.,
Corcoran, T., Newman, R., Robertson, J.S.,
Yates, P., and Oxford, J.S. 1993. The haemagglutinins of influenza A (H1N1) viruses in the O
or D phases exhibit biological and antigenic
differences. Epidemiol. Infect. 111:135-142.
Inuenza:
Propagation,
Quantication,
and Storage
15G.1.22
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UNIT 15J.1
Of the retroviruses, human immunodeficiency viruses (HIVs) have the largest human
health impact. The two types of HIV, HIV-1 and -2, are the causative agents of acquired
immune deficiency syndrome (AIDS). Since the epidemic began over 20 years ago,
20 million people have died from AIDS worldwide. In December 2004, nearly 40 million
people were estimated to be infected with HIV (WHO, 2004).
HIV-1 is divided into three groups, M (major), N (new), and O (outlier). The vast
majority of infections worldwide are caused by M-group viruses, which are subdivided
into subtypes or clades depending on sequence diversity. Clades are termed A, B, C, D,
F, G, H, and J. There are also recombinant viruses (circulating recombinant forms or
CRFs) that are epidemic in particular regions, e.g., AE in Thailand, AB in Russia and the
Ukraine, and BF in Argentina and Uruguay. Note that there are no clades E or I, since
these subtypes were found to be recombinants with other clades. Protocols provided here
are appropriate for all HIV-1 clades; however, as noted below, not all of these techniques
can be applied to HIV-2.
HIV-1 requires interactions with CD4 and either CCR5 or CXCR4 coreceptors on the cell
surface in order to infect cells. CXCR4-using (X4) variants can be isolated from 50% of
AIDS patients and are associated with a more rapid loss of CD4+ T cells and faster disease
progression (Asjo et al., 1986; Tersmette et al., 1989; Connor and Ho, 1994; Scarlatti
et al., 1997). CXCR4 is more widely expressed on different CD4+ T cell populations
compared to CCR5, and X4 viruses thus have a broader T cell tropism (Ostrowski et al.,
1999; Blaak et al., 2000). Nevertheless, CD4 depletion and AIDS occur in patients
from whom only CCR5-using viruses can be isolated (de Roda Husman et al., 1999;
Cecilia et al., 2000). CCR5-using (R5) viruses are the predominantly transmitted form,
and individuals homozygous for a defective CCR5 gene (32 CCR5) are substantially
protected from infection (Carrington et al., 1999).
The protocols described in the following pages represent the basic tools for HIV research.
Basic Protocols 1 to 5 describe methods to obtain and propagate the virus for various
applications, while Basic Protocols 6 to 10 describe methods for quantitation of viral
preparations. More specific or adapted protocols are beyond the scope of this unit,
although relevant procedures are discussed briefly.
CAUTION: Regulations regarding HIV research vary around the world. Consultation with
local Institutional Biosafety Committees or equivalent oversight body is strongly advised.
Institutional Biosafety Committee review will usually be required prior to commencing
work with HIV. In the laboratory, human immunodeficiency virus is most likely to be
transmitted via cuts and skin abrasions. Removal of all sharp objects from the laboratory is
essential. Disposable gowns and double gloves must be worn. HIV should be manipulated
in a class II biosafety cabinet or a cabinet that provides equivalent or greater protection
to the operator. In the United States, HIV is designated as a Biosafety Level 2 (BSL-2)
pathogen provided that additional practices and containment equipment recommended for
BSL-3 are used. However, for procedures that may generate high-infectivity titers of HIV,
BSL-3 conditions are required. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B)
for more information.
Animal RNA
Viruses
Contributed by Kathryn H. Richards and Paul R. Clapham
Current Protocols in Microbiology (2006) 15J.1.1-15J.1.22
C 2006 by John Wiley & Sons, Inc.
Copyright
15J.1.1
Supplement 2
CAUTION: Follow all appropriate guidelines and regulations for the use and handling
of human-derived materials. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
STRATEGIC PLANNING
Many HIV-1 primary isolates, replication-competent molecular clones, and HIV plasmid
DNA constructs, as well as other reagents, are available via the NIH AIDS Research and
Reference Reagent Program (http://www.aidsreagent.org) or from the European Centre
for AIDS Reagents (http://www.nibsc.ac.uk/catalog/aids-reagent).
A number of protocols described in this unit require preparation of peripheral blood
mononuclear cell (PBMC) or macrophage cultures from blood. Protocols for both of
these procedures can be found in Kanof et al. (1996).
Uninfected CD4+ cell lines can become contaminated with HIV. Contamination will
result in cytopathic effects that include the formation of multinucleated giant cells (syncytia). However, the extent of cytopathicity depends on the cell line infected and the HIV
strain involved. Chronically infected cell lines may not be readily distinguished from
their uninfected counterparts by light microscopy. Uninfected cells should be cultured
in a laboratory separate from the HIV laboratory. If this is not possible, uninfected cells
should be cultured separately from infected cells and stored in separate incubators. Uninfected cells that have been cultured in an HIV laboratory should not be supplied to other
laboratories.
Materials
HIVs:
Propagation,
Quantication,
and Storage
15J.1.2
Supplement 2
9. Optional: For more efficient removal of cellular debris, filter viral supernatants
through 0.45-m low-protein-binding filters after the centrifugation in step 8.
10. Divide supernatants into 0.5- to 1-ml aliquots in cryovials. Snap-freeze in a dry-ice
ethanol bath, then transfer to a 80 or 152 C freezer for long-term storage.
Animal RNA
Viruses
15J.1.3
Current Protocols in Microbiology
Supplement 2
Repeated freeze-thaw cycles will reduce infectivity. Storage at 152 C may preserve
infectivity better than 80 C, particularly if the 80 C freezer is frequently accessed,
undergoing cycles of elevated temperature.
BASIC
PROTOCOL 2
Materials
Blood from uninfected individual
Complete RPMI medium/20% FBS (see recipe) containing 0.5 g/ml
phytohemagglutinin (PHA; Sigma; store in aliquots at 20 C and add to
medium just before use)
Complete RPMI medium/20% FBS (see recipe) containing 20 U/ml interleukin 2
(IL-2; Roche Applied Science; store in aliquots at 20 C and add to medium
just before use)
Cell-free virus isolate seed stock (viral supernatant; Basic Protocol 1)
RPMI 1640 medium containing 20% FBS
Tabletop centrifuge (e.g., Sorvall Legend RT)
Plastic hemacytometer (preferred for safety reasons)
25- or 75-cm2 tissue culture flasks
0.45-m low-protein-binding filters (Millipore; optional)
Cryovials
Dry ice/ethanol bath
80 or 152 C freezer
Additional reagents and equipment for preparing PBMC (Kanof et al., 1996),
counting cells with a hemacytometer (APPENDIX 4A) and p24 ELISA (Basic
Protocol 10) or RT activity assay (Basic Protocol 9)
Prepare uninfected PBMC
1. Isolate PBMC from uninfected individuals blood (Kanof et al., 1996). Dilute to
22.5 106 cells/ml in RPMI 1640 medium containing 20% FBS and 0.5 g/ml
PHA to induce T cells to grow. Incubate for 2 days.
2. Centrifuge cells 5 min at 300 g (1000 rpm), room temperature, without braking.
Remove medium. Resuspend cells in RPMI 1640 medium containing 20% FBS and
20 U/ml IL-2. Incubate 2 days.
3. Count cells using a plastic hemacytometer (APPENDIX 4A).
6. Centrifuge cells 5 min at 300 g (1000 rpm), room temperature, without braking.
Remove supernatant and resuspend cells in 5 ml RPMI 1640 containing 20% FBS and
20 U/ml IL-2. Transfer cells to 25-cm2 tissue culture flasks and continue incubation.
15J.1.4
Supplement 2
10. Optional: For more efficient removal of cellular debris, filter viral supernatants
through 0.45-m low-protein-binding filters after the centrifugation in step 9.
11. Divide supernatants into 0.5- to 1-ml aliquots in cryovials. Snap-freeze in a dry
ice/ethanol bath, then transfer to a 80 or 152 C freezer for long-term storage.
Repeated freeze-thaw cycles will reduce infectivity. Storage at 152 C may preserve
infectivity better than 80 C, particularly if the 80 C freezer is frequently accessed,
undergoing cycles of elevated temperature.
BASIC
PROTOCOL 3
Materials
CD4+ T cell line, e.g., H9 (Popovic et al., 1984) or MOLT-4 cl.8 (Kikukawa et al.,
1986) or C8166 (Salahuddin et al., 1983)
Seed stocks of T cell lineadapted viruses (available from the NIH AIDS Research
and Reference Reagent Program; http://www.aidsreagent.org)
RPMI medium/10% FBS (see recipe)
25- or 75-cm2 tissue culture flasks
Cryovials
Tabletop centrifuge (e.g., Sorvall Legend RT)
Plastic hemacytometer (preferred for safety reasons)
Dry ice/ethanol bath
80 or 152 C freezer
Additional reagents and equipment for counting cells with a hemacytometer
(APPENDIX 4A), quantification by TCID50 (Basic Protocol 6), and p24 ELISA
(Basic Protocol 10) or RT activity assay (Basic Protocol 9)
1. In a 25-cm2 tissue culture flask, infect 5 106 cells from the CD4+ T cell line with
0.5 to 1 ml seed stock of virus at a multiplicity of infection of 0.01 TCID50 per cell
(see Basic Protocol 6 for quantification by TCID50 ). Incubate for 3 hr.
2. Centrifuge culture 5 min at 300 g (1000 rpm), room temperature, without braking.
Remove supernatant, and resuspend cells in 5 ml of RPMI 1640 medium containing
10% FBS.
Note that T cell lines require 10% FBS for growth.
Animal RNA
Viruses
15J.1.5
Current Protocols in Microbiology
Supplement 2
Figure 15J.1.1 Typical syncytia and cytopathic effect in C8166 T-cells infected with a TCLA
HIV-1 strain. (A) Infected C8166 cells. (B) uninfected C8166 cells.
3. Incubate cultures, monitoring for cytopathic effect (Fig. 15J.1.1) at daily intervals.
HIV usually causes cell death and the formation of syncytia in cultures of CD4+ T cell
lines which can be distinguished from smaller single cells.
4. Maintain cell concentration at 1 106 cells/ml by counting cells (APPENDIX 4A) and
adding growth medium to appropriate volumes every 3 to 4 days.
5. When cultures show extensive cytopathic effects, mix infected cells with uninfected
cells at a 1:4 ratio.
6. Harvest viral supernatant 48 to 72 hr after adding cells, or at peak RT or p24 values
(see Basic Protocols 9 and 10), by centrifuging cells 5 min at 600 g (1500 rpm),
room temperature, without braking, and transferring the supernatant in 0.5- to 1-ml
aliquots into cryovials.
7. Snap-freeze in dry ice/ethanol bath, then transfer to a 80 or 152 C freezer for
long-term storage.
Repeated freeze-thaw cycles will reduce infectivity. Storage at 152 C may preserve
infectivity better than 80 C, particularly if the 80 C freezer is frequently accessed,
undergoing cycles of elevated temperature.
BASIC
PROTOCOL 4
HIVs:
Propagation,
Quantication,
and Storage
15J.1.6
Supplement 2
Materials
293T cells (human embryonic kidney cell line transformed by sheared adenovirus
5 DNA and expressing SV40 T antigen; Pear et al., 1993)
DMEM medium (e.g., Invitrogen) containing 10% FBS (prepare with nuclease-free
H2 O)
HIV cDNA: plasmid DNA containing HIV proviral cDNA (pNL4.3, pYU-2, or
pJRCSF (available from the NIH AIDS Research and Reference Reagent
Program; http://www.aidsreagent.org/)
2 M CaCl2 (prepare with nuclease-free H2 O)
Nuclease-free (DEPC-treated) H2 O (APPENDIX 2A)
2 HEPES-buffered saline (HBS), pH 7.05 (see recipe)
6-well tissue culture dishes or 60- or 100-mm tissue culture plates
Clear plastic tubes
15- or 50-ml centrifuge tubes
Cryovials
Dry ice/ethanol bath
80 or 152 C freezer
1. Plate 293T cells in 6-well tissue culture plates or 60-mm or 100-mm tissue culture
dishes at 1 105 cells/ml the day before transfection using DMEM medium containing 10% FBS. On the day of transfection, change medium on cells 3 hr before
beginning transfection.
2. Referring to Table 15J.1.1 for volumes of reagents needed, mix plasmid DNA with
water in a clear plastic tube and mix by gently vortexing on a low setting.
Clear plastic tubes are used in order to more easily see the precipitate described in step 4.
3. Again referring to Table 15J.1.1 for the appropriate volumes, add 2 M calcium
chloride to the DNA/water mixture and mix by gently vortexing on a low setting. In
another tube, pipet the appropriate volume of 2 HBS.
4. While gently vortexing the tube containing the 2 HBS, add the DNA/water/CaCl2
mixture dropwise. Incubate tube at room temperature for 30 min.
The mixture may appear slightly opaque after 30 min because of the formation of
DNA/calcium phosphate precipitate.
5. Gently vortex the mixture, then add dropwise directly to the medium on the cells
(see step 1). Gently swirl the plate of cells while adding the mixture. Incubate cells
overnight.
6. At 12 to 16 hr post-transfection, change the medium on the cells.
Reagent
6-well plate
60-mm dish
100-mm dish
HIV cDNA
2.5 g
6-12 g
10-20 g
2 M CaCl2
10.34 l
37 l
62 l
Nuclease-free water
To 83.4 l
To 300 l
To 500 l
2 HBS, pH 7.05
83.4 l
300 l
500 l
Total
166.8 l
600 l
1000 l
Animal RNA
Viruses
15J.1.7
Current Protocols in Microbiology
Supplement 2
HIVs:
Propagation,
Quantication,
and Storage
Figure 15J.1.2 Vectors used to produce envelope+ reporter viruses. (A) pSVIIIenv expression
vector encodes envelope and rev proteins. (B) env pNL4.3 is a full-length clone of HIV-1 which
contains a premature stop codon in the envelope gene. Reporter genes are often incorporated
into the nef gene. Thus, env pNL4.3 encodes all the viral genes required for particle production
except for envelope. Cotransfection of 293T cells with env pNL4.3 and pSVIIIenv carrying the
envelope of choice results in the production of high-titer envelope+ pseudotype virions.
15J.1.8
Supplement 2
Materials
293T cells (human embryonic kidney cell line transformed by sheared Adenovirus
5 DNA and expressing SV40 T antigen; Pear et al., 1993)
DMEM medium (e.g., Invitrogen) containing 10% FBS (prepare with nuclease-free
H2 O)
Reporter construct DNA (i.e., pNL4.3env , pNL4.3env nef GFP+ , pNL4.3env
nef luc+ (available from the NIH Reagent Program;
http://www.aidsreagent.org/)
pSVIIIenv (available with various primary isolate envelopes from the NIH Reagent
Program; http://www.aidsreagent.org/), or pVSV-G
Calcium phosphate transfection kit (optional; Promega)
6-well tissue culture plates
15-ml centrifuge tubes
Tabletop centrifuge (e.g., Sorvall Legend RT)
0.45-m low-protein-binding filters (Millipore; optional)
Cryovials
Dry ice/ethanol bath
80 or 152 C freezer
Additional reagents and equipment for transfection of cells (see Basic Protocol 4)
and quantification of virus (Basic Protocol 7)
1. Plate 293T cells at 1 105 cells/ml in DMEM containing 10% FBS, at a volume of
2 ml per well in a 6-well plate.
Other appropriate cell densities and volumes can be used for larger plates or flasks.
15J.1.9
Current Protocols in Microbiology
Supplement 2
QUANTIFICATION OF HIV
There are many different methods for quantifying the infectivity of HIV particles. The
infectivity assay of choice depends on the purpose of the experiment, as well as the cell
type used. Tissue culture infectious dose 50 (TCID50 ) assays can be readily applied to
almost any cell type including cells that grow in suspension (e.g., PBMC, lymphocyte cell
lines). Focus-forming unit (FFU) assays, where foci are identified by immunostaining for
viral antigens, are a useful tool for evaluating infectivity of HIV, which does not readily
form plaques (see Commentary for explanation). FFU assays are best applied to adherent
cell types including cell lines and primary cell cultures. For instance, HIV infection of
primary macrophages can easily be evaluated by estimation of FFUs following in situ
immunostaining. Advantages of FFU assays include an evaluation of the number of cells
infected and sensitivity that allows identification of individual HIV+ cells. FFU assays
that detect very few infected cells can be used to evaluate infection of cell types that are
less permissive to HIV-1 (e.g., primary astrocytes; Willey et al., 2003).
ELISAs that quantify p24 antigen or RT activity provide an estimate of the number of
physical virus particles, but do not evaluate viral infectivity. The viral load (number of
virus particles) in patient plasma is routinely quantified by assays that measure viral RNA
present in virions.
NOTE: All solutions and equipment coming into contact with live cells must be sterile,
and proper aseptic technique should be used accordingly.
NOTE: All culture incubations are performed in a humidified 37 C, 5% CO2 incubator
unless otherwise specified.
BASIC
PROTOCOL 6
Materials
HIVs:
Propagation,
Quantication,
and Storage
15J.1.10
Supplement 2
Figure 15J.1.3 TCID50 calculation. The table shows the virus dilutions that resulted in viral replication. Viral
replication may be detected by RT or p24 estimation of virus particles produced in the cell supernatant (see
Basic Protocols 9 and 10).
1. In a 96-well flat-bottom plate, make 10-fold serial dilutions of virus to 106 in growth
medium. Place 50 l of each dilution into appropriate wells. Add 100 l of a 5 105
cells/ml cell suspension to each well.
Each virus dilution should be tested in replicates of at least four.
2. Incubate plate. Examine or test cultures for infection at an appropriate time that
allows for a single infection event to be sufficiently amplified by subsequent rounds
of replication for detection (e.g., up to 18 days for PBMC).
Infection of PBMC can be determined by estimation of RT or p24 antigen in supernatants
(see Basic Protocols 9 and 10). Infection of CD4+ T cell lines by TCLA strains can be
detected by direct observation for cytopathic effects (e.g., syncytia; Fig. 15J.1.1).
3. Calculate the dilution where 50% of wells are positive (i.e., the tissue culture infectious dose 50 or TCID50 ) using the Karber method (McKnight et al., 1995):
Animal RNA
Viruses
15J.1.11
Current Protocols in Microbiology
Supplement 2
Materials
CD4+ , coreceptor+ cells (e.g., GHOST/CCR5; available from NIH AIDS Research
and Reference Reagent Program; http://www.aidsreagent.org)
Complete DMEM medium/10% FBS (see recipe)
Virus stock to be assayed (see Basic Protocols 1 to 4)
Phosphate-buffered saline (PBS; APPENDIX 2A)
1:1 methanol:acetone, 20 C
PBS (APPENDIX 2A) containing 1% (v/v) FBS and 0.05% (w/v) sodium azide
Monoclonal antibody specific for HIV-1 p24 antigen (available from UK
Centralized Facility for AIDS Reagents (CFAR,
http://www.nibsc.ac.uk/spotlight/aidsreagent/index.html): ARP#365 and CFAR
ARP#366 for HIV-1 or HIV-2+ human sera (WHO Panel C for HIV-2; also
available from CFAR)
Secondary antibody: goat anti-mouse -galactosidase conjugate for HIV-1 or goat
anti-human -galactosidase conjugate for HIV-2 (Southern Biotechnology)
0.5 mg/ml Xgal in N,N-dimethylformamide
Yellow PBS (see recipe)
PBS (APPENDIX 2A) containing 0.05% (w/v) sodium azide
48-well tissue culture plate
Infect cells with virus
1. Plate GHOST/CCR5 cells at 46 104 cells/ml in a 48-well tissue culture plate
in complete DMEM/10% FBS the day before infection, placing 500 l of cell
suspension in each well. Incubate cells overnight.
2. Thaw virus stocks quickly in a 37 C water bath until just thawed. Serially dilute
virus stocks 10-fold in complete DMEM/10% FBS, taking into account the expected
infectivity titer when deciding on the range of dilutions.
3. Remove medium from cells. Place 100 l virus or virus dilution into each well.
Incubate 3 hr.
4. Remove virus from cells. Wash cells once by adding 500 l complete DMEM/10%
FBS to each well and then aspirating, then place 500 l fresh DMEM containing
10% FBS onto cells. Incubate 72 hr.
HIVs:
Propagation,
Quantication,
and Storage
5. Remove medium and wash cells once with 500 l/well PBS using the technique
described in the previous step.
15J.1.12
Supplement 2
Figure 15J.1.4 Infectivity of HIV-1 R5 viruses detected as foci. HIV-1 reporter viruses were
used to infect NP2 cells with and without CD4 and the coreceptor CCR5. Reporter viruses were
prepared as described in Basic Protocol 5. In this assay, infection was detected (in this case
without using the reporter gene) by immunostaining for intracellular p24 antigen as described in
Basic Protocol 7.
Animal RNA
Viruses
15J.1.13
Supplement 2
BASIC
PROTOCOL 8
Materials
Target cells for titrating virus with a luciferase reporter gene: PBMC, macrophages,
or other target cells that carry a luciferase reporter gene, e.g., HeLa TZM-bl
(CD4+ , CXCR4+ , CCR5+ ; Wei et al., 2002); available from the NIH AIDS
Reagent Program at http://www.aidsreagent.org
Complete DMEM medium/10% FBS (see recipe)
Reporter virus (see Basic Protocol 5) carrying a luciferase reporter cloned into the nef
gene of pNL4.3 env construct (available from the NIH AIDS Reagent Program
at http://www.aidsreagent.org) or HIV virus stock (Basic Protocols 1 to 4)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Bright-Glo substrate (Promega)
96-well luminometer culture plate (Corning)
Luminometer (e.g., Clarity from Bio-Tek)
1. Set up target cells at 46 104 cells/ml in DMEM containing 10% FBS. Add 100 l
of cell suspension per well to a 96 well-luminometer plate the day before titration.
Use HeLa/CD4/CCR5 TZM-bl cells for titrating HIV without a reporter gene. Use other
cell types for titrating HIVs that carry a luciferase reporter.
2. Thaw virus stocks quickly in a 37 C water bath until just thawed. Serially dilute
virus stocks 10-fold in complete DMEM/10% FBS, taking into account the expected
infectivity titer when deciding on the range of dilutions.
3. Remove medium from cells. Place 100 l virus or virus dilution into each well.
Incubate 3 hr.
4. Remove virus from cells. Wash cells twice with DMEM containing 10% FBS, then
place 100 l fresh DMEM containing 10% FBS onto cells. Incubate 72 hr.
5. Aspirate medium, then place 50 l PBS into each well.
6. Add 50 l Bright-Glo substrate to each well. Read plate with luminometer.
BASIC
PROTOCOL 9
HIVs:
Propagation,
Quantication,
and Storage
15J.1.14
Supplement 2
to score wells as positive or negative in a TCID50 estimation (see Basic Protocol 6), or can
be used to compare the production of viral particles released from different cultures. To
compare RT levels between assays done at different times, a standard control supernatant
needs to be included. The standard should be a single virus-containing supernatant that
is stored in aliquots at 80o C. Each time an RRT assay is done, a freshly thawed control
supernatant aliquot can be tested alongside experimental samples.
CAUTION: Radioactive materials require special handling. Follow the guidelines provided by your local radiation safety adviser. See UNIT 1A.4 for more information.
Materials
RRT assay solution A (see recipe)
Cell culture supernatants (infected and uninfected) for testing RT activity (Basic
Protocols 1 to 4)
10Ci/ml [3 H]TTP (sp. act. 10 to 25 Ci/mmol)
RRT assay solution B (see recipe)
RRT stop solution (see recipe)
5% (w/v) Na2 HPO4
70% and 100% ethanol
Scintillation fluid (Emulsifier Safe from Perkin-Elmer)
Plastic flexible 96-well plate (Falcon)
Cell harvester (e.g., Skatron)
DE-81 chromatography paper
Fan for air drying filters
Polystyrene board
1. Pipet 10 l RRT assay solution A into each well of a plastic 96-well flexible plate.
2. Add 2 l cell culture supernatant from infected cells to the appropriate wells and
add 2 l supernatant from uninfected cells to appropriate wells as controls.
3. Incubate at 37 C for 15 min.
4. Add 12 l [3 H]TTP to 3 ml RRT assay solution B. Add 25 l of this mixture to each
well. Mix gently, then incubate 6 hr at 37 C.
5. Add 20 l of RRT stop solution to each well. Mix by swirling.
6. Filter reaction mix on to DE-81 chromatography paper using a cell harvester.
7. Wash filters five times, each time with 2 ml of 5% Na2 HPO4 , then once with 2 ml
of distilled water, followed by 2 ml of 70% ethanol, and then 2 ml of 100% ethanol.
Dry filters by pinning them to a polystyrene board and placing in front of a fan.
8. Transfer filters to scintillation vials and add 5 ml scintillation fluid to each. Quantify
incorporation of [3 H]TTP by RT onto the p(dT)12-18 primer by counting 3 H using
a scintillation counter.
BASIC
PROTOCOL 10
Animal RNA
Viruses
15J.1.15
Current Protocols in Microbiology
Supplement 2
Materials
Coating antibody: sheep antiHIV p24-I/II/III mixture (Cliniqa)
Coating buffer: 0.1 M NaHCO3 (pH 8.5)/0.15 M NaCl
Supernatants to be assayed for HIV (Basic Protocols 1 to 4)
5% (w/v) benzalkonium chloride
Tris-buffered saline (TBS), pH 7.4 (APPENDIX 2A)
TBS, pH 7.4 (APPENDIX 2A), containing 0.05% (w/v) benzalkonium chloride
TBS, pH 7.4 (APPENDIX 2A), containing 2% (w/v) nonfat dry milk
10 g/ml p24 standard (National Institute of Biological Standards and Controls;
http://www.nibsc.ac.uk/)
Nonfat dry milk
Sheep serum (Sigma)
10% (v/v) Tween-20
Mouse anti-p24 alkaline phosphatase (AP)conjugated antibody (Cliniqa)
TBS (APPENDIX 2A) containing 0.05% Tween-20
AMPAK enzyme amplification kit (Dako)
AMPAK wash buffer
Substrate
Amplifier
Stop solution
Maxi-Sorp 96-well plate (Nunc) with lid
Automated ELISA plate washer (optional)
Spectrophotometer with microtiter plate reader
Prepare coated plates and viral supernatants for testing
1. Dilute coating antibody 1:200 in coating buffer. Add 100 l to each well of a MaxiSorp 96-well ELISA plate. Incubate at room temperature overnight or at 4 C for
2 days.
2. Inactivate virus in the supernatants to be tested by adding 80 l to 20 l of 5%
benzalkonium chloride. Prepare a 1:20 dilution by adding 10 l of the inactivated
sample to 190 l TBS. Add 20 l of the 1:20 dilution to 180 l TBS containing
0.05% benzalkonium chloride for a 1:200 dilution.
Only the 1:20 and 1:200 dilutions need to be assayed. All samples should be assayed in
triplicate.
3. After coating, wash plate twice, each time by filling the wells with TBS and then
hitting the inverted plate over paper towels. Alternatively, if available, use an automated ELISA plate washer. Block plate by adding 200 l fresh TBS containing 2%
nonfat dry milk per well and incubating plate 5 to 60 min at room temperature.
15J.1.16
Supplement 2
7.5 ml TBS
0.4 g nonfat dry milk
2 ml sheep serum
500 l 10% Tween-20
5 l mouse anti-p24AP conjugate.
9. Add 100 l of this solution to each well. Cover the plate. Incubate at room temperature
for 1 hr.
10. Wash plate twice with TBS containing 0.05% Tween-20.
11. Wash plate four times with AMPAK wash buffer according to the manufacturers
instructions for the AMPAK kit.
12. Add 50 l per well of the reconstituted substrate from the AMPAK kit. Incubate at
room temperature for 30 to 75 min (incubation time will depend on the age of the
AMPAK kit).
13. Add 50 l per well of reconstituted amplifier from the AMPAK kit. Incubate at room
temperature for 30 min, or until color develops.
14. Add 25 l per well of stop solution from the AMPAK kit. Read plate at 492 nm in a
spectrophotometer.
15J.1.17
Current Protocols in Microbiology
Supplement 2
HIVs:
Propagation,
Quantication,
and Storage
Plaque assays
Plaque assays can be used to quantify a variety of cytopathic viruses, e.g., adenoviruses
(UNIT 14C.1), herpesviruses (UNIT 14E.1), and
rhabdoviruses. Retroviruses are generally less
15J.1.18
Supplement 2
high reproducibility. Reporter viruses are readily adapted to different experimental systems.
For example, reporter viruses carrying VSVG envelopes have been exploited to investigate the post-entry TRIM-5 restriction (Ikeda
et al., 2004). The authors of this unit have
used reporter viruses carrying different envelopes derived from HIV-1 in brain and immune tissue to investigate viral tropism and
receptor use (Peters, 2004). Another reporter
virus system that has been adapted to evaluate the first events in HIV entry utilizes
a -lactamase-vpr fusion protein (Cavrois
et al., 2002). This fusion protein is coexpressed with an envelope expression vector
and an env construct encoding the other
viral proteins (e.g., pNL4.3env ). The virions produced contain the -lactamase-vpr fusion protein, which is delivered into the cytoplasm of cells upon virus entry. Target cells
can be prelabeled with CCF2, a cytoplasmic
green fluorescent dye. Upon infection and delivery of -lactamase-vpr, the lactamase enzyme cleaves CCF2, changing its fluorescence
from green (520 nm) to blue (447 nm). The
-lactamase-vpr fusion protein system is useful for confirming HIV entry and distinguishing entry from attachment of virions to the cell
surface and their endocytosis.
Plasma viral load
The amount of virus or viral load present
in the plasma of HIV-infected individuals is
routinely estimated for HIV+ individuals. The
most widely used method quantifies HIV RNA
present in virus particles in plasma to provide
an estimate of plasma viral load (PVL). The
specific protocol is beyond the scope of this
chapter. Briefly, HIV-specific primers are used
to amplify viral RNA by RT-PCR. Several
kits are available commercially with specificities for different subtypes and with different
sensitivities.
Animal RNA
Viruses
15J.1.19
Current Protocols in Microbiology
Supplement 2
Anticipated Results
HIVs:
Propagation,
Quantication,
and Storage
Time Considerations
Isolation of HIV into PBMC cultures usually takes 2 to 3 weeks, with daily monitoring,
before virus is detected in the cell supernatant.
Similarly, the production of new viral stocks
from PBMC involves up to 2 weeks of culture. In contrast, replication-competent virus
or reporter viruses are harvested just 48 hr after transfection of 293T cells with appropriate
plasmids.
Infectivity titrations vary in length considerably. TCID50 assays on PBMCs take up to
18 days to allow virus at endpoint dilutions to
expand sufficiently for detection. FFU assays
on cell lines are complete within 4 days, while
FFU assays on macrophages can be completed
within 7 days. Reporter viruses with a luciferase readout can usually be read 48 hr after
infection.
Acknowledgements
The authors thank Paul Peters for advice
on protocols that measure luciferase activity. The authors current work is supported
by grants from the NIH (R01MH64408 and
AI062514) and the Elizabeth Glaser Pediatric
AIDS Foundation. Dr. Clapham is an Elizabeth
Glaser Research Scientist. Dr. Richards is
supported by NIH grant R01MH64408. The
authors thank the NIH AIDS Research and
Reference Reagent Program and the Central
Facility of AIDS Reagents, U.K. for supplying many reagents used in their research, some
of which are described in this section.
Literature Cited
Asjo, B., Morfeldt Manson, L., Albert, J., Biberfeld,
G., Karlsson, A., Lidman, K., and Fenyo, E.M.
1986. Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet 2:660-662.
Bjorndal, A., Deng, H., Jansson, M., Fiore, J.R.,
Colognesi, C., Karlsson, A., Albert, J., Scarlatti,
G., Littman, D.R., and Fenyo, E.M. 1997.
Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J. Virol. 71:74787487.
Blaak, H., vant Wout, A.B., Brouwer, M.,
Hooibrink, B., Hovenkamp, E., and
Schuitemaker, H. 2000. In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is
established primarily by syncytium-inducing
variants and correlates with the rate of CD4(+)
T cell decline. Proc. Natl. Acad. Sci. USA
97:1269-1274.
Carrington, M., Dean, M., Martin, M.P., and
OBrien, S.J. 1999. Genetics of HIV-1 infection: Chemokine receptor CCR5 polymorphism
and its consequences. Hum. Mol. Genet. 8:19391945.
15J.1.20
Supplement 2
Animal RNA
Viruses
15J.1.21
Current Protocols in Microbiology
Supplement 2
HIVs:
Propagation,
Quantication,
and Storage
15J.1.22
Supplement 2
UNIT 16B.1
Stewart M. Gray
1
USDA, ARS, Department of Plant Pathology, Cornell University, Ithaca, New York
ABSTRACT
A majority of plant viruses are transmitted between hosts by insect vectors, and it is
often important to use insect transmission in the laboratory to maintain virus isolates
or to study virus-vector-plant interactions. Although many of these viruses can also be
mechanically transmitted in the laboratory using infected sap, maintenance by mechanical transmission can often lead to changes in the virus, either minor changes in gene
sequences or, in some cases, major deletions of genome sequences. These can affect both
virus-vector and virus-host interactions. This unit describes some simple and practical
methods for conducting virus transmission experiments using sap-sucking insects. Curr.
C 2008 by John Wiley & Sons, Inc.
Protoc. Microbiol. 10:16B.1.1-16B.1.10.
Keywords: plant virus r transmission
INTRODUCTION
This unit describes how to use aphids, or other sap-sucking insects, to transmit viruses
either from infected plant material (Basic Protocol) or by using an in vitro membrane
system that allows insects to feed on purified virus alone or in combination with other
purified proteins (Alternate Protocol). Transmission methods are provided for both the
short duration transmission of nonpersistent viruses and the longer duration transmission
of semipersistent or persistent viruses. Additionally, general information is provided on
rearing aphids for use in virus transmission experiments (Support Protocol).
STRATEGIC PLANNING
Choosing the Host Plant
The host range of most species of aphids is described in the literature (Blackman and
Eastop, 2000, 2006; Dixon, 2005). Ideally, it is best to rear the aphids on a plant that is not
susceptible to the virus of interest in order to avoid virus infection in the plants used to
rear aphids. However, it is more important to determine whether the aphids will transfer
easily from the maintenance host to the host used for virus transmission studies. Keep in
mind that not all varieties of a host plant are equally palatable to an aphid. Aphids reared
on one host may not readily feed on another. Reduced or absent virus transmission may
be due to the lack of proper feeding on the virus recipient host, rather than an inability to
transmit the virus. Check the survival and reproduction on both hosts prior to the start of
the virus transmission experiments. In addition, virus infection may alter the suitability
of a host.
Confining the Aphids on Test Plants
Cages may enclose an entire plant or group of plants, or they may confine aphids to
a leaf or area of a single plant. Adequate ventilation is required to reduce the relative
humidity and free moisture within the cage that can lead to disease development and
aphid entrapment. Cages must also be able to prevent escape of the aphids from the cage
or intrusion of foreign pests into it. A mesh aperture size of 210 m or less will prevent
ingress or egress of most first instar aphids. Certain plastics, glues, and sealers can have
Plant RNA
Viruses
Current Protocols in Microbiology 16B.1.1-16B.1.10, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16b01s10
C 2008 John Wiley & Sons, Inc.
Copyright
16B.1.1
Supplement 10
Figure 16B.1.1 Examples of insect cages appropriate for small insects. Cages are used to retain
aphids on healthy plants during inoculation. They may enclose an entire plant or group of plants,
or they may confine aphids to a leaf or area of a single plant.
Aphid
Transmission of
Plant Viruses
toxic effects on the aphids and/or the host plant. Thus, any new material should be tested
for toxic/adverse effects to both the plant and the insect prior to use. Commercially
available insect cages are rarely appropriate for working with small insects (e.g., aphids,
thrips, whiteflies), and most researchers construct their own cages. Cages can be as
simple as a plastic container covered with muslin cloth or more elaborate plastic and
mesh containers. Some examples are shown in Figure 16B.1.1.
16B.1.2
Supplement 10
Facilities
Design
If you are fortunate enough to be able to design aphid rearing and working facilities,
there are several features to consider. The physical layout should minimize the possibility
of contamination, both viral and insect. If possible, rearing facilities should be planned
so that there are separate areas for working with virus and virus-free materials, as well
as a third space for a plant nursery. All work areas should be free of other insect pests,
pathogens, or insecticide contamination.
Minimizing contamination
It is best to grow all plants used for rearing aphids and for use in the transmission studies
in an isolated greenhouse or growth room that is not treated with any pesticides and with
limited access by nonessential personnel, to minimize the introduction of insects and
diseases.
Colony maintenance activities or collection of aphids for use in virus transmission
tests are conducted in a no virus work area. Caged colonies are removed from the
rearing chambers, aphids collected, and plants (and extra aphids) disposed of within the
workroom, without contaminating the rearing chambers or outside areas with any aphids.
A freezer is an effective and environmentally friendly way to dispose of the unwanted
aphids remaining on the plants and in the pots.
Recaging a colony once the cage is removed often leads to the escape of individual
aphids, which can contaminate other colonies. Additionally, once an aphid colony is
removed from a cage or the cage is removed from the plant, there is the possibility of
introducing either contaminating aphids or pests. A one use only colony is the best
insurance against contamination problems.
Aphid transfer
The transfer of aphids exposed to virus is conducted in a separate facility consisting of an
isolated workroom directly connected to plant growth rooms. This arrangement allows
viruliferous aphids to be transferred from virus source plants or tissues to recipient plants
and held in isolation during the inoculation access period. All plants in the work room
and growth rooms are caged at all times except during the brief periods that aphids are
placed on the plants.
TRANSMISSION FROM INFECTED PLANT MATERIAL
Insect transmission of plant viruses from infected plant material to healthy plants is
useful for determining the identity of insect vectors and the transmission efficiency of
individual vectors. Additionally, this technique is useful for generating additional infected
plant material if the virus is not transmitted by mechanical, bombardment (UNIT 16B.3),
or agroinfiltration (UNIT 16B.2) methods. Insects are allowed to feed on infected material
for an appropriate length of time (acquisition access period), then moved to a healthy
host for an appropriate inoculation access period. This seemingly simple process can
present many challenges if the proper protocols are not followed. For example, the use
of improper feeding times could prevent or reduce the probability of transmission, or
damaging insect mouthparts during improper handling could prevent virus transmission.
This protocol describes methods involving aphid vectors, but the general techniques are
useful for other insects with piercing sucking mouthparts (e.g., whiteflies, leafhoppers).
NOTE: If the virus or vector is not found in your state or geographical area, regulations
may apply. If uncertain, check with the United States Department of Agriculture, Animal
and Plant Health Inspection Service (USDA APHIS; http://www.aphis.usda.gov) plant
BASIC
PROTOCOL
Plant RNA
Viruses
16B.1.3
Current Protocols in Microbiology
Supplement 10
protection and quarantine (PPQ) manuals for information about permits for insects and
plant pests.
Materials
Aphids (Support Protocol) or other insects
Infected plant material
1.5-ml tubes, optional
Parafilm, optional
Healthy plants
Appropriate insecticide: insecticidal soaps, contact insecticides (e.g., pyrethroids),
or systemic insecticides (e.g., organophosphates and carbamates)
Fine-tip paint brush (e.g., round camel hair #2)
Shallow plastic or glass dishes with tight-fitting lids, 10 cm in diameter and 2 cm
deep
Cages to retain aphids on healthy plants during inoculation (e.g., see Fig. 16B.1.1)
Fine white sand (sandbox sand, available in garden supply stores), optional
1. Collect healthy aphids that have not been exposed to virus by gently tapping the
leaf with the handle of a paint brush and letting the aphids fall onto a clean sheet
of paper. Alternatively, remove individual aphids with a moistened paint brush by
gently prodding the aphid from behind until it begins to move and then gently picking
it up with the brush. Transfer the aphids to the collection dish.
Handle the aphids gently. Pulling them off a leaf while they are feeding may damage
their stylets (a small feeding tube that is inserted into the leaf tissue). Aphids adopt a
characteristic posture while feeding. The antennae are laid back over the body and the
posterior end is raised. Tapping a feeding aphid with a paintbrush usually causes it to
withdraw its stylet; the antennae will be raised and moved forward, and the insect may
begin to walk.
4a. For stylet-borne, nonpersistently transmitted viruses: Introduce five to ten aphids
onto the infected tissue and observe for feeding. If the insects walk off the leaf,
discard them (see step 5, second annotation, or step 8 below, for disposal methods).
Use only those aphids that actually probe the leaf tissue for 15 to 90 sec for transfer
to healthy plants (step 5).
Transmission experiments involving stylet-borne, nonpersistently transmitted viruses require short acquisition feeding periods (<2 min). Nonpersistently transmitted viruses
are carried on the very distal tips of the insect mouthparts (stylets). These viruses are
acquired during very brief probes of epidermal or mesophyll cells, usually 15 to 60 sec
in duration. Transfer of the aphids should take place after 30 to 90 sec of feeding activity
because longer feeding times actually decrease the probability that an insect will transmit
these viruses.
4b. For circulative persistently or semipersistently transmitted viruses: Add whole leaves
or leaf sections from infected plant material to the dishes containing starved aphids.
Allow the aphids a 24 to 48 hr acquisition access period. Keep the dish at 18 C to
22 C.
Aphid
Transmission of
Plant Viruses
If the infected plant material is from herbaceous plants prone to wilting, the petiole of the
leaf can be immersed in water in 1.5-ml tube sealed with Parafilm and placed in a dish
with a tight-fitting lid. It is best if the leaf material does not wilt during the acquisition
access period.
16B.1.4
Supplement 10
Younger symptomatic tissue is usually a good source of virus, but some aphid-virus
combinations may require the use of older tissue.
Persistently or semipersistently transmitted viruses require longer acquisition feeding
periods (minutes to hours; actual feeding time during the acquisition access period);
generally, increased feeding times means increased transmission efficiency. Semipersistently transmitted viruses are carried in the foregut of the insects and are regurgitated
back into the plant during prolonged feeding, i.e., not during brief probes.
Persistently transmitted viruses actually circulate through the insect body, being taken up
through gut tissues and eventually winding up in salivary tissues where they are secreted
back into the plant during the salivary phase of feeding. These viruses will require one to
several days to complete the circulative pathway that starts with ingestion and ends with
salivation.
See Background Information for a discussion of mechanisms of virus infection (e.g.,
circulatively, semipersistently, and nonpersistently transmitted viruses).
7. Observe the aphids at the end of the inoculation feeding period to be sure they are
on the plant and alive.
8. Kill all the aphids (including newly produced aphids) by treating the plants with an
appropriate insecticide.
If adult aphids are used in this transmission experiment, they will produce young aphids
during the inoculation access period; Observing young aphids is a good indication that
the adults were feeding well.
Contact insecticides such as pyrethroids work well for many insects and do not remain
active on or in the plant for more than a few days, so the infected plants can be used as
source plants for subsequent transmission experiments.
Systemic insecticides (e.g., organophosphates and carbamates) are slower acting, but
longer lasting, so the plants will not be useful for future transmission experiments.
Insecticidal soaps will also work to kill many sap-sucking insects, and they are relatively
safe to use.
9. Allow the plants to grow in the greenhouse and observe for symptoms.
Plant RNA
Viruses
16B.1.5
Current Protocols in Microbiology
Supplement 10
ALTERNATE
PROTOCOL
IN VITROTRANSMISSION ASSAY
In vitro techniques to test for insect transmission of plant viruses were first developed in
the 1920s when it was discovered that insects with piercing sucking mouthparts would
feed on solutions through an animal membrane. This technique later evolved to use
synthetic membrane-like materials, most notably Parafilm. In vitro techniques are useful
if the vectors will not feed on the natural hosts of the virus. They are especially useful for
investigations using purified preparations of virus. For example, these techniques have
enabled the testing of purified (or semipurified) virus preparations from plants or from
cloned virus inoculated into cell culture. They also allow the mixing of virus with other
proteins to determine the need for and identification of transmission helper factors. This
protocol describes methods involving aphid vectors, but the general techniques are useful
for other insects with piercing, sucking mouthparts, e.g., whiteflies, leafhoppers. A dated
but useful reference is Kunkel (1977).
d. Stretch a second sheet of Parafilm and carefully lower it over the liquid. Wrap the
Parafilm onto the sides of the cylinder, trapping the liquid between the two sheets.
It is best to stretch the second sheet of Parafilm and lower it onto the top of the liquid
and then slowly relax the edges of the Parafilm and allow contact with the edges of the
cylinder.
Kunkel (1977) provides excellent schematics for preparing these chambers.
e. Attach the screening to the foam, using contact cement or similar glue.
3. Place the aphids on the screen covering the hole in the bottom of the foam block.
Quickly lower the cylinder into the foam block before the aphids climb out.
Add the aphids as a clump rather than individually.
Do not crush any of the aphids and do not overcrowd the membrane.
Aphid
Transmission of
Plant Viruses
See Video 1 at http://www.currentprotocols.com for instructions on how to assemble the feeding chamber shown in Figure 16B.1.2. See Video 2 at http://www.
currentprotocols.com for assembly of an alternate type of feeding chamber.
16B.1.6
Supplement 10
virus solution
stretched Parafilm
foam block
screen
Figure 16B.1.2
4a. For stylet-borne, nonpersistently transmitted viruses: Allow the aphids a <2 min
acquisition.
In the case of the short acquisition feeding periods involving stylet-borne, nonpersistently
transmitted viruses, it is best to actually observe the aphids feeding and transfer only
those that actually probe the membrane for 30 sec or more.
See Background Information for a discussion of mechanisms of virus infection (e.g.,
circulatively, semipersistently, and nonpersistently transmitted viruses).
5. Carefully dislodge (see Basic Protocol) the aphids that were observed to be feeding
(nonpersistent viruses) or that were actively feeding on the membrane at the end
of the acquisition feeding period (persistent, semipersistent viruses) and transfer to
healthy plants. Cage aphids on the plant using either a whole plant cage or some type
of cage that clips onto a leaf without damaging the tissue.
6. Allow a suitable inoculation access period.
Hours (usually overnight) are convenient for nonpersistent viruses; 2 to 4 days is appropriate for persistent and semipersistent viruses.
7. Observe the aphids at the end of the inoculation feeding period to be sure they are
on the plant and alive.
8. Kill all the aphids, including the newly produced aphids, by treating the plants with
an appropriate insecticide (see Basic Protocol, step 8 annotation).
9. Allow plants to grow in the greenhouse and observe for symptoms.
Plant RNA
Viruses
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SUPPORT
PROTOCOL
REARING APHIDS
Aphids are among the easiest of insects to maintain in the laboratory; however, certain
rules need to be strictly followed in order to generate physiologically normal insects that
will behave in a normal manner. The following are some specifics of one system that
has been in use for over 40 years to maintain multiple aphids used in virus transmission
studies (see Katsar and Gray, 1999; also contains chapters about rearing other types of sapsucking insect vectors of plant viruses), although there are numerous systems described
that are equally effective. Details on facilities, maintenance schedules, handling, and
potential pitfalls are provided in the Strategic Planning section. In addition, general
information on starting new colonies and selecting appropriate rearing conditions are
provided. The system outlined here is easily adaptable for use with many aphid species
and host plants. In general, pathogens and parasites are not transovarially transmitted
to the progeny, although this rule does not apply to viruses that replicate in aphids. To
minimize the chance of parasite or pathogen contamination, start all colonies with first
instar nymphs. Most aphids reproduce via parthenogeneisis and give birth to live young.
There are four instar (immature) developmental stages prior to the adult stage. The first
instars are the live-borne young; they will usually molt to second instars within 2 to
3 days.
Materials
Aphids, either collected from the wild or obtained from other researchers colonies
Leaves from healthy plants (or moist filter papers)
Healthy plants
Fine-tip paint brush (e.g., round camel hair #2)
Small containers with tight-fitting lids
Plant cages (see Fig. 16B.1.1)
1. Using a fine-tip paint brush, place apterous, parthenogenic, female adults in small
containers with tight-fitting lids containing moist filter paper or leaves from healthy
host plants.
2. Allow the adult aphids to produce progeny over a 12 to 24 hr period at a temperature
appropriate for the aphid and plant species used.
A temperature of 18 C to 22 C is a good place to start.
3. Transfer (using a paint brush) five to ten newly emerged first instar nymphs to
healthy plants in new cages to initiate the colony. Dispose of the remaining adults
and nymphs (see Basic Protocol).
4. Allow colonies to develop for 2 to 3 weeks.
Depending on the aphid species, the plant, and environmental conditions, the plant will
usually begin to decline in 3 to 4 weeks, and aphid colonies are not as robust on declining
plants. In addition, more winged forms of the aphids will develop.
5. Collect aphids (see Basic Protocol, step 1 annotation) for experimental use or to start
new colonies.
COMMENTARY
Background Information
Aphid
Transmission of
Plant Viruses
become more difficult if there is limited information about efficient vectors and if the viruses
are not easily maintained by mechanical transmission. Fortunately, the vectors of most plant
viruses or virus families are known, and taxonomic and mechanistic specificity exists at the
16B.1.8
Supplement 10
Critical Parameters
It should be noted that viruses maintained
for extended periods by mechanical transmission or vegetative propagation may loose their
ability to be transmitted by their natural vector.
This can occur because of selection for mutations or deletions in genes for critical components of the virus required for vector transmission, but dispensable for host plant infection.
Additional information on the mechanics of
plant virus transmission by insects is reviewed
in Ammar and Nault (2002), Brown and Czosnek (2002), Gray and Gildow (2003), Hogenhout et al. (2003), Ng and Perry (2004), and
Ullman et al. (2005).
Troubleshooting
Insects die or do not feed on virus-infected
tissue
Be sure that plant material has not been
treated with any type of pesticide that may act
as an antifeedant or is toxic to the insects.
Insects may not feed well on infected plant
material; try another host or another cultivar
of the same host.
Be sure the dishes do not contain any detergent or chemical residues.
Insects die or do not feed on membranes
Insects will vary in their ability to feed and
preference for feeding on membranes. Try to
feed them on a 15% sucrose solution. It may be
necessary to increase or decrease the sucrose
content (10% to 20% range). Be sure to use
fresh solutions and filter sterilize.
If the insects feed on sucrose, but not the
virus solution, it may be the choice of buffer,
concentration of virus, or impurities in the solution. Filter sterilize the solution, change the
concentrations of the virus and/or buffer, or
use a different buffer (or water).
Insects feed well on tissue/membranes but
not on healthy plants
Many insects do not acclimate well to different hosts. It may be best if the healthy plants
are of the same host and variety as the virus
source material and/or the same host on which
the aphids are reared.
Insects do not transmit virus
The insect may not be a vector, or it may be
an inefficient vector. Try increasing the number of aphids placed on each healthy plant.
Alternatively, try another source of the same
insect species or a different species.
Anticipated Results
Virus symptoms should develop on inoculated plants within 2 to 3 weeks. Transmission efficiency can range from 0% to 100%,
depending on the vector species and population, source and recipient plants, and number
of vectors used to inoculate the host. If an ideal
system is developed, it is rare that 100% of the
individual insects will transmit virus, and in
many cases, the transmission efficiency is low
enough that transmission tests using individual
insects will yield negative results. Therefore, it
is best to start the transmission experiments by
Plant RNA
Viruses
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Time Considerations
Transmission experiments can take considerable time, much of which is related to rearing
insects. For example, it will generally take 3
to 4 weeks to establish a sizable colony of
most aphid species on a suitable host, longer
if they are reared on a marginal host. Other
insects will take longer or shorter times to
establish colonies, depending on generation
times.
The actual transmission experiments are
relatively quick to set up and conduct, but
this depends on the acquisition and inoculation times required, as well as the number of
treatments and plants used in each experiment.
Then, there is the time required for the inoculated virus to infect and spread in the plant
until it can be detected either by symptoms
or some other diagnostic method. In short, a
transmission experiment can take months if it
includes establishing insect colonies and determining all the suitable parameters.
Literature Cited
Ammar, E.D. and Nault, L.R. 2002. Virus transmission by leafhoppers, planthoppers, and treehoppers (Auchenorrhyncha, Homoptera). Adv. Bot.
Res. 36:141-167.
Aphid
Transmission of
Plant Viruses
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UNIT 16B.2
The majority of plant viruses, if not all, are experimentally introduced into epidermal
cells of intact plant leaves by simple mechanical inoculation. However, abrasives commonly used to create surface injury to facilitate virus entry often cause extensive cell
damage, exposing RNA inoculum to nucleases. This constraint, however, does not apply
to inoculation with infectious virions purified from symptomatic leaves. Despite the fact
that mechanical inoculation of whole plants is the simplest approach, it suffers from the
difficulty of attaining a synchronous and uniform delivery of the inoculum to the majority
of plant cells. Attainment of a synchronized infection is not applicable to viruses having
monopartite genomes (e.g., tobacco mosaic tobamovirus; TMV); whereas, replication
and gene expression studies for viruses having multipartite genomes (e.g., brome mosaic
bromovirus; BMV) requiring the presence of all genome components in the same cell
are hindered by this approach.
The advent of recombinant DNA technology has greatly enhanced approaches in handling RNA-based viral genomes as well as for delivering one or multiple foreign genes
into a wide range of plant cells for gene expression studies. The literature is replete
with a variety of delivery approaches, such as electroporation, microinjection, or particle
bombardment (UNIT 16B.3), that successfully deliver nucleic acids to plant cells. However,
these approaches often require highly sophisticated equipment and skilled personnel.
Research conducted over the years has demonstrated that plant pathogens could serve as
potential tools for effective delivery of genes into plant cells. One such pathogen that has
been exploited for gene delivery is the bacterial pathogen Agrobacterium tumefaciens.
Delivery of cultures of Agrobacterium transformants into whole leaves by infiltration,
referred to as agroinfiltration (Bendahmane et al., 2000; Voinnet et al., 2003), is a simple,
user-friendly, highly reproducible approach to transient gene expression. Agroinfiltration
results in transfer of transgenes from the transfer DNA (T-DNA) region of the bacterial
tumor-inducing (Ti) plasmid into plant cells. The nonintegrated T-DNA copies remaining
transiently present in the nucleus can be transcribed, leading to transient expression of
T-DNA genes (Fig. 16B.2.1A). Agrobacterium-mediated transient gene expression has
been widely used in plant biology for identification of disease-resistance genes (Bendahmane et al., 2000) and induction and suppression of post-transcriptional gene silencing
(PTGS; Johansen and Carrington, 2001; Voinnet et al., 2003). Most importantly, agroinfiltration facilitates delivery and coexpression of multiple genes to the same cell. Although
this technique was developed originally for use with DNA plant viruses (Grimsley et al.,
1987), it subsequently proved to be useful for initiating infection with RNA viruses
(Leiser et al., 1992).
To facilitate agroinfiltration, the complementary DNA (cDNA) copy of a given viral RNA
genome initially has to be placed under the control of a cauliflower mosaic virus 35S
promoter (CaMV 35S) by subcloning into a binary vector, i.e., a two-plasmid system
in Agrobacterium for carrying the virulence genes on one plasmid and the engineered
T-DNA region on the other. Creation of the binary vector is followed by transformation
into a suitable Agrobacterium strain. The presence of a natural 5 viral sequence is an
essential prerequisite for achieving wild typelevel biological activity (Boyer and Haenni,
1994). Therefore, cloning of cDNA copies into a suitable binary vector should be designed
such that the transcription by the CaMV 35S promoter should initiate precisely at the
authentic viral 5 end. Unlike the 5 end, the effect of 3 nonviral nucleotide extensions on
the biological activity of viral RNAs is varied (Boyer and Haenni, 1994; Ishikawa et al.,
Contributed by Padmanaban Annamalai and A. L. N. Rao
Current Protocols in Microbiology (2006) 16B.2.1-16B.2.15
C 2006 by John Wiley & Sons, Inc.
Copyright
Plant RNA
Viruses
16B.2.1
Supplement 1
1997; Annamalai and Rao, 2005). However, if viral mRNAs with correctly terminated
3 ends are required, a self-cleaving ribozyme cassette from either the satellite tobacco
ringspot virus (Bruening et al., 1988) or hepatitis virus (Been, 1994) can also be
engineered. A variety of binary vectors suitable for agroinfiltration have been developed
(Hellens et al., 2000; Annamalai and Rao, 2005): some common features of a typical
binary vector are shown schematically in Figure 16B.2.1B.
Several T-DNA vectors suitable for agroinfiltration are now available and include the
following: pBIN19, pBINPLUS, pC22, pCAMBIA series, pCGN series, pPZP series,
and pGREEN series. The choice of vector depends on the application and considerations of convenience (Hellens et al., 2000). Additional information concerning the
characteristics of various binary vectors can be obtained from the following web sites:
http://www.cambia.org or http://www.sainsbury-laboratory.ac.uk.
Delivery and
Expression of
Functional Viral
RNA Genomes
In Planta by
Agroinltration
16B.2.2
Supplement 1
Numerous Agrobacterium strains suitable for transformation of binary plasmids carrying the gene of interest are also available and the choice of an Agrobacterium
strain depends on the host plant to be infiltrated. Some commonly used strains include LBA4404, C58C1, GV3100, GV2260, GV3101(pMP90), GV3101(pMP90RK),
EHA101, or EHA 105 (Hellens et al., 2000; Weigel and Glazebrook, 2002). Most
Agrobacterium strains (except C58C1 and GV3100) carry a rifampicin resistance
gene for selection. Strain GV3101(pMP90) carries gentamycin resistance on the Ti
plasmid, whereas GV3101(pMP90RK) carry gentamycin and kanamycin resistance.
Earlier techniques used strain LBA 4404, but this strain often appeared to be avirulent.
Current Protocols in Microbiology
Alternatively, more virulent strains derived from C58 such as GV3101(pMP90) and GV
3101(pMP90RK) or EHA105 are also available (Hellens et al., 2000).
In the methods described here, transformed Agrobacterium culture is grown to log phase,
collected by low-speed centrifugation, and resuspended in an infiltration solution. The
suspension is then infiltrated to the abaxial (back) surface of a leaf using a syringe (without
the needle). Expression of the gene of interest can be analyzed 2 to 6 days postinfiltration.
Given the fact that agroinfiltration facilitates transformation of a single cell with multiple
genes, the approach is ideal for not only delivering DNA-based genomes of monopartite
RNA viruses but also for coexpression of individual viral RNA components comprising
either bipartite or tripartite (i.e., the genome divided among two or three distinct RNA
species, respectively) viruses for a synchronized high-level gene expression within the
same cell (Vlot et al., 2001; Liu and Lomonossoff, 2002; Annamalai and Rao, 2005).
This inherent trait associated with agroinfiltration is also suitable for co-delivery of either
defective interfering RNAs (DI RNAs) or satellite RNAs along with their respective helper
viral genomes.
This unit describes procedures for the preparation of competent cells of Agrobacterium
(Basic Protocol 1) and their transformation using a freeze-thaw approach (Basic Protocol
2), preparation of competent cells (Basic Protocol 3 and Alternate Protocol 1) and their
transformation using electroporation (Basic Protocol 4), and finally, preparation of transformed cultures of Agrobacterium for infiltration of a plant species (Basic Protocol 6).
In addition, Basic Protocol 5 and Alternate Protocol 2 provide methods for verifying
the presence of the gene of interest in the transformed cells. Both transformation procedures are effective, and the decision for choosing one largely depends on convenience
and availability of required laboratory equipment. The basic approaches described in
this unit are generally applicable not only to analyzing transient gene expression but
also to the study of high-level expression of biologically active viral genomes in virus
replication, movement, and assembly.
CAUTION: Agrobacterium is a Biosafety Level 1 (BSL-1) organism. Such organisms
are not known to consistently cause disease in healthy adult humans, and are of minimal
potential hazard to laboratory personnel and the environment. Standard microbiological
practices should be followed when working with these organisms. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for more information.
BASIC
PROTOCOL 1
Since DNA is a very hydrophilic molecule, it would not normally pass through a bacterial cells membrane. Bacteria that are able to take up DNA are called competent.
Competency can be induced by treatment with calcium chloride in the early log phase of
growth. The bacterial cell membrane is permeable to chloride ions but is nonpermeable
to calcium ions. As the chloride ions enter the cells, water molecules accompany the
charged particles. This influx of water causes the cells to swell and is necessary for uptake of DNA. Competent cells must always be maintained in a cold environment, during
storage and use. Competent cells cannot be defrosted more than once. This protocol
describes procedures for preparing competent Agrobacterium cells for transformation
with a binary plasmid carrying the gene of interest using a freeze-thaw approach (see
Basic Protocol 2).
Materials
Agrobacterium strain (e.g., LBA 4404, C58C1, GV2260, or EHA 105; see Hellens
et al., 2000)
LB plates (see recipe)
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Viruses
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Current Protocols in Microbiology
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BASIC
PROTOCOL 2
Delivery and
Expression of
Functional Viral
RNA Genomes
In Planta by
Agroinltration
16B.2.4
Supplement 1
Materials
Competent Agrobacterium cells (Basic Protocol 1), 80 C
Plasmid DNA: binary vector containing the gene of interest (e.g., see Sambrook
and Russell, 2001)
LB medium (see recipe)
LB plates with 50 g/ml kanamycin and 50 g/ml rifampicin (see recipe) or other
appropriate antibiotic
28 C orbital shaker
28 C incubator
1. Thaw one of the microcentrifuge tubes containing competent cells on ice.
2. Add 2 to 5 g plasmid DNA (binary vector containing the gene of interest).
3. Mix gently and incubate 45 min on ice.
4. Freeze contents of the microcentrifuge tube by placing in liquid nitrogen for 1 min;
immediately transfer to 37 C. Incubate 3 min.
5. Add 0.9 ml LB medium and incubate on an orbital shaker 3 hr at 28 C, 200 to
300 rpm.
This will allow recovery of cells from frozen state and expression of the marker gene.
9. Incubate plates 48 hr at 28 C.
After 48 hr, significant numbers of colonies are visible on the plate. Generally these
colonies appear compact compared to normal bacterial colonies.
BASIC
PROTOCOL 3
Since DNA is a very hydrophilic molecule, it would not normally pass through a bacterial
cells membrane. The bacterial cells are competent when they are amenable to uptake of
the plasmid DNA. Competent cells prepared for the freeze-thaw method of transformation
(Basic Protocols 1 and 2) are not suitable for electroporation because the methods are
different. In transformation by electroporation, DNA is forced into cells by initially
incubating on ice the reaction mixture containing the competent cells and the DNA,
followed by electroporation.
Materials
Agrobacterium strain (e.g., LBA 4404, C58C1, GV2260, or EHA 105; see Hellens
et al., 2000)
LB plates (see recipe)
LB medium (see recipe)
1 mM 4-(2-Hydroxyethyl)piperazine-1-ethane sulfonic acid (HEPES), pH 7: pass
through a 0.45-m filter to sterilize; store up to 3 months at room temperature
Glycerol, sterile
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Viruses
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Current Protocols in Microbiology
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28 C incubator
28 C orbital shaker
250-ml flask
50-ml polypropylene centrifuge tubes with tops, sterile (e.g., Fisher)
1.5-ml microcentrifuge tubes, sterile
1. Streak desired strain of Agrobacterium from a glycerol stock onto an LB plate
supplemented with the appropriate antibiotic. Incubate at 28 C for 2 days.
2. Pick a discrete single colony and inoculate a tube containing 5 ml LB medium.
3. Incubate the tube on an orbital shaker overnight at 28 C, 200 to 300 rpm.
4. In a 250-ml flask, inoculate 100 ml LB medium with 1 ml of overnight culture.
Incubate 6 hr at 28 C or until the culture reaches an optical density (OD600 ) of 0.5
to 0.6.
5. Transfer the flask containing the grown bacterial cells to a bucket of ice and incubate
30 min.
6. Transfer bacterial cells to 50 ml-polypropylene centrifuge tubes and centrifuge
5 min at 2000 g (5000 rpm), 4 C. Discard supernatant.
7. Resuspend pellet in 20 ml prechilled 1 mM HEPES, pH 7.0. Centrifuge 5 min at
2000 g, 4 C. Discard supernatant.
8. Wash pellet with 20 ml prechilled 1 mM HEPES, pH 7.0, and repeat centrifugation.
Discard supernatant.
9. Resuspend the final pellet in 2 ml prechilled 1 mM HEPES, pH 7.0, and add sterile
glycerol to a final concentration of 10% (v/v).
10. Dispense 50-l aliquots of the electrocompetent cells into sterile 1.5-ml microcentrifuge tubes.
11. Use cells immediately or store up to 3 months at 80 C.
ALTERNATE
PROTOCOL 1
16B.2.6
Supplement 1
BASIC
PROTOCOL 4
7. Centrifuge cells 5 min at 500 g, room temperature. Discard most of the supernatant,
leaving 100 l for resuspension.
8. Gently mix pellet by tapping with a finger.
9. Spread the transformed mixture onto YEP plates supplemented with 50 g/ml
kanamycin and 50 g/ml rifampicin.
Generally, the contents of one tube are spread onto one plate.
28 C.
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Viruses
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After 48 hr, significant numbers of colonies are visible on the plate. Generally, these
colonies appear compact compared to normal bacterial colonies.
Materials
Transformed Agrobacterium cultures (Basic Protocol 2 or Basic Protocol 4)
LB medium with appropriate antibiotics (see recipe)
Plasmid DNA extraction kit (e.g., QIAprep Spin miniprep kit; Qiagen; optional)
28 C orbital shaker
Additional reagents and equipment for isolation (Engebrecht et al., 1991) and
digestion (Sambrook and Russell, 2001) of plasmid DNA, and agarose gel
electrophoresis (Voytas, 2000)
1. Pick a single discrete colony of transformed Agrobacterium and inoculate a tube
containing 5 ml LB medium with appropriate antibiotics.
Generally ten to fifteen individual colonies should be randomly picked for screening.
Materials
Delivery and
Expression of
Functional Viral
RNA Genomes
In Planta by
Agroinltration
16B.2.8
Supplement 1
1. Pick fully grown colonies from the plates containing the transformed Agrobacteria
and suspend in 10 l distilled water.
Normally 10 to 20 such colonies should be selected for analysis.
1 min
1 min
1 min
10 min
94 C
55 C
72 C
72 C
(denaturation)
(annealing)
(extension)
(final extension).
6. Analyze the PCR products on a 1% agarose gel (Voytas, 2000) to verify presence or
absence of the gene of interest.
The size of the PCR product can be used to confirm the presence of the gene of interest in
the transformant.
Once a positively transformed bacterial colony has been identified, it can be stored
permanently as a glycerol stock for future agroinfiltration experiments.
BASIC
PROTOCOL 6
Generally young seedlings of Nicotiana benthamiana having two to three well expanded
leaves (five-leaf stage, three- to four-week-old plants) are ideal for infiltration.
Materials
Positively identified, transformed Agrobacterium cultures (Basic Protocol 5 or
Alternate Protocol 2)
LB plates supplemented with 50 g/ml kanamycin and 50 g/ml rifampicin (see
recipe) or other appropriate antibiotic
LB medium supplemented with 50 g/ml kanamycin and 50 g/ml rifampicin (see
recipe) or other appropriate antibiotic
1 M 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.6: pass through a 0.45-m
filter to sterilize and store up to 3 months at room temperature in a brown bottle
100 mM 5 dimethoxy-4 -hydroxyacetophenone (acetosyringone) in DMSO
10 mM MgCl2 : prepare from filter-sterilized (0.45-m filter) 1 M MgCl2 and
sterile H2 O
Nicotiana benthamiana seedlings: five-leaf stage, i.e., three- to four-week-old
plants having two to three well expanded leaves, or other appropriate seedling
28 C incubator
28 C orbital shaker
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12. Keep the Agrobacterium cultures to be used for infiltration at room temperature for
at least 3 hr without shaking.
13. Using a 1-ml syringe, gently press the syringe onto one half of the abaxial surface
of a fully expanded leaf of a Nicotiana benthamiana (or other appropriate plant)
seedling. Repeat the process on the opposite side of the main vein.
16B.2.10
Supplement 1
There is not a specific volume for each infiltration. Depending on the type of assay, either
the entire leaf or a 1- to 2- cm spot can be infiltrated with liquid culture.
A fully infiltrated part of the leaf gives a water-soaked appearance.
Generally infiltration is very efficient in N. benthamiana, requiring only one infiltration
site. However, depending on the age of the leaf, more than one infiltration may be
required. Depending on the nature of experiment, the extent of infiltrated area can be
controlled.
14a. For immediate transfer: After agroinfiltration, transfer plants to greenhouse, growth
room, or large growth chamber.
14b. For delayed transfer: Hold agroinfiltrated plants in a growth chamber 1 day under
mild light conditions (1,000 lux) with 16 hr light and 8 hr dark photoperiod cycle,
20 C to 23 C, and then transfer plants to a greenhouse.
Incubating infiltrated plants at higher temperatures can inhibit T-DNA transfer.
15. Collect leaf samples from 2 days post-infiltration onwards to screen for transient
expression of the gene of interest.
Kanamycin, 50 mg/ml
Dissolve 50 mg kanamycin in 1 ml water. Sterilize by passing through a 0.22-m
filter. Dispense 100-l aliquots into sterile 1.5-ml microcentrifuge tubes and store
up to 3 months at 20 C.
LB medium with and without 50 g/ml kanamycin and 50 g/ml rifampicin
Dissolve the following in 1 liter H2 O with mixing:
5 g yeast extract
10 g bacto-tryptone
10 g sodium chloride
Dispense 100 ml medium into 250-ml Erlenmeyer flasks and autoclave 20 min at
15 psi on liquid cycle. Store up to 1 month at room temperature or up to 4 months
at 4 C. If antibiotics are required, before use add 50 l of 100 mg/ml rifampicin
(see recipe) and 100 l of 50 mg/ml kanamycin (see recipe) or other appropriate
antibiotics to each 100 ml medium.
Plant RNA
Viruses
16B.2.11
Current Protocols in Microbiology
Supplement 1
SOB medium
Dissolve the following with shaking in 950 ml H2 O:
20 g tryptone
5 g yeast extract
0.5 g sodium chloride
Add 10 ml 250 mM CaCl2 . Adjust pH to 7.0 with 5 N NaOH. Adjust final volume
to 1 liter with water. Autoclave 20 min at 15 psi. Dispense 100 ml medium into
250-ml Erlenmeyer flasks and autoclave 20 min at 15 psi on liquid cycle. Store up
to 1 month at room temperature or up to 4 months at 4 C. Just before use add 0.5 ml
sterile 2 M MgCl2 to 100 ml medium.
SOC medium
Dissolve the following with shaking in 950 ml H2 O:
20 g tryptone
5 g yeast extract
0.5 g sodium chloride
Add 10 ml of 250 mM CaCl2 . Adjust pH to 7.0 with 5 N NaOH. Adjust final volume
to 1 liter with water. Autoclave 20 min at 15 psi. Add 20 ml sterile 1 M glucose
solution. Dispense 100 ml medium into 250-ml Erlenmeyer flasks and autoclave
20 min at 15 psi on liquid cycle. Store up to 1 month at room temperature or up to
4 months at 4 C. Just before use add 0.5 ml sterile 2 M MgCl2 to 100 ml medium.
YEP plates with and without 50 g/ml kanamycin and 50 g/ml rifampicin
Dissolve the following in 1 liter H2 O with mixing:
10 g Bacto peptone
5 g yeast extract
15 g NaCl
15 g agar powder
Dispense 100 ml medium into 250-ml Erlenmeyer flasks and autoclave 20 min at
15 psi on liquid cycle. Cool the medium to 55 C in a water bath and add 50 l of
100 mg/ml rifampicin (see recipe) and 100 l of 50 mg/ml kanamycin (see recipe)
or other appropriate antibiotics to each 100 ml medium. Dispense 25 to 30 ml into
sterile 15 100mm disposable petri dishes. When the medium has completely
solidified, store plates in a covered plastic box or wrapped with Parafilm up to 6
weeks at 4 C.
COMMENTARY
Background information
Delivery and
Expression of
Functional Viral
RNA Genomes
In Planta by
Agroinltration
Agroinfiltration is simple, efficient, and robust compared to conventional mechanical inoculation or other delivery methods. Most importantly, it results in synchronous delivery
of Agrobacterium transformants to a majority
of the cells encompassing the infiltrated area.
Furthermore, agroinfiltration is ideal for delivering multiple Agrobacterium transformants
to a single cell. Because of the high transformation rate and efficient accumulation of
mRNAs, the method is ideal for analyzing biological activities of viral genomes with defective replication traits. One caveat with this
approach is that not all host plants are suitable for infiltration. Among several tobacco
species tested, Nicotiana benthamiana was
found to be the easiest to infiltrate. Likewise,
16B.2.12
Supplement 1
Troubleshooting
Possible causes of problems related to
agroinfiltration and potential solutions are
listed in Table 16B.2.1.
Anticipated Results
The high level of Agrobacterium-mediated
transformation generally results in the production of an abundant amount of mRNA from
each transformed cell. As a result, mRNA levels and the expression of respective genes can
be verified with ease using routine detection
assays such as northern blots, immunoblots,
and confocal laser scanning microscopy
(UNIT 2C.1), if reporter genes such as GFP are
used.
Time Considerations
Critical Parameters
A fresh culture must be grown for 48 hr
from the initial stock each time an infiltration is to be performed. Furthermore, a 16-hr
culture should be grown using a 48-hr-old primary culture. This will eliminate the presence of dead cells in the inoculum. Verify
the final optical density at OD600 of the culture, which must be 0.8 to 1.0. When two
or more Agrobacterium transformants are to
be co-infiltrated, grow each culture independently as described above. When two Agrobac-
Plant RNA
Viruses
16B.2.13
Current Protocols in Microbiology
Supplement 1
Problem
Possible Cause
Solution
Agrobacterium cells
did not grow
Concentration of
rifampicin too high
Overnight culture
did not reach OD600
of 0.5
No transformants
Reduce voltage
No transformants
Reduced quality or
quantity of plasmid DNA
No transformants
No insert
No transformants
Use of inappropriate
antibiotic
No activation of virulence
genes
No transient
expression
No insert
No transient
expression
No activation of virulence
genes
No transient
expression
Activation of RNA
silencing
Literature Cited
Annamalai, P. and Rao, A.L.N. 2005.
Agrobacterium-mediated DNA expression
of functional genome components of brome
mosaic virus in planta: Transient expression of
viral capsid protein and RNA packaging independent of replication. Virology 338:96-111.
Balucombe, D.C. 1999. Fast forward genetics based
on virus induced gene silencing. Curr. Opin.
Plant Biol. 2:109-113.
Been, M.D. 1994. Cis-and trans-acting ribozymes
from human pathogen, hepatitis delta virus.
Trends Biochem. Sci. 19:251-256.
Bendahmane, A., Querci, M., Kanyuka, K., and
Baulcombe, D.C. 2000. Agrobacterium transient expression system as a tool for the isolation
of disease resistance genes: Application to the
R2 locus in potato. Plant J. 21:73-81.
Boyer, J.C. and Haenni, A.L. 1994. Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415-426.
Delivery and
Expression of
Functional Viral
RNA Genomes
In Planta by
Agroinltration
Bruening, G., Buzayan, J., Gerlach, W., and Hampel, A. 1988. Small RNA autolytic cleavage and
ligation. In From Proteins to Ribosomes, Vol. 1
(R.H. Sarma and M.H. Sarma, eds.) pp. 239-248.
Adenine Press, Schenectady, New York.
Daros, J.A. and Flores, R. 2004. Arabidopsis
thaliana has the enzymatic machinery for replicating representative viroid species of the family pospiviroidae. Proc. Natl. Acad. Sci. U.S.A.
101:6792-6797.
Engebrecht, J., Brent, R., and Kaderbhai, M.A.
1991. Minipreps of plasmid DNA. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.6.11.6.10. John Wiley & Sons, Hoboken, N.J.
Grimsley, N., Hohn, T., Davies, J.W., and Hohn,
B. 1987. Agrobacterium mediated delivery of
infectious maize streak virus into maize plants.
Nature 325:177-179.
Hellens, R., Mullineaux, P., and Klee, H. 2000.
A guide to Agrobacterium binary Ti vectors.
Trends Plant Sci. 5:446-451.
Ishikawa, M., Janda, M., Krol, M.A., and Ahlquist,
P. 1997. In vivo DNA expression of functional
brome mosaic virus RNA replicons in Sacchromyces cerevisiae. J. Virol. 71:7781-7790.
Johansen, L.K. and Carrington, J.C. 2001. Silencing
on the spot. Induction and suppression of RNA
16B.2.14
Supplement 1
Plant RNA
Viruses
16B.2.15
Current Protocols in Microbiology
Supplement 1
UNIT 16B.3
This unit addresses particle bombardment, a method for the acceleration of microscopic
particles of metal coated with viral material into plant tissue by the use of pressurized
gas. It is primarily used for inoculating intact plants with full-length infectious cDNA
or transcripts of RNA viruses. Particle bombardment has been used to insert DNA into
living plant cells for both transient and permanent gene expression since the pioneering
work of Klein et al. (1987). Additionally, Klein et al. were the first to show the expression
of tobacco mosaic virus (tobamovirus) in onion epidermal cells following bombardment
with RNA. Inoculating RNA viruses by bombardment requires full-length, infectious,
viral cDNA which has been cloned or PCR-amplified and has appropriate promoters and
termination signals. Bombardment with RNA transcripts refers here to in vitroproduced
transcripts.
Bombardment does not replace the use of other mechanical or natural vector inoculation
methods described in this section; however, it permits cDNA inoculation of plants without
the production of viral transcripts, resulting in lower costs, less work, improved stability
of transcripts, and greater efficiency (for potyviruses). Inoculation of cDNA can also be
accomplished by infiltration with Agrobacterium tumefaciens (UNIT 16B.2), although this
lengthier method inserts another pathogen into the plant-virus system.
There are many types of particle bombardment equipment, including the particle inflow
gun (PIG; Finer et al., 1992), the plastic PIG (Grey et al., 1994), and the Bio-Rad PBS
(Klein et al., 1987), all of which may require technical assistance from outside the
laboratory to construct a vacuum chamber for their use (Finer et al., 1992; Gray et al.,
1994). Here, the authors discuss two types of bombardment equipmentthe Blowpipe
and the HandGunneither of which require a vacuum chamber and both of which can
be assembled in the laboratory with little assistance, using commonplace, commercially
available parts. The more complex apparatus, the HandGun, should cost $200 to $300
in parts, excluding the cost of a gas cylinder. Consumables, e.g., gas, metal particles,
and chemicals, are also low cost with this bombardment method. Other advantages
of homemade equipment are ease of maintenance and the ability to alter the device,
according to need, without concern for a manufacturers warranty.
In Basic Protocol 1, the authors describe the parts required for the Blowpipe, a simple
but useful instrumenteffectively the same as the device described by Takeuchi et al.
(1992)and the HandGun, a partially automated version of the Blowpipe, which is
easier to handle and therefore generally preferable but which requires more parts, cost,
and construction effort (Gal-On et al., 1997). Both of these devices share a common
component, the discharge assembly.
In addition, instructions for the use of the equipment are provided (Basic Protocol 2),
along with information about preparation of cDNA or RNA transcripts (Support
Protocol 1) and the metal mixtures for bombardment (see Reagents and Solutions).
Bombardment with plant sap, virus preparation, or viral RNA extract is also discussed
(Support Protocol 2).
The technology described here is very efficient. Particle bombardment without a vacuum
is 105 more efficient than mechanical inoculation (UNIT 16B.1) with a plasmid encoding a
full-length clone driven by a promoter, and particle bombardment in a vacuum is 10-fold
Plant RNA
Viruses
Contributed by Victor Gaba and Amit Gal-On
Current Protocols in Microbiology (2005) 16B.3.1-16B.3.14
C 2005 by John Wiley & Sons, Inc.
Copyright
16B.3.1
Bombardment material
Reference
RNA-virus preparation
Table 16B.3.2 Techniques and Investigations Enabled by the Blowpipe and HandGun
Bombardment Technology
Technique
Reference
more efficient than without a vacuum (Gal-On et al., 1995, 1997). The materials described
in Table 16B.3.1. can be used for plant RNA virus inoculation by particle bombardment.
This technology allows for the techniques and investigations detailed in Table 16B.3.2,
among them the convenience of mass bombardment methods (Alternate Protocol) and
particle bombardment in the greenhouse or the field.
BASIC
PROTOCOL 1
Inoculation of
Plants Using
Bombardment
16B.3.2
Current Protocols in Microbiology
Figure 16B.3.1 The assembled HandGun. (a) HandGun. (b) The trigger switch taped in position.
(c) Wall-mounted timer. (d) Secondary wall-mounted gas pressure gauge. (e) Secondary gas
regulator. (f) Clamp stand to hold the HandGun. (g) Plant to be bombarded. The tubing on the
right hand side of the picture connects to the He gas cylinder. The lter holder is not attached.
Materials
Materials for construction of the discharge assembly (Fig. 16B.3.2):
Luer adapter, male luer lock to 1/4 -in. (6.35-mm) unified fine threads (UNF; e.g.,
Cole-Parmer cat. no. 31507-73; a)
Luer adapter, male (M) 1/4 -in. (6.35-mm) fitting attached to a machined UNF
female thread to accept the luer lock adapter (b)
13-mm-diameter plastic filter holder (Pall-Gelman cat. no. 4312; c)
Materials for construction of the Blowpipe apparatus (Fig. 16B.3.3):
7-mm rubber vacuum hose, 5 m (a)
Plastic two-way tap, 8 to 10 mm (b)
Barbed fitting to connect rubber tubing to a metal fitting (c)
1
/4 -in. (6.35-mm) coupling adapter female piping thread (FPT)/FPT, if required
Worm-driven hose clips for fixing rubber tubing onto metal pipes (d)
Materials for construction of the HandGun apparatus:
Materials for assembling the HandGun device:
Electrically operated miniature solenoid valve (Dynamco cat. no. D3533KL0;
Fig. 16B.3.4a)
Base plate (Dynamco cat. no. B03B2B) with 1/4 -in. (6.35-mm) apertures
(Fig. 16B.3.4b).
Plant RNA
Viruses
16B.3.3
Current Protocols in Microbiology
Figure 16B.3.2 Components of the discharge assembly. (a) Leur adapter male, luer lock to UNF
thread. (b) Leur adapter, male 1/4 -in. BSP tting with a machined UNF female thread. (c) Filter
holder (open): top on left, lower part on right, showing the grid.
1
Construct the discharge assembly for both Blowpipe and HandGun (Fig. 16B.3.2)
1. Screw the UNF thread of the luer adapter into the UNF female thread of the adapter
machined to accept it and having a 1/4 -in. fitting on the other end.
16B.3.4
Current Protocols in Microbiology
Figure 16B.3.3 Materials for constructing the Blowpipe apparatus. (a) Rubber vacuum hose,
7-mm diameter. (b) Two-way plastic tap. (c) Barbed tting to connect rubber hose to a pipetting. (d) Worm-drive hose clips. Four are required. (e) Assembled discharge assembly (from
Fig. 16B.3.2). Filter holder is not attached.
2. Twist the 13-mm-diameter plastic filter holder into the luer fitting at the end of the
assembly.
The filter holders have an internal grid on which to place the drop of nucleic acidcoated
metal, a screw fitting to attach to the bombardment device, and an exit nozzle shape well
suited to the bombardment process. It is advisable to purchase at least ten units.
4. Insert the plastic two-way tap between the lengths of tubing beside the discharge
assembly. Connect the barbed fitting to the discharge assembly (see steps 1 and 2),
with a 1/4 -in. coupling adapter FPT/FPT, if required. Secure all joints with wormdriven hose clips.
The assembled discharge assembly and coupling adapter are shown in Figure 16B.3.3.
Plant RNA
Viruses
16B.3.5
Current Protocols in Microbiology
Figure 16B.3.4 The HandGun apparatus. (a) Electrically operated miniature solenoid valve.
(b) Base plate. (c) Pipe-tubing elbow (also see Fig. 16B.3.5c). (d) 1/4 -in. BSP adapter FPT/M
(40 mm). (e) A miniature DIN plug. (f) Discharge assembly (from Fig. 16B.3.2). Filter holder is
not attached.
Figure 16B.3.5 Assorted components required for constructing the bombardment devices. (a)
1
/4 -in. BSP coupling adapter FPT/FPT. (b) 1/4 -in. BSP nipple adapter M/M. (c) Pipe-tubing elbow
(see Fig. 16B.3.4). (d) 1/4 -in. BSP plugs (non-protruding). (e) T-adapter, MFF, 1/4 , 1/2 , 1/4 in. (see
Fig. 16B.3.7b). If the HandGun is constructed with a secondary regulator, six BSP coupling
adapters, six BSP nipple adapters, and four pipe-tubing elbows will be required.
Inoculation of
Plants Using
Bombardment
Figure 16B.3.6 Components for the timer assembly. (a) 220 V variable (0 to 100 msec) timer.
(b) Push-button electrical switch. (c) Plastic box for mounting the timer with electrical connections.
16B.3.6
Current Protocols in Microbiology
Figure 16B.3.7 Components for the secondary gas regulator. (a) Pressure regulator (0.05 to
0.85 MPa), with female 1/4 -in. BSP ttings. (b) T-adapter, MFM, 1/4 , 1/2 , 1/4 in. (see Fig. 16B.3.5e).
(c) 0- to 10-bar gas pressure gauge, 100 mm diameter, 1/2 -in. BSP male tting. Pipe-tubing elbows
(Fig. 16B.3.5c) are at each end of the secondary gas regulator. Adapters (couplings and nipples;
see Fig. 16B.3.5) connect the pieces when necessary. Direction of helium gas ow is indicated by
arrows. (d) Two-way tap; 1/4 -in. female BSP ttings.
11. Place the timer in a plastic box, which may be wall mounted. Make sure that both of
the wires are long enough to place the box with the timer where necessary, and that
both wires reach the position where the HandGun will be operated (Fig. 16B.3.1).
Plant RNA
Viruses
16B.3.7
Current Protocols in Microbiology
These hoses will run from the gas cylinder to the secondary gas regulator and from there
to the HandGun.
19. Set the exit port from the gas cylinder to a maximum of 5 bars (500 kPa), so that
the miniature solenoid valve will not be under too much pressure.
20. Tape the trigger button of the timer to the right-hand side of the HandGun
(Fig. 16B.3.1).
This allows the hand holding the HandGun to operate the switch and the other hand to
hold the plant.
BASIC
PROTOCOL 2
Materials
1.25 M Ca(NO3 )2 4H2 O, pH 10.5 or 7.8: adjust pH slowly with HCl, autoclave,
aliquot, and store up to 2 years at 20 C
Tungsten or gold particle stock (see recipes)
cDNA or RNA transcripts in water (Support Protocol 1) or
Plant sap, purified virus, viral RNA extract, or total RNA (Support Protocol 2)
Target plants at correct stage, e.g., cotyledon
Microcentrifuge tubes, sterile
Blowpipe or HandGun (Basic Protocol 1)
Disposable gloves (UNIT 1A.3)
10-ml syringe
NOTE: Use normal precautions for working with nucleic acids, including sterile solutions
and autoclaved utensils.
16B.3.8
Current Protocols in Microbiology
Vary the total amount according to need, calculating for 2.5 l per shot, preparing
only enough mixture for bombardment of five to ten plants.
The unadjusted pH of the Ca(NO3 )2 solution is 9 to 11, appropriate for precipitation of
cDNA onto the metal particles. However, RNA should be precipitated at pH 7 to 8. Lower
the pH with careful addition of HCl, since the solution is not buffered.
Use of Eppendorf-brand microcentrifuge tubes and tips can reduce the clumping on
addition of DNA. Additionally, the use of siliconized microcentrifuge tubes and tips, while
optional, can help avoid loss of tungsten- or gold/DNA on vessel walls. See APPENDIX
2A for silanization procedure.
The authors use a tungsten particle size of 1.6 m, but other groups have used other sizes
successfully.
2. At the beginning, middle, and end of a 5- to 10- min period, mix by tapping.
7a. Screw the filter holder closed and attach it to the luer-lock adapter at the end of the
Blowpipe.
8a. Support the cotyledon or leaf of the target plant with a gloved hand.
CAUTION: Use gloves so that the metal is not bombarded into the experimenters hand.
Tungsten and gold are considered toxic metals.
9a. Hold the discharge assembly close to the target plant, and sharply open and close
the tap beside it, discharging the Blowpipe onto the plant.
10a. Repeat steps 3a to 9a until no bombardment mixture is left. Discard the microcentrifuge tube.
11a. Make a fresh bombardment mixture. Use a fresh, autoclaved filter and holder every
time a different cDNA or RNA transcript is used (steps 1 to 10a).
Plant RNA
Viruses
16B.3.9
Current Protocols in Microbiology
6b. Screw the filter holder closed and attach it to the luer-lock adapter at the end of the
HandGun.
7b. Adjust the clamp stand so that the end of the filter holder is 2 to 3 cm away from
the target leaf or cotyledon of the target plant.
8b. Support the cotyledon or leaf to be bombarded with a gloved hand and press the
button to fire.
CAUTION: Use gloves so that the metal is not bombarded into the experimenters hand.
Tungsten and gold are considered toxic metals.
9b. Fire the HandGun again at a second cotyledon or leaf on the same plant to discharge
entirely.
10b. Repeat steps 3b to 9b until no bombardment mixture remains.
11b. Discard the microcentrifuge tube and make a new bombardment mixture, using a
clean, autoclaved, filter holder for each different cDNA or mixture of cDNAs (steps
1 and 2, and 3b to 10b).
1. Drill a small hole in the top of a filter holder (Fig. 16B.3.2.c, left) with a 2-mm bit.
A pipet tip can be pushed through the drilled hole to touch the grid, and release the
bombardment mixture.
16B.3.10
Current Protocols in Microbiology
SUPPORT
PROTOCOL 1
In this unit bombardment with cDNA refers only to the use of infectious cloned DNA of a
plant RNA virus under control of any constitutive plant-active promoter (e.g., the CaMV
or SVBV promoter; Wang et al., 2000). The authors have been successful in using a plant
terminator at the end of the viral sequence. Alternatively, the viral cDNA (Gal-On et al.,
1995) or PCR-produced fragments (Fakhfakh et al., 1996) can be linearized at the 3 end,
resulting in effective termination. The efficiency of a viral poly(A) signal as a terminator
is not yet clear. Bombardment with RNA transcripts here refers to in vitroproduced
transcripts.
Prepare plasmids for cDNA bombardment, preferably using a commercial column (e.g.,
Qiagen; also see Heilig et al., 1998) or any miniprep method (Engebrecht et al., 1991).
The plasmid preparation can be bombarded directly onto plants from the column eluate or
from the miniprep. However, sometimes dilution of the plasmid preparation is necessary
to obtain better results. It is also possible to bombard with cDNA amplified by PCR
without cloning or purification (Fakhfakh et al., 1996).
For RNA transcripts (both naturally capped and naturally uncapped) produced using
cDNA cloned under the control of a nonplant promoter (e.g., phage promoter T7), which
allows in vitro transcription, 5 -capping is advisable, although uncapped transcripts are
infectious but less efficient (Gal-On et al., 1997). DNase treatment and phenol/chloroform
extraction are not necessary prior to bombardment.
Dilute the cDNA or transcripts to a range of 50 to 250 ng/l. High inoculation efficiency
can also be obtained with 1050 ng per/l. The best results are obtained with 50 ng/ml
of nucleic acids. Excess cDNA could reduce efficiency due to aggregation (clumping) in
the bombardment mixture.
SUPPORT
PROTOCOL 2
Plant RNA viruses (nonphloem limited) are usually inoculated mechanically, using infected plant sap or natural vectors. Using bombardment methods with sap extracted from
plant tissue can save plant material but has an efficiency similar to mechanical inoculation. However, using bombardment methods with purified virus and viral RNA is more
efficient than mechanical inoculation, as demonstrated by potyvirus inoculation (Gal-On
et al., 1995). Extra preparation is unnecessary with plant sap, purified virus, RNA extract,
or total RNA from an infected plant, except to adjust the calcium nitrate to a pH of 7 to
8 in these cases (Basic Protocol 2).
Plant RNA
Viruses
16B.3.11
Current Protocols in Microbiology
COMMENTARY
Background Information
The procedures described in this unit require nucleic acids (cDNA or RNA) to be
precipitated onto heavy metal (tungsten, gold)
particles. The coated particles are deposited in
a small volume on the grid of a filter holder and
accelerated into plant tissue by a blast of pressurized gas. The apparatus enables inoculation
with full-length infectious clone cDNA, PCR
products, virus from sap or virus preparation,
cloned viral transcripts, and some phloemlimited RNA viruses. Additionally, this technology allows the inoculation of large numbers
of plants, the inoculation of soft plants that
do not survive bombardment inoculation by
other means, inoculation in the greenhouse, the
study of viral recombination in planta, rapid
promoter analysis, and the monitoring of virus
movement using an infectious clone bearing a
reporter gene.
Troubleshooting
Critical Parameters
Inoculation of
Plants Using
Bombardment
Following the parameters listed here is critical to the success of the procedure:
1. Secure the gas cylinder according to local
safety rules.
2. Ensure that the electrical output of the
timer (current and voltage) is suitable to run
the solenoid gas valve.
Anticipated Results
Visible leaf damage due to bombardment
with tungsten particles is commonly observed
a day or two after bombardment but does not
16B.3.12
Current Protocols in Microbiology
Problem
Possible Cause
Solution
Tissue specificity
Tungsten clumping
Particle clumping
Time Considerations
The longest period of the procedure is waiting for the ordered components, and this will
depend entirely on the suppliers. Assembling
the Blowpipe will take a few minutes, assembly of the HandGun will take 20 min, and
wiring up the timer and inserting into a suitable box may take 1.5 to 2 hr. Bombardment
will be at the pace of 20 to 30 plants/hr for
a single worker making a metal-DNA preparation for five shots, bombarding five plants,
and then making the next bombardment mixture. This process can be accelerated by two
operators working together, one making prepa-
Acknowledgements
Contribution from the Agricultural Research Organization, The Volcani Center, Bet
Dagan, Israel, No. 502/05. The authors work
was supported by Research Grants from
BARD, The United States-Israel Binational
Agricultural Research and Development Fund,
and from the Chief Scientist of the Ministry
of Agriculture (Israel). The authors thank Dr.
Alex Lipsky, Department of Ornamental Horticulture, ARO Volcani Center, for permission
to publish his method for preparation of gold
powder.
Literature Cited
Engebrecht, J., Brent, R., and Kaderbhai, M.A.
1991. Minipreps of plasmid DNA. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G.
Seidman, J.A. Smith, and K. Struhl, eds.).
pp. 1.6.1-1.6.10. John Wiley & Sons, Hoboken,
N.J.
Fakhfakh, H., Vilaine, F., Makni, M., and Robaglia,
C. 1996. Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y. J.
Gen. Virol. 77:519-523.
Finer, J.J., Vain, P., Jones, M.W., and McMullen,
M.D. 1992. Development of the particle inflow
gun for DNA delivery to plant cells. Plant Cell
Rep. 11:323-328.
Gal-On, A., Meiri, E., Huet, H., Wu, J.H., Raccah,
B., and Gaba, V. 1995. Particle bombardment
Plant RNA
Viruses
16B.3.13
Current Protocols in Microbiology
Shiboleth, Y.M., Arazi, T., Wang, Y., and GalOn, A. 2001. A new approach for weed
control in a cucurbit field employing an attenuated potyvirus-vector for herbicide resistance.
J. Biotechnol. 92:37-46.
Takeuchi, Y., Dotson, M., and Keen, N.T. 1992.
Plant transformation: A simple particle bombardment device based on flowing helium. Plant
Mol. Biol. 18:835-839.
Yang, G., Mawassi, M., Ashoulin, L., Gafny, R.,
Gaba, V., Gal-On, A., and Bar-Joseph, M. 1997.
A cDNA clone from a defective RNA of citrus
tristeza virus is infective in the presence of the
helper virus. J. Gen. Virol. 78:1765-1769.
Wang, Y., Gaba, V., Wolf, D., Xia, X.D., Zelcer, A.,
and Gal On, A. 2000. Identification of a novel
plant virus promoter using a potyvirus infectious
clone. Virus Genes 20:11-17.
Key References
Gal-On, et al., 1995. See above.
Particle bombardment as an inoculation method using a vacuum device.
Gal-On, et al., 1997. See above.
Particle bombardment as an inoculation method using handheld devices.
Inoculation of
Plants Using
Bombardment
16B.3.14
Current Protocols in Microbiology
UNIT 16C.1
ABSTRACT
A one-step reverse transcriptionpolymerase chain reaction (RT-PCR) is used to detect
two cherry flexiviruses, Cherry green ring mottle virus (CGRMV) and Cherry necrotic
rusty mottle virus (CNRMV), in Prunus species. This unit presents procedures for collection of plant samples, preparation of total nucleic acids, viral RNA-rich or total RNA
extracts from plant tissues, and subsequent amplification of the viral targets by one-step
RT-PCR using a pair of consensus primers. The PCR amplicons are visualized by electrophoresis in a 1% agarose gel containing ethidium bromide in TAE buffer and viewed
under ultraviolet light. This procedure is rapid, sensitive, reliable, and cost-effective and
is generally useful on a wide variety of plant/virus systems. The use of a semi-automatic
homogenizer for sample preparation and one-tube RT-PCR for virus detection makes
this approach ideal for screening large numbers of samples. Curr. Protoc. Microbiol.
C 2007 by John Wiley & Sons, Inc.
6:16C.1.1-16C.1.9.
Keywords: RT-PCR r plant viruses r Cherry green ring mottle virus (CGRMV) r
Cherry necrotic rusty mottle virus (CNRMV)
INTRODUCTION
Advances in genomic characterization of plant viruses have made reverse transcription
polymerase chain reaction (RT-PCR) a common technique for the detection of plant
RNA viruses, due to its high sensitivity and specificity. Viral nucleotide sequence is
a prerequisite for the RT-PCR-based detection method. If the nucleotide sequence is
not available for a particular virus, the viral genome will first have to be cloned and
sequenced. Nucleotide sequences of Cherry green ring mottle virus (CGRMV, AF017780,
AJ291761) and Cherry necrotic rusty mottle virus (CNRMV, AF237816), two closely
related flexiviruses infecting Prunus species, are available, and are used as the basis of
this protocol which illustrates a general procedure that may be useful for other RNA
viruses. The sequence data are used to design primers that anneal to highly conserved
regions in the genomes to enable RT-PCR-based detection of a wide range of different
isolates of these viruses (Zhang et al., 1998; Rott and Jelkmann, 2001b; Gentit et al.,
2002). The availability of commercial one-step RT-PCR kits makes the RT-PCR more
efficient and convenient. Suitable RT-PCR templates can be prepared (see Template
preparation) from different plant tissues year round by using the basic protocols, or the
alternate protocols can be used to prepare template from young leaves in the spring.
The amplified PCR products are electrophoresed through 1% agarose gels containing
ethidium bromide (0.1 g/ml) in TAE buffer and visualized under UV light. When a
large number of samples must be tested, a 96-well PCR plate and a 96-well E-Gel system
(Invitrogen) may be used to replace PCR strips and standard agarose gels.
NOTE: Information about commercial reagents and equipment is provided solely to assist
the identification of suitable sources of such items. No specific endorsement or approval
is intended and equivalents from other manufacturers may be equally suited.
Plant RNA
Viruses
Current Protocols in Microbiology 16C.1.1-16C.1.9, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16c01s6
C 2007 John Wiley & Sons, Inc.
Copyright
16C.1.1
Supplement 6
STRATEGIC PLANNING
The time of year and the growth stage of the host are critically important for virus testing.
In general, virus titer typically falls in response to high ambient temperature. Therefore,
detection of viruses including CGRMV and CNRMV in Prunus species is best done in
early spring when plant tissues are succulent and temperatures are mild. The low concentration of phenolic compounds and polysaccharides in young tissues and high concentration of the viruses at this time make the isolation of the viral nucleic acids easier (Demeke
and Adams, 1992; Rott and Jelkmann, 2001a). Samples, usually young leaves, are collected from five different shoots of a plant and pooled because the distribution of these
viruses in plants, especially field trees, is not uniform (Parker et al., 1976; Li and Mock,
2005). Flowers, bark, and root tips can also be used. The extraction can also be done in
early fall from leaves, and in winter from the buds and bark of dormant budwood, if either
the Basic Protocol or the QIAGEN RNeasy plant mini kit is used (Li and Mock, 2005).
BASIC
PROTOCOL 1
Materials
Plant sample
CTAB buffer (extraction buffer; see recipe)
24:1 (v/v) chloroform/isoamyl alcohol
Isopropanol, room temperature
70% (v/v) ethanol
20 mM TrisCl, pH 8.0
RT-PCR-Based
Detection of Plant
Viruses
16C.1.2
Supplement 6
5. Cool the tube at 20 C for 15 min until the tubes are partially frozen.
Cooling time may vary. Make sure the buffer in the tubes is half frozen. This step is critical
to reduce RNA degradation during homogenization.
6. Process the cooled tube with a FastPrep bead-mill homogenizer two times, for 30 sec
at speed setting 4.5.
If a FastPrep bead-mill homogenizer or similar bead mill is not available, a mortar and
pestle cooled to 20 C can be used to grind the sample. However, this method is not as
efficient as using the FastPrep homogenizer.
7. Incubate the tube in a water bath, a dry-bath incubator, or a hybridization oven for
15 to 60 min at 65 C.
8. Microcentrifuge the tube 5 min at maximum speed, room temperature.
9. Transfer 650 l supernatant to a labeled 1.5-ml microcentrifuge tube.
The supernatant may contain pieces of plant debris.
10. Add an equal vol (650 l) of 24:1 chloroform/isoamyl alcohol to the tube and vortex
vigorously for 30 sec.
11. Microcentrifuge 10 min at maximum speed, room temperature.
12. Carefully remove 500 l of the upper (aqueous) phase to a labeled 1.5-ml microcentrifuge tube.
13. Add 70% vol (350 l) of isopropanol to the tube.
14. Incubate the tube 10 min on ice.
15. To pellet the nucleic acid, microcentrifuge 10 min at maximum speed, room
temperature.
16. Note the position of the pellet and carefully remove the supernatant by pipet. Be
careful not to disturb the pellet.
17. Add 0.5 ml of 70% ethanol, and invert several times.
18. Microcentrifuge 5 min at maximum speed, room temperature.
19. Note the position of the pellet and carefully remove the ethanol using a pipet.
Centrifuge again to collect remaining ethanol.
20. Carefully remove the remaining ethanol by pipet and air dry the pellet on the bench
for 10 to 15 min.
Plant RNA
Viruses
16C.1.3
Current Protocols in Microbiology
Supplement 6
21. Add 100 l of 20 mM TrisCl, pH 8.0, and put the tube on ice for 10 to 15 min.
This will soften the pellet.
22. Pipet up and down to dissolve the pellet. Use immediately or store at 20 C for
future use (up to 3 months).
ALTERNATE
PROTOCOL 1
Materials
Prepared tissue (Basic Protocol 1, steps 1 to 6)
Extraction buffer (see recipe)
Sterile distilled water
3% (v/v) Triton X-100 (Sigma-Aldrich)
ELISA microplate (Dynatech Laboratories)
Additional reagents and solutions for preparing plant tissue (Basic Protocol 1)
1. Prepare tissue as described in Basic Protocol 1, steps 1 to 6.
2. Microcentrifuge tube 5 min at 12,000 to 15,000 g (depending on the microcentrifuge used), 4 C.
3. Load 175 l of the supernatant containing virions into wells of an ELISA microplate.
To avoid contamination, it may be necessary for the novice to include empty wells between
samples.
16C.1.4
Supplement 6
9. Use the viral RNA extract immediately for RT-PCR assays or store at 20 C or
80 C until use.
The viral RNA extract cannot be stored at 20 C for more than two months.
Current Protocols in Microbiology
ALTERNATE
PROTOCOL 2
The RNeasy Plant Mini Kit (QIAGEN) has been found suitable to extract total RNA from
young leaf tissues. The manufacturers instructions should be followed for this purpose,
and pure template is obtained by this method. However, three factors limit its use when
large numbers of samples must be tested. First, the high concentration of polysaccharides
in mature leaves clogs the flow of extract through the columns, making sample recovery
difficult. Second, plant tissues are ground in liquid nitrogen with a mortar and pestle,
which does not easily scale to large numbers of samples. Finally, it is more expensive
than other methods.
RNA samples should be stored at 20 C following extraction until RT-PCR is carried
out.
BASIC
PROTOCOL 2
Primers determine the specificity of the RT-PCR, and primers used in virus detection are
usually based on conserved regions in their genomic sequences to be able to detect a wide
range of different strains or isolates. In our example using CGRMV and CNRMV, the high
nucleotide sequence identity among two viruses and their isolates (>67%) results in many
pairs of potential primers. Primer pairs may be selected using the Oligonucleotide Properties Calculator available at (http://www.basic.northwestern.edu/biotools/oligocalc.html).
Primers should have an annealing temperature (Tm ) of 60 C and lack sequences with
self complementarity. Primer pairs that meet these criteria may be compared using
RT-PCR to detect the viruses of interest (Li and Mock, 2005). The consensus primers
used in the protocol are based on two highly conserved regions adjacent to the coat protein
(CP) genes and have been used successfully to detect all CGRMV and CNRMV isolates
tested. Another advantage of this primer pair is that the amplicon can be used to obtain
the complete nucleotide sequence of the CP gene, which is very useful in determining
the taxonomic position of the target virus or isolate.
Materials
QIAGEN OneStep RT-PCR Kit (QIAGEN)
Sense primer: 5 - CCTCATTCACATAGCTTAGGTTT-3
Antisense primer: 5 -ACTTTAGCTTCGCCCCGTG-3
Total nucleic acid extract (Basic Protocol 1 or Alternate Protocols 1 or 2)
40 U/l RNase OUT (Invitrogen)
1% agarose gel
0.1 g/ml ethidium bromide (APPENDIX 2A)
PCR tube, PCR strip, or PCR plate
Thermal cycler
Gel imaging system
Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000)
Plant RNA
Viruses
16C.1.5
Current Protocols in Microbiology
Supplement 6
RT-PCR template
Total nucleic acids or
total RNA
8.88 l
4.88 l
5.0 l
5.0 l
5.0 l
5.0 l
1.0 l
1.0 l
10 M sense primer
1.5 l
1.5 l
10 M antisense primer
1.5 l
1.5 l
0.12 l
0.12 l
1.0 l
1.0 l
Reagents
RNase-free watera
5 reaction buffer
Q solution
10 mM dNTPs
Enzyme
mixc
1. Prepare RT-PCR master mix according to the recipes given in Table 16C.1.1.
2. Add 1 l of total nucleic acid extract or 5 l of viral RNArich extract to a PCR
tube, PCR strip, or well of a PCR plate.
3. To the tube, strip, or well, add 24 l PCR master mix if using total RNA, or 20 l if
using viral RNArich extract, and mix thoroughly by pipetting.
Avoid generation of air bubbles during mixing. A brief centrifugation of the PCR strip or
PCR plate at low speed can remove air bubbles.
4. Close the tube or PCR strip tightly, or seal the plate well.
This step is important in preventing evaporation of the PCR solution during PCR cycling.
5a. For most commercially available thermal cyclers: Run the PCR reaction with the
following thermal cycling conditions:
1 cycle:
45 min
5 min
50 C
95 C
13 cycles:
1 min
1 min
94 C
(72-1) C
90 sec
30 sec
30 sec
60 sec
10 min
72 C
94 C
60 C
72 C
72 C
22 cycles
1 cycle
RT-PCR-Based
Detection of Plant
Viruses
(reverse transcription)
(activation of HotStar Taq
DNA polymerase)
(denaturation)
(touch-down annealing
temperature)
(extension)
(denaturation)
(annealing)
(extension)
(final extension).
These conditions are appropriate for most commercially available thermal cyclers. A
touch-down thermal cycling program that includes a series of sequentially lower annealing temperatures as used here ensures optimal amplification of target sequences.
The calculated optimal annealing temperature, based on the nucleotide content of the
oligonucleotides, can be used to replace those in the touch-down cycles if the thermal
cycler used does not have this function. Amplification of the viral target has been
16C.1.6
Supplement 6
obtained in the short thermal cycling conditions listed below using a Peltier thermal
cycler (PTC-200, MJ Research). However, optimization of the conditions is recommended
for different thermal cycles.
5b. For Peltier thermal cycler: Run the PCR reaction with the following thermal cycling
conditions:
1 cycle:
45 min
5 min
50 C
95 C
13 cycles:
30 sec
30 sec
94 C
(72-1) C
60 sec
20 sec
20 sec
30 sec
5 min
72 C
94 C
60 C
72 C
72 C
22 cycles
1 cycle
(reverse transcription)
(activation of HotStar
Taq DNA polymerase)
(denaturation)
(touch-down
annealing
temperature)
(extension)
(denaturation)
(annealing)
(extension)
(final extension).
7. View the gel under UV light to determine if the expected amplicon (959 bp) is
present.
CTAB buffer
Dissolve 2% cetyltrimethylammonium bromide (CTAB) in distilled water with
stirring. Use gentle heat (60 C), if necessary. Add 2% polyvinylpyrollidone 40,000
(PVP 40,000), 1.4 M NaCl, 20 mM EDTA (from 0.5 M solution, pH 8.0; APPENDIX 2A)
and 100 mM TrisCl (from 1 M solution, pH 8.0; APPENDIX 2A) to the solution.
Stir until dissolved. Add water to make final volume. Store 2 to 3 months at
room temperature (CTAB will precipitate at 4 C). Add 2-mecaptoethanol to 0.2%
immediately before use.
Extraction buffer
Dissolve 20% polyvinylpyrollidone 40,000 (PVP 40,000), 10% sucrose, and 0.15%
chicken egg albumin in distilled water with stirring. Then add 100 mM potassium
phosphate (from 1 M solution, pH 7.6; APPENDIX 2A), to the solution. Stir to mix.
Add water to make final volume. Store 2 to 3 months at 4 C. Add ascorbic acid to
30 mM immediately before use.
COMMENTARY
Background Information
Cherry green ring mottle virus (CGRMV)
and Cherry necrotic rusty mottle virus (CN-
RMV), unassigned members of the family Flexiviridae (Fauquet et al., 2005) are
flexuous filamentous plant viruses with a
Plant RNA
Viruses
16C.1.7
Current Protocols in Microbiology
Supplement 6
RT-PCR-Based
Detection of Plant
Viruses
Isolation of template that is free of compounds inhibitory to PCR is critical. To prevent oxidation during tissue grinding, precool
the tube containing the sample and buffer in a
freezer and work fast. Nucleic acids or virus
particles are protected once they are released
into buffer. A translucent white pellet should
be obtained from Basic Protocol 1 if the conditions are right; the quality of the extract is
poor if a brown pellet is obtained. The disruption of some plant cells may release reactive
phenolic compounds which oxidize rapidly
to form oxidized phenolics that undergo irreversible electrophilic substitution reactions
with proteins and nucleic acids. Basic Protocol 1 and the accompanying alternative protocols emphasize simplicity, low cost, and broad
options. However, the extracts obtained by
these methods contain host proteins including
nucleases, which slowly degrade the nucleic
acids even when frozen; therefore, the template preparations cannot be stored for more
than 2 months. Further purification of the
extracts is necessary if long-term storage is
required.
There are several one-step RT-PCR kits
commercially available. The protocol presented here has been optimized for the
QIAGEN OneStep RT-PCR kit. The RT-PCR
reaction volume is reduced to minimize the
cost per reaction. However, the reduction of
the recommended reaction volumes may affect
the efficiency of RT-PCR. Therefore, reaction
conditions should be optimized if other kits
are used. The dNTP mixtures provided in the
RT-PCR kit should be distributed in aliquots
and stored frozen. Frequent freeze-thaw cycles can degrade dNTP mixtures and reduce
the efficiency of RT-PCR.
Anticipated Results
Time Considerations
Basic Protocol 1 can be completed in 3 hr
if twelve samples are processed. Forty-eight
samples can be processed easily in a single day.
The alternative protocol for template preparation is simpler and faster in term of handson time, usually 2 hr per twelve samples, but
overnight incubation is required. RT-PCR can
be completed in 3.5 hr.
16C.1.8
Supplement 6
Literature Cited
Clark, M.F. 1981. Immunosorbent assays in plant
pathology. Ann. Rev. Phytopathol. 19:83-106.
Clark, M.F. and Adams, A.N. 1977. Characteristics of the microplate method of enzyme-linked
immunosorbent assay for the detection of plant
viruses. J. Gen. Virol. 34:475-483.
Demeke, T. and Adams, R.P. 1992. The effect of
plant polysaccharides and buffer additives on
PCR. Biotechniques 12:332-334.
Doyle, J.J. and Dickson, E.E. 1987. Preservation of
plant samples for DNA restriction endonuclease
analysis. Taxon 36:715-722.
Fauquet, C.M., Mayo, M.A., Maniloff, J.,
Desselberger, U., and Ball, L.A. 2005. Virus
Taxonomy-Eighth Report of the International
Committee on Taxonomy of Viruses. Elsevier
Academic Press.
Foissac, L., Gentit, P., Svanetia-Dumas, L., Dulucq,
M.J., and Candresse, T. 2002. Polyvalent detection of fruit tree tricho, capillo, and foveaviruses
by nested RT-PCR using degenerated and inosine containing primers (PDO RT-PCR). Acta
Hortic. 550:37-43.
Gentit, P., Foissae, X., Svanetia-Dumas, L.,
Peypelut, M., Macquaire, G., and Candresse,
T. 2002. Molecular characterization of foveaviruses associated with the cherry necrotic mottle
leaf disease and complete sequencing of a European isolate of Cherry green ring mottle virus.
Arch. Virol. 147:1033-1042.
Jones, A.S. and Walker, R.T. 1963. Isolation and
analysis of the deoxyribonucleic acid of Mycoplasma mycoides var. capri. Nature 198:588589.
Jones, A.T. and McGavin, W.J. 2002. Improved
PCR detection of Blackcurrant reversion virus
in Ribes and further evidence that it is the causal
agent of reversion disease. Plant Dis. 86:13331338.
Li, R. and Mock, R. 2005. An improved reverse transcription-polymerase chain reaction
Plant RNA
Viruses
16C.1.9
Current Protocols in Microbiology
Supplement 6
UNIT 16D.1
Protoplast inoculations, unlike infection of whole plants, can provide a quantitative assay
for measuring viral RNA accumulation. Protoplasts are generated by enzymatically
treating plant tissue to produce cells lacking cell walls. Infectious viral RNA can be
inoculated into a calculated number of protoplasts by permeabilizing the cell membrane
and then culturing the cells in a medium that supports their growth. There are three
main sources of tissue for the preparation of protoplasts: (1) fresh tissue such as excised
leaves or cotyledons, (2) tissue that is maintained in long-term suspension culture, and
(3) undifferentiated callus that is cultured on solid media. The use of fresh tissue requires
time-consuming peeling of the epidermal layer, and protoplasts produced in different
experiments can vary in their ability to be infected by viruses. Suspension cultures are a
more common choice, but initiating and maintaining the cultures can be problematic. The
authors have found that callus cultures prepared from sterilized seeds are advantageous
because they require little effort to initiate and maintain, and upon enzymatic treatment
they produce uniform protoplasts that are nearly always competent for virus infection.
This unit describes how to generate protoplasts prepared from callus and inoculate them
with infectious viral RNA. Basic Protocol 1 outlines generation of callus from seeds
of Arabidopsis thaliana ecotype Col-0. Basic Protocol 2 describes the production of
protoplasts from the callus using cellulase and pectinase and the inoculation of protoplasts
using polyethylene glycol. Basic Protocol 3 addresses the extraction of total RNA from
infected protoplasts.
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aeseptic technique should be used accordingly.
BASIC
PROTOCOL 1
Materials
Arabidopsis thaliana (ecotype Col-0) seeds
70% ethanol
Bleach containing 4% to 6% sodium hypochlorite
10% (w/v) sodium dodecyl sulfate (SDS; APPENDIX 2A)
H2 O, sterile
Callus maintenance medium (CM) 1% agar plates (see recipe)
1.5-ml microcentrifuge tubes, sterile
Forceps, sterile
20 C incubator with light control (e.g., Percival Scientific I-36LL;
http://www.percival-scientific.com)
Plant RNA
Viruses
Contributed by John C. McCormack and Anne E. Simon
Current Protocols in Microbiology (2005) 16D.1.1-16D.1.9
C 2005 by John Wiley & Sons, Inc.
Copyright
16D.1.1
1. Aliquot 300 Arabidopsis thaliana seeds into a 1.5-ml microcentrifuge tube and
wash twice with 1 ml of 70% ethanol by vortexing vigorously. Pulse centrifuge and
remove the alcohol by pipetting.
2. Add 1 ml bleach containing 4% to 6% sodium hypochlorite followed by 50 l of 10%
SDS. Vortex vigorously. Incubate 5 to 10 min at room temperature. Pulse centrifuge
and remove supernatant.
3. Wash the seeds five times by suspending them in 1 ml sterile water, vortexing, and
pulse centrifuging. After the final centrifugation, resuspend the seeds in sterile water.
For steps 3 and 4, all work should be conducted in a sterile environment. Surface-sterilized
seeds should be placed immediately onto CM plates.
4a. To create new callus: Spread 100 surface-sterilized seeds onto each callus maintenance CM agar plate. Seal lids of individual plates with Parafilm
Callus Cultures
of Arabidopsis
Figure 16D.1.1 Appearance of callus cultures in sterile 100 15mm petri dishes at several
stages of growth. (A) Plant tissue appears green and has not undergone dedifferentiation at 2
weeks into the rst passage. (B) At 1 week into the second passage, callus appears green with
tinges of yellow and becomes undifferentiated. (C) Callus appears mostly yellow and friable at 1 12
weeks into the third passage. This plate contains 1 g callus and is the earliest time point for
digestion of callus into protoplasts. (D) Callus clumps become larger as tissue grows within each
passage. This plate is 3 weeks into the third passage and is ready to be split to begin the next
passage. Appearance of callus in the fourth through sixth stages should not change. For the color
version of this gure go to http://www.currentprotocols.com.
16D.1.2
Current Protocols in Microbiology
4b. To passage existing callus: Use sterilized forceps to mechanically break up the callus
and transfer onto new CM plates. Seal individual plates with Parafilm.
Callus should be passaged every 3 to 4 weeks for up to six passages. During passages one
through three, the color of the callus should change from green to yellow. For passages
three through six, tissue should be light yellow and friable (Fig. 16D.1.1). Browning
or softening of callus indicates dead or dying tissue and should be discarded. When
passaging, break up the intact callus into smaller clumps of 0.2 to 0.4 cm in diameter; do
not simply move intact callus from old to new CM plates. One plate of developing callus
can be passaged onto three to four new plates.
BASIC
PROTOCOL 2
In this protocol, protoplasts are generated by treating callus with pectinase and cellulase
to separate cells from one another and to degrade the cell wall. Protoplasts are then
washed with 0.6 M mannitol to remove the enzymes and cells are enumerated using
a hemacytometer. Protoplasts (5 106 ) are then inoculated with in vitro transcripts of
infectious viral RNA using polyethylene glycol (PEG) and calcium chloride, which make
cell membranes permeable to RNA. Finally, inoculated protoplasts are washed with 0.6
M mannitol containing 1 mM calcium chloride to remove the PEG and then suspended
in protoplast culture medium (PCM), a medium that supports cell growth. Protoplasts
can be incubated for a finite time, generally <50 hr before regeneration of the cell wall
begins.
Materials
0.6 M mannitol, room temperature and 4 C
Arabidopsis callus (in the third to sixth passage; Basic Protocol 1)
Protoplast inoculation medium (PIM; see recipe)
Cellulase, Trichoderma viride (10 KU/g dry weight; Calbiochem)
Pectinase, Rhizopus sp. (3 KU/g dry weight; Calbiochem)
RNA of interest: transcribe in vitro (see UNIT 16D.4)
1.0 M CaCl2
50% polyethylene glycol (PEG; see recipe)
0.6 M mannitol containing 1 mM CaCl2 , 4 C
Protoplast culture medium (PCM; see recipe)
14.6-cm Pasteur pipet: melt into L-shape
Rotating shaker
50-ml polypropylene centrifuge tubes, sterile
125-ml glass bottles: sterilize by autoclaving
Refrigerated centrifuge with bucket rotor appropriate for 50-ml tubes
Funnel: sterilze by autoclaving
53-m nylon mesh (Small Parts): sterilize by autoclaving
Digest the callus
1. Add 15 ml of 0.6 M mannitol (room temperature) to each plate of callus in a sterile
environment. Break the callus into smaller pieces using a Pasteur pipet that has been
melted into an L-shape.
Callus is friable; very little manipulation is necessary to break it up without breaking the
agar matrix.
2. Pour callus and mannitol from plates into a sterile 50-ml polypropylene centrifuge
tube. Shake on a rotating shaker 20 min at 100 rpm, room temperature.
Current Protocols in Microbiology
Plant RNA
Viruses
16D.1.3
3. For each plate of callus being processed, dispense 50 ml PIM into a sterile 125-ml
glass bottle along with 0.5 g cellulase and 0.1 g pectinase. Incubate mixture on a
rotating shaker at 100 rpm, room temperature, until completely dissolved.
Cellulase and pectinase dissolve more quickly if PIM is added to the bottle first, followed
by the powdered enzymes.
4. Using a refrigerated centrifuge with a bucket rotor, centrifuge the callus from step 1
for 4 min at 900 g (brake off), room temperature, and pour off supernatant.
5. For each plate of callus being processed, pour 25 ml PIM containing dissolved
enzymes into the polypropylene centrifuge tube containing the callus and transfer to
a sterile 125-ml glass bottle.
6. Add another 25 ml PIM containing dissolved enzymes to the polypropylene tube to
collect any remaining callus and transfer to the glass bottle. Wrap bottle completely
in aluminum foil.
7. Incubate glass bottle containing callus and PIM with dissolved enzymes 3.5 hr on a
rotating shaker at 100 rpm, room temperature.
Solution should become turbid after 3.5 hr as the callus is digested and cells are released.
8. Filter the turbid solution into a 50-ml polypropylene tube using a sterile funnel and
53-m nylon mesh.
10. Add 20 ml cold (4 C) 0.6 M mannitol and gently invert the centrifuge tube several
times to wash the protoplasts. Centrifuge as in step 9. Repeat for a total of three
washes.
11. After the final wash, centrifuge cells, resuspend protoplasts in cold 0.6 M mannitol
up to a volume of 20 ml, and keep on ice.
12. Enumerate protoplasts using a hemacytometer (APPENDIX 4A).
Intact protoplasts should appear round with a large vacuole under a light microscope.
Each plate of callus (1 g) after digestion should yield at least 3 107 protoplasts.
13. Aliquot 5 106 cells into 50-ml polypropylene centrifuge tubes, one tube for each
inoculation. Collect protoplasts by centrifuging 5 min at 228 g (brake off), 4 C.
Pour off supernatant leaving 100 l. Keep protoplasts on ice until inoculation (up
to 1 hr).
For turnip crinkle virus (TCV), the authors routinely add 15 pmol of in vitrotranscribed
RNA to each inoculation mix; the amount of inoculated RNA may differ for other viral
16D.1.4
Current Protocols in Microbiology
systems. Subviral RNAs can be co-inoculated with the helper virus; for the TCV system,
an equimolar amount of viral and subviral RNA is added to the inoculation mix. All
inoculation mixes should be prepared at the same time and within an hour of inoculating
protoplasts; failing to do so may result in uneven or poor inoculations.
15. Add the inoculation mix to the tube containing the protoplasts and pipet gently up
and down twice. Add 2.2 ml of 50% PEG, mix well by gently shaking the tube
15 sec, and incubate at room temperature for 20 sec.
16. Add cold 0.6 M mannitol containing 1 mM CaCl2 to a volume of 30 ml and incubate
15 min on ice.
17. Centrifuge protoplasts 5 min at 228 g (brake off), 4 C. Decant the supernatant
leaving 10 ml residual supernatant.
18. Add 10 ml cold 0.6 M mannitol containing 1 mM CaCl2 . Centrifuge protoplasts 5 min
at 228 g (brake off), 4 C. Decant the supernatant. Repeat wash and centrifugation
two more times. After the final centrifugation completely decant the supernatant.
19. Resuspend each tube of protoplasts in 3 ml PCM and pour into a 60 15mm petri
dish.
20. Cover petri dishes in aluminum foil and incubate at room temperature.
For the turnip crinkle virus system, protoplasts are generally incubated between 24 and
40 hr.
BASIC
PROTOCOL 3
Materials
Inoculated protoplasts (Basic Protocol 2)
1:1 phenol/chloroform (Tris-buffered phenol; APPENDIX 2A)
RNA extraction buffer (see recipe)
3 M sodium acetate, pH 5.2 (APPENDIX 2A)
100% and 70% ethanol
H2 O, sterile
Microcentrifuge, refrigerated
1. Check the condition of the inoculated protoplasts by examining plates under a light
microscope. Tilt plates to evenly distribute cells prior to removal.
Tilting the plates prior to pipetting allows for an equal number of cells to be dispensed
when the protoplast-containing medium is divided into two tubes (step 3).
Intact protoplasts should appear round with a large vacuole under a light microscope.
2. At 24 to 40 hpi, split each plate of infected cells into two microcentrifuge tubes.
Centrifuge 3 min at 1500 g (brake off), room temperature.
3. Remove one-half of the supernatant using a pipet. Centrifuge 3 min at 1500 g
(brake off), room temperature. Remove the remainder of the supernatant, leaving
100 l of medium. Place one tube at 80 C as a backup for each transformation.
Proceed with total RNA extraction using the second tube.
Do not pipet off all the supernatant after the second centrifugation; leave 100 l in
each tube to avoid excessive loss of cells.
Current Protocols in Microbiology
Plant RNA
Viruses
16D.1.5
4. Add 200 l of 1:1 phenol/chloroform and 200 l RNA extraction buffer to each
tube and vortex vigorously for 10 sec. Microcentrifuge 2 min at 10,400 g, 4 C.
Transfer the upper layer to a new tube.
To phenol, add 8-hydroxyquinoline to a final concentration of 0.1% for partial inhibition
of RNases. Avoid removing any of the lower layer to minimize RNA degradation.
2,4-D, 2 mg/ml
Fully dissolve 0.02 g of 2,4-dichlorophenoxyacetic acid (2,4-D) in 5 ml of 100%
ethanol. Adjust volume to 10 ml with sterile water. Store up to 6 months in the dark
at 20 C.
Do not autoclave solution.
Callus Cultures
of Arabidopsis
16D.1.6
Current Protocols in Microbiology
Kinetin, 2 mg/ml
0.02 g kinetin
667 l 1 N NaOH
Fully dissolve kinetin in 1 N NaOH
Adjust volume to 10 ml with sterile H2 O
Store up to 6 months in the dark at 20 C
Do not autoclave solution.
Plant RNA
Viruses
16D.1.7
Current Protocols in Microbiology
COMMENTARY
Background Information
Callus Cultures
of Arabidopsis
16D.1.8
Critical Parameters
and Troubleshooting
Callus culture contamination
Fungal or bacterial contamination of callus
may appear one to two weeks after passaging. Contamination may arise from improper
preparation of CM plates, inadequate surfacesterilization of Arabidopsis thaliana seeds, or
poor aseptic technique during passaging.
Dead or dying callus cultures
Healthy callus is mostly light yellow and
friable, whereas dying tissue appears brown
and should not be used for passaging. Excess condensation on callus culture plates may
contribute to tissue death. To reduce condensation, CM plates should be dried at room temperature for one day before use. Plates containing excess condensation can still be used
Current Protocols in Microbiology
Anticipated Results
Each plate of callus culture should yield at
least 3 107 protoplasts upon treatment with
cellulase and pectinase. For the turnip crinkle
virus (TCV) system, the efficiency of protoplast infection has been found to be 30%
using the polyethylene glycol method. Extraction of total RNA from 5 106 inoculated
Time Considerations
Preparation of CM plates and surfacesterilization of seeds can be accomplished
within 2 days. Once the seeds are plated, callus should be ready for enzymatic digestion in
the middle of the third passage, which takes
8 weeks. Digestion of callus into protoplasts
and inoculations of protoplasts with viral RNA
can be accomplished in 9 hr. At the desired
time point, extraction of total RNA from protoplasts can be performed in 2 to 3 hr.
Literature Cited
Brown, T., Mackey, K., and Du, T. 2004. Analysis
of RNA by northern and slot blot hybridization.
In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. 4.9.1-4.9.14. John Wiley & Sons, Hoboken,
N.J.
Cocking, E.C. 1960. Method for the isolation of
plant protoplasts and vacuoles. Nature 187:927929.
Gallagher, S.R. 2004. Quantitation of DNA and
RNA with absorption and fluorescence spectroscopy. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K.
Struhl, eds.) pp. A.3D.1-A.3D.12. John Wiley
& Sons, Hoboken, N.J.
Kreps, J.A. and Simon, A.E. 1997. Environmental and genetic effects on circadian clockregulated gene expression in arabidopsis. Plant
Cell 9:297-304.
Nagy, P.D., Pogany, J., and Simon, A.E. 1999. RNA
elements required for RNA recombination function as replication enhancers in vitro and in vivo
in a plus strand RNA virus. EMBO J. 18:56535665.
Takebe, I., Otsuki, Y., and Aoki, S. 1968. Isolation
of tobacco mesophyll cells in intact and active
state. Plant Cell Physiol. 9:115-124.
Wang, J. and Simon, A.E. 1997. Analysis of the two
subgenomic RNA promoters for turnip crinkle
virus in vivo and in vitro. Virology 232:174-186.
Zhang, F. and Simon, A.E. 2003. A novel procedure
for the localization of viral RNAs in protoplasts
and whole plants. Plant J. 35:665-673.
Plant RNA
Viruses
16D.1.9
Current Protocols in Microbiology
UNIT 16D.2
Studies leading to an increased understanding of various aspects of the plant-virus infection cycle in whole plants are hindered by two major limitations. First, it is often
difficult to achieve infection of substantial numbers of primary cells during mechanical
inoculation of a leaf; consequently, the inability to attain synchronous infection makes
it difficult to characterize viral-specific events, most importantly during early stages of
infection. Second, the infection cycle in initially infected cells is randomized because
of cell-to-cell movement of the replicating virus. These limitations were, however, circumvented when Takabe and Otsuki (1969) discovered that single plant cells, namely
protoplasts, can be isolated by enzymatic treatment of intact leaves and synchronously
infected with either purified virus or viral RNA. The protoplast system has been regarded
as a hallmark in plant virology since it broke barriers that had prevented plant virus
replication studies at the level attainable with bacterial and animal viruses. Merits of
the protoplast system include (1) establishment of synchronous infection, (2) delivery
of a known concentration of the inoculum to a defined number of cells, (3) flexible
control of experimental conditions, (4) uniform sampling, as well as facile isolation
and characterization of virus-related products, and (5) the ability to study replication of
viruses that cannot be transmitted mechanically. The only disadvantage of this system
is that cell-to-cell movement functions cannot be analyzed. Nevertheless, over the past
decade experiments performed with plant protoplasts have significantly advanced our
understanding of virus replication. A majority of earlier studies involved infection of
protoplasts with purified virus preparations. However, the advent of recombinant DNA
technology and our ability to manipulate RNA viral genomes at the DNA level provided
new directions. The following sections describe isolation and infection of protoplasts obtained from leaves of monocotyledonous and dicotyledonous hosts. Note that the basic
protocol for isolating protoplasts from either monocotyledonous or dicotyledonous hosts
is similar except the enzyme solutions used are different.
NOTE: A complimentary procedure for preparing protoplasts using callus cultures is
presented in UNIT 16D.1.
BASIC
PROTOCOL 1
This section describes a basic procedure, adapted from Loesch-Fries and Hall (1980), for
the preparation and inoculation of mesophyll protoplasts from barley plants (Hordium
vulgare; Rao et al., 1994). This procedure can be used for isolating mesophyll protoplasts
from other dicotyledonous hosts such as rice. Various steps required for successful isolation of viable protoplasts are presented in the first half of the protocol, while the second
half describes inoculation of protoplasts with viral RNA using polyethylene glycol.
Materials
5- to 6-day-old barley leaves
Enzyme solution for barley (see recipe)
0.55 M sucrose (see recipe)
0.55 M mannitol, pH 5.9 (see recipe)
5 mg/ml fluorescein diacetate: dissolve 5 mg flourescein diacetate (Sigma) in 1 ml
acetone; store in an opaque container for up to 6 months at 4 C
Contributed by A.L.N. Rao
Current Protocols in Microbiology (2007) 16D.2.1-16D.2.8
C 2007 by John Wiley & Sons, Inc.
Copyright
Plant RNA
Viruses
16D.2.1
Supplement 4
28 C incubator
100-ml beakers
300- to 350-m gauze
15- and 50-ml polypropylene tubes
Beckman Model J6B centrifuge (preferred) or clinical tabletop centrifuge
Large-bore pipet
Microscope equipped with UV light source (also see UNIT 2A.1)
Additional reagents and solutions for counting cells in a hemacytometer
(APPENDIX 4A)
Prepare leaves for protoplast extraction
1. Harvest 0.5 g of 5- to 6-day-old barley leaves.
Avoid harvesting dry or yellow leaves.
2. With a sharp razor, slice leaves lengthwise and then crosswise into 1-mm2 sections.
3. Transfer sliced material into 25 ml enzyme solution for barley and incubate 3 to
3.5 hr at 28 C, without shaking.
Completely digested leaf material will have a transparent appearance.
4. Decant the solution containing the enzyme-digested leaf material into a 100-ml
sterile beaker.
At this stage, a gentle swirling will facilitate release of protoplasts from the digested leaf
material.
5. Filter the solution through gauze into another 100-ml sterile beaker and transfer the
filtrate into a sterile 50-ml polypropylene tube.
6. Add 12.5 ml of the enzyme solution containing the digested material into each 50-ml
polypropylene tube containing enzyme-digested leaf material.
7. Underlay 10 ml of 0.55 M sucrose beneath the digested material in each tube.
12. Remove most of the supernatant and gently resuspend protoplasts in the remaining
liquid.
13. Add 10 ml of 0.55 M mannitol, pH 5.9, and repeat wash as described above.
16D.2.2
Supplement 4
18. Determine the number of viable protoplasts per cubic centimeter (10 cells/mm2 =
105 cells/cm3 ) and estimate the number of viable protoplasts per milliliter.
19. Dispense desired number of protoplasts (1 105 ) to be inoculated into a 15-ml
polypropylene tube and add 1 ml of 0.55 M mannitol, pH 5.9.
From this stage, absence of enzyme solution enhances cell wall formation that will
significantly affect uptake of the inoculum. Therefore, speed and accuracy are essential
to achieve high infection rate.
BASIC
PROTOCOL 2
A wide range of plant viruses infect dicotyledonous hosts such as tobacco species (e.g.,
Nicotiana benthamiana). Even some viruses that are restricted to monocotyledonous
hosts, such as Brome mosaic virus, can also infect Nicotiana benthamiana. There is little
host restriction at the protoplasts level. Nevertheless, a comparison of the replication
profiles of a given virus accumulating in transfected protoplasts from natural and nonhost
systems will be useful in assessing the basic events that regulate the infection pattern.
NOTE: The procedure used for isolating protoplasts from intact tobacco leaves is the
same as that described above for barley protoplasts with some modifications.
Materials
3-week-old N. benthamiana leaves
0.55 M mannitol, pH 5.9 (see recipe)
Carborundum powder (Fisher)
Enzyme solution for tobacco (see recipe)
100 15-mm petri dish
Cotton swabs
28 C incubator
Additional reagents and equipment for preparing the leaves for protoplasts
extraction and removing and counting the protoplasts (Basic Protocol 1, steps 4
to 20)
1. Harvest 1 g of 3-week-old N. benthamiana leaves.
Select only well expanded leaves, avoiding dry or yellow ones.
16D.2.3
Current Protocols in Microbiology
Supplement 4
6. Transfer leaf to a petri dish containing 0.55 M mannitol, pH 5.9, and gently wash off
carborundum powder.
7. With a sharp razor, slice the leaf on either side of the midrib portion into two halves.
Then slice crosswise into 1-mm2 sections.
8. Float the sliced materials in enzyme solution for tobacco such that the bottom
(abrasive side) of the sliced leaf is touching the enzyme solution. Incubate 3 to 3.5 hr
at 28 C.
After this step, follow Basic Protocol 1, steps 4 to 20.
BASIC
PROTOCOL 3
Materials
Protoplasts in 15-ml polypropylene tubes (Basic Protocol 1 or 2)
Viral RNA or transcripts (UNIT 16K.2)
40% polyethylene glycol (PEG; see recipe)
0.55 M mannitol, pH 5.9 (see recipe)
Culture medium for mesophyll protoplasts (see recipe)
24-well plate (Corning)
Fluorescent lamp
1. Centrifuge tubes containing desired number of protoplasts (from Basic Protocol 1)
for 3 min at 1000 g, room temperature.
2. Remove most of the supernatant and add desired concentration of either viral RNA
or transcripts, keeping the volume small (5 to 10 l; 1 to 5 g of RNA).
3. Gently mix the protoplast pellet to resuspend in the inoculum, then add 150 l of
40% PEG and gently shake the contents for 10 sec.
4. Add 2 drops of 0.55 M mannitol, pH 5.9, to the side of the tube and mix gently.
5. Continue to add mannitol dropwise over the next 5 to 10 min until the volume has
reached 1.5 ml.
Preparation and
Inoculation of
Mesophylll
Protoplasts from
Monocotyledenous
and
Dicotyledenous
Hosts
16D.2.4
Supplement 4
10. Place inoculated protoplasts in a culture plate (24-well plate from Corning) and keep
under a fluorescent lamp for 20 to 24 hr or desired length of time.
An alternative procedure over PEG mediated transfection is electroporation (UNIT 16D.4).
Since application of an electric pulse is likely to damage the majority of protoplasts, it is
advisable to use significantly higher number of cells (a minimum of 106 per sample).
Ca solution, 100
Weigh 14.70 g CaCl2 and dissolve in 100 ml of 0.55 M mannitol, pH 5.9 (see
recipe). The pH of this solution should be around 6.5. Sterilize the solution by
autoclaving. Store up to 1 to 3 months at room temperature.
Plant RNA
Viruses
16D.2.5
Current Protocols in Microbiology
Supplement 4
KK solution, 1000
Dissolve 2.72 g KH2 PO4 and 10.11 g KNO3 in 100 ml sterile water and adjust the
pH to 6.5 with 1 N KOH. Sterilize the solution by autoclaving. Store up to 1 to 3
months at room temperature.
Sucrose, 0.55 M
Weigh 18.82 g sucrose and dissolve in 80 ml sterile distilled water. Adjust volume
to 100 ml and sterilize by filtration. Store up to 1 to 3 months at 4 C.
COMMENTARY
Background Information
Preparation and
Inoculation of
Mesophylll
Protoplasts from
Monocotyledenous
and
Dicotyledenous
Hosts
Critical Parameters
The kind of leaf material used will have
a profound influence on the quality of protoplasts and subsequent infection to a given inoculum source. Therefore, the following criteria will help in isolating protoplasts that yield
consistent results: (1) grow plants under optimum light and humidity conditions; (2) avoid
using leaves that are dry, yellow, or damaged;
(3) while slicing leaf material, always use a
sharp blade and do not exert pressure minimizing release of leaf extract; (4) during initial
stages of protoplast preparations, be gentle and
16D.2.6
Supplement 4
Problem
Possible Cause
Solution
No digestion of leaf
slices
No dark interface
containing protoplasts
Centrifugation
Incubation temperature
is too high
Rough handling
procedures
Quality of protoplasts
Quality of RNA
PEG
Culture medium
Poor infectivity
Troubleshooting
Commonly encountered problems and their
solutions are found in Table 16D.2.1.
Anticipated Results
From the specified isolation procedure,
generally 1 g leaf tissue should yield 1 to
2 106 protoplasts. Infection of high quality and viable protoplasts generally results in
a high level of infectivity. This would offer
advantage of characterizing viral progeny at
various time post-inoculation with ease.
Time Considerations
Incubation of protoplasts in the enzyme
solution requires 2.5 to 3 hr. Alternatively,
overnight enzyme digestion at room temperature can be performed by reducing the concentration of the enzyme by 5- to 10-folds. This
however should be normalized depending on
the experimental conditions. The time required
for isolation and inoculation with RNA samples require a nonstop operation of 2 hr. So,
the entire process takes about 5 hr.
Plant RNA
Viruses
16D.2.7
Current Protocols in Microbiology
Supplement 4
Literature Cited
Hall, R.D. 1991. The initiation and maintenance
of plant cell suspension cultures. In Plant Tissue Culture Manual (K. Lindsey, ed.) A3:121. Kluwer Academic Publishers, Dordrecht,
Netherlands.
Hibi, T., Rezelman, G., and Van Kamen, A. 1975.
Infection of cowpea mesophyll protoplasts
with cowpea mosaic virus. Virology 64:308318.
Howell, S.H. and Hull, R. 1978. Replication of
cauliflower mosaic virus and transcription of
its genome in turnip leaf protoplasts. Virology
86:468-481.
Loesch-Fires, L.S. and Hall, T.C., 1980. Synthesis,
accumulation and encapsidation of individual
brome mosaic virus RNA components in barley protoplasts. J. Gen. Virol. 47:323-332.
Maule, A.J., Boulton, M.I., and Wood, K.R. 1980.
Poyethylene glycol mediated infection of cucumber protoplasts by cucumber mosaic virus
and RNA. J. Gen. Virol. 47:199-203.
Motoyoshi, F. and Oshima, N. 1979. Standardization in inoculation procedure and effect of a resistance gene on infection of tomato protoplasts
with tobacco mosaic virus RNA. J. Gen. Virol.
44:801-806.
Rao, A.L.N., Duggal, R., Lahser, F., and Hall,
T.C. 1994. Analysis of RNA replication in plant
viruses. In Methods in Molecular Genetics:
Molecular Virology Techniques (K.W. Adolph,
ed.) Vol. 4, pp. 216-236. Academic Press,
Orlando, Fla.
Takabe, I. and Otsuki, Y. 1969. Infection of tobacco
mesophyll protoplasts by tobacco mosaic virus.
Proc. Natl. Acad. Sci. U.S.A. 64:843-848.
Preparation and
Inoculation of
Mesophylll
Protoplasts from
Monocotyledenous
and
Dicotyledenous
Hosts
16D.2.8
Supplement 4
UNIT 16D.3
Plant protoplasts provide a rapid means of introducing nucleic acids into cells. Thus,
the use of protoplasts is a valuable tool to investigate gene expression control or virus
replication in vivo, in a very short time frame (<48 hr).
In the plant, each cell is surrounded by a wall consisting of a thick matrix of polysaccharides, proteins, and polyphenolic compounds. These walls are impervious to macromolecules. To allow rapid uptake of nucleic acids, the walls must be removed using cellulases. This yields delicate, spherical cells (protoplasts) held intact only by the plasma
membrane. Protoplasts are free floating, and do not attach to surfaces like most cultured
animal cells. They are highly vulnerable to changes in osmotic pressure, so the medium
in which they are obtained must be iso-osmotic with the cell interior. Protoplasts live
only a few days. Upon longer incubation they either die or regenerate cell walls. In fact,
whole plants can be regenerated from some protoplast types. Therefore, protoplasts must
be prepared fresh for each experiment.
Protoplasts can be derived from whole plants, stationary undifferentiated tissue (callus
cultures; refer to UNIT 16D.1), or from undifferentiated tissue grown in solution (suspension
culture), in which it is suspended by constant shaking. The authors find that large quantities of uniform protoplasts can be obtained more rapidly and reliably from suspension
cultures than from plants. This unit describes a simple, rapid, and reliable method to generate large amounts of protoplasts from an oat (Avena sativa) suspension culture. Basic
Protocol 1 is divided into sections that describe: (1) isolation of protoplasts by digestion
with cellulases, (2) preparation of protoplasts for electroporation, (3) electroporation of
protoplasts with RNA to assay gene expression or RNA replication, and (4) culture of
cells post-electroporation and collection for reporter gene assays or RNA extraction. A
Support Protocol is provided for subculturing oat suspension cultures.
Oat protoplasts have been used commonly for viruses that infect monocots. However, it is
noteworthy that the host range of viruses is greatly expanded at the protoplast level. Basic
Protocol 2 describes methods for maintaining and storing the oat suspension culture cells
on solid medium. More details on electroporation are described in UNIT 16D.4. Methods
for extraction of RNA from protoplasts are described in UNIT 16E.2.
BASIC
PROTOCOL 1
The oat cell suspension culture system described herein allows one to follow the activity of
both stably and transiently transformed cells. Plant protoplasts are prepared by overnight
digestion of plant cell walls, followed by washing of the cells. An electrical pulse is
applied to cells in the presence of DNA or RNA, and the cells are transferred to growth
medium. This unit describes the steps for preparation of cells through their harvest.
Basic Protocol 1 outlines digestion and handling of protoplasts for viral replication
studies. Minor differences in the handling of the cells for reporter gene expression assays
are described at specific steps in the protocol and discussed in the Commentary. Methods
for generating and maintaining suspension cultures are described in the Support Protocol.
Plant RNA
Viruses
Contributed by Aurelie M. Rakotondrafara, Jacquelyn R. Jackson, Elizabeth Pettit Kneller, and
W. Allen Miller
16D.3.1
Supplement 5
Materials
7-day-old oat suspension culture (Avena sativa cv. Stout; see Support Protocol)
Oat protoplast enzyme solution (see recipe)
Artificial sea water (ASW)/0.6 M mannitol (1:1 ratio; see recipe)
Oat protoplast electroporation buffer (see recipe)
1 M spermidine: filter sterilize using a 0.2-m filter and divide into 1-ml aliquots
(store up to 1 year at 20 C)
Murashige and Skoog (MS) medium with 0.4 M mannitol (see recipe)
RNA sample
50-ml conical centrifuge tubes
145 20 or 100 15mm sterile plastic petri dishes
Gyrotory shaker (e.g., New Brunswick Scientific Model G2)
Motorized pipet filler/dispenser (e.g., Eppendorf EasyPet)
10-ml serological pipets
Centrifuge (e.g., Sorvall RC-5C Plus) with SH-3000 or comparable
swinging-bucket rotor with inserts for 15-ml conical tubes
4-mm electroporation cuvettes
1-ml wide-bore polypropylene pipet tips
6-well cell culture plates
Electroporator with square wave pulse (e.g., BTX T820 ElectroSquare Porator or
Bio-Rad GenePulser Xcell with CE module)
Additional reagents and equipment for counting cells with a hemacytometer
(APPENDIX 4A)
NOTE: All work should be carried out under sterile conditions. Unless otherwise noted,
all solutions should be autoclaved and then stored at 4 C. Equipment which will come in
direct contact with the protoplasts should be sterilized before use. Keep solutions sterile
at all times.
2. Remove supernatant and gently resuspend cells in 20 ml freshly prepared oat protoplast enzyme solution.
Always use a freshly prepared enzyme solution.
Protoplasts are very fragile to mechanical manipulation. Resuspend cells by gently inverting the tube or by pipetting up and down slowly.
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
50 ml of the enzyme solution (also see step 3) is sufficient for the digestion of up to 10 ml
packed cells. If the amount of packed cells exceeds 10 ml, adjust volume of cells to 10 ml
or increase the total amount of enzyme solution to maintain a 5:1 ratio of enzyme solution
to packed cells.
16D.3.2
Supplement 5
3. Transfer cell/enzyme mix into a large petri dish (145 20mm) and add the remaining 30 ml enzyme solution.
If using a small petri dish (100 15mm), transfer the cell/enzyme mix into three sterile
petri dishes and divide the remaining enzyme solution into each petri dish in equal
aliquots. Final volume in each dish should not exceed 20 ml.
4. Seal the petri dish with Parafilm and cover it with aluminum foil to protect it from
light.
At this point, the cell/enzyme suspension has a granulated appearance.
11. Aspirate off supernatant and resuspend cells again in 10 ml of ASW/0.6 M mannitol.
12. Repeat wash by centrifuging as in step 8.
If cell debris is still observed under the light microscope, one additional wash is recommended. Protoplasts are very sensitive to osmotic pressure. Osmotic differences due to
improperly prepared buffer can contribute to the constant presence of broken cells. Such
a protoplast preparation should not be used for electroporation.
Plant RNA
Viruses
16D.3.3
Current Protocols in Microbiology
Supplement 5
16. Inspect a drop of the protoplast suspension under a light microscope. Proceed to
step 17 only if protoplast suspension is largely free of cell debris.
19. When ready to electroporate, add RNA to the cells and mix by gently inverting the
cuvette.
To minimize degradation of the RNA sample, electroporate sample as soon as RNA is
added.
The amount of RNA to be added depends on the type of assay. For replication studies, it
is recommended that as much as 10 g RNA per sample be used. Higher amounts can be
used for virion purification. For transient expression, 1 g or less of RNA is sufficient.
Efficient co-inoculation of more than one RNA simultaneously can be performed simply
by mixing more than one RNA sample prior to electroporation. Alternatively, a two-step
electroporation approach can be used if RNAs are to be introduced into the same cells at
different times (see Commentary).
20. After adding RNA to the cells, place cuvette immediately into the holder of the
electroporator device and electroporate cells at a defined voltage and capacitance
setting, depending on the electroporator available.
If using the BTX Electro Square Porator T820, the optimal setting for electroporating oat
protoplasts is one pulse for 6 msec at 300 V.
If using the Bio-Rad GenePulser Xcell, the optimal setting is 300 V/500 F.
Both electroporators perform with similar efficiencies at the above settings.
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
21. After electroporation, place cuvette back on ice until the remaining samples have
been electroporated (step 18).
22. Under a sterile hood, transfer each sample into one well on the 6-well cell culture
plate containing 5 ml MS medium with 0.4 M mannitol.
16D.3.4
Supplement 5
For maximum recovery of cells, remove 1 ml MS medium from the well prior to transferring
the electroporated sample into the well. Use this 1-ml aliquot of medium to rinse the
cuvette.
White threads of debris resulting from cell lysis after electroporation are commonly
observed in the sample.
For transient expression assays, it is not critical to work under sterile conditions. The
sample can be poured into the MS medium immediately after electroporation.
23. Seal the plates with Parafilm and cover with aluminum foil. Incubate the plates 24
to 48 hr at room temperature.
The quality of the protoplast preparation determines the viability of protoplasts for at
least 48 hr after electroporation.
For transient expression, cells can be harvested 30 min post-transfection. Optimal expression is observed after 4 hr, although individual experiments may require some optimization.
Harvest cells
24. Inspect electroporated samples in culture plates under a light microscope.
A mixture of intact protoplasts and cell debris clumps is commonly observed.
25. Carefully collect cells from the culture plate and gently transfer each sample into a
separate 15-ml centrifuge tube.
26. Pellet cells by centrifuging as in step 8.
For virion purification, centrifuge cells 5 min at 700 g, 4 C.
SUPPORT
PROTOCOL
Plant RNA
Viruses
16D.3.5
Current Protocols in Microbiology
Supplement 5
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
16D.3.6
Supplement 5
Plant RNA
Viruses
16D.3.7
Current Protocols in Microbiology
Supplement 5
COMMENTARY
Background Information
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
16D.3.8
Supplement 5
require any special equipment. Electroporation, as described in this unit, is generally the
most efficient, rapid, and reproducible technique to introduce nucleic acids into plant
and mammalian cells (See UNIT 16D.4). It involves passing an electric pulse through a sample of cells which momentarily opens pores
within the plasma membrane to allow macromolecules to enter (or exit) the cell (Fromm
et al., 1985, 1987). Although it is a harsh procedure, a sufficient number of cells recover to
be used for downstream applications. Viability
and successful inoculation of the protoplasts
by electroporation depend on the nucleic acid
concentration, the ionic strength of the buffer,
the voltage, and the duration of the electrical
pulse applied (see Critical Parameters; Fromm
et al., 1985, 1987). The same conditions can
be used for electroporation of RNA or DNA.
If appropriate care is taken, it is possible
to electroporate the same batch of protoplasts
more than once, for example to introduce different macromolecules at 24 hr intervals (Shen
and Miller, 2004). To do this, critical changes
in the procedure include the use of higher concentrations of protoplasts per sample and a decrease of voltage during electroporation from
300 to 280 V (or lower) to minimize cell death.
Twenty-four hours after the first electroporation, cells are harvested, washed, and resuspended in electroporation buffer. After addition of the second RNA (or DNA) sample, the
cells are electroporated at 280 V. Satisfactory
cell recovery was observed under such
conditions.
Critical Parameters
The use of cell suspension cultures instead of plants as starting material for making protoplasts, offers the advantage of having a source material that is already aseptic.
For the protocols described above, it is important that these sterile conditions be maintained throughout the course of the experiment. This is especially true for replication
assays, which require long incubation periods
in sucrose-containing culture medium that is
an ideal breeding ground for fungal and bacterial contaminants. All media should be prepared, autoclaved, and opened only under a
laminar-flow hood.
Before preparing protoplasts from oat cell
suspension cultures, it is vitally important that
the protoplasts be generated from a cell suspension culture that has been maintained properly. The age of the cell suspension culture is
critical when using it as a source for isolating
Plant RNA
Viruses
16D.3.9
Current Protocols in Microbiology
Supplement 5
Problem
Possible cause(s)
Solution(s)
Contamination of suspension
culture/buffers
Poor-quality medium/buffer
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
16D.3.10
Supplement 5
Troubleshooting
Table 16D.3.1 provides some commonly
encountered problems as well as their solutions and possible causes.
Anticipated Results
Seven-day-old suspension cultures should
appear as fine clumps. Inspection of the cells
under a light microscope following digestion
should show that the majority of cells are
spherical, freely floating individual cells that
are uniform in size. The washing procedure
should remove most of the debris. Following
electroporation, viability of the protoplasts is
high (80%), with a small amount of debris resulting from cell lysis observed in the medium.
At 24 to 48 hr post-electroporation, there will
be a mixture of intact cells and clumps of debris visible under the light microscope.
From one 50-ml culture of oat suspension
culture, it is possible to electroporate up to
10 samples for viral replication studies, and
up to 25 samples for transient expression assays. The washing and electroporation steps
are carried out routinely with one to two
50-ml cultures at a time.
Time Considerations
The process of digestion of plant cells requires 30 min to 1 hr for set up, followed by
overnight digestion. Washing and electroporation takes <2 hr, depending on the number of
samples handled. For translation assays, 4 hr
incubation of electroporated cells is optimal,
although reporter gene activity is above background within 15 to 30 min following electroporation. To study viral replication, a 24 to
48 hr incubation is usually allowed before harvesting of cells, but this may vary depending
on the replication of the virus.
Literature Cited
Barnett, A., Hammond, J., and Lister, R.M. 1981.
Limited infection of Cereal Leaf Protoplasts by
Barley yellow dwarf virus J. Gen. Virol. 57:397401.
Birsin, M., Onde, S., and Ozgen, M., 2001. Callus
induction and plant regeneration from mature
embryos of oat (Avena sativa L.). Turk. L. Biol.
25:427-434.
Chen, Z., Klockare, R., and Sundqvist, C. 1994.
Origin of somatic embryogenesis is proliferating root primordia in seed derived oat callus.
Hereditas 120:211-216.
Cocking, E.C. 1960. A method for the isolation of
plant protoplasts and vacuoles. Nature 187:962963.
Plant RNA
Viruses
16D.3.11
Current Protocols in Microbiology
Supplement 5
Contributed by Aurelie M.
Rakotondrafara, Jacquelyn R. Jackson,
Elizabeth Pettit Kneller, and W. Allen
Miller
Iowa State University
Ames, Iowa
Preparation and
Electroporation of
Oat Protoplasts
from Cell
Suspension
Culture
16D.3.12
Supplement 5
UNIT 16D.4
This unit presents protocols for inoculating protoplasts prepared from tobacco (Nicotiana
tabacum) and the related species Nicotiana benthamiana with viroid RNA by electroporation in order to study viroid replication. The unit covers maintenance of cultured
cells, preparation of protoplasts, electroporation parameters, and culturing of electroporated protoplasts. It also highlights critical aspects of these steps and discusses common
problems and their solutions.
BASIC
PROTOCOL
Materials
Tobacco BY-2 (N. tabacum L. var Bright Yellow 2) cultured cells; available from
many sources including authors laboratory (ding.35@osu.edu)
N. benthamiana cultured cells (Qi and Ding, 2002; Sunter and Bisaro, 2003);
available from authors laboratory (ding.35@osu.edu)
Murashige & Skoog (MS) medium (see recipe)
Digestion and washing buffer (see recipe) containing 1.5% (w/v) cellulase
(Onozuka RS; Yakult) and 0.2% (w/v) macerase (Calbiochem-Novabiochem)
Digestion and washing buffer (see recipe), prechilled to 4 C
Electroporation buffer (see recipe), prechilled to 4 C
RNA prepared from plasmid containing PSTVd cDNA by in vitro transcription
(see Support Protocol 1)
Protoplast culturing solution (see recipe)
Protoplast culturing solid medium (see recipe) in 6-well culture plates
RNeasy Plant Mini Kit (Qiagen; also see Support Protocol 2)
250-ml flasks
50-ml sterile polypropylene conical tubes (e.g., Fisher)
Swinging-bucket centrifuge (e.g., Marathon 21000R, Fisher)
Platform shaker (New Brunswick Scientific)
100- and 40-m nylon mesh (Spectrum)
400-ml beakers
Electroporator Plus and 4-mm-gap electroporation cuvettes (all available from
BTX; also see Internet Resources)
Disposable sterile 1.5-ml microcentrifuge tubes (Fisher)
UV spectrophotometer (e.g., Thermo Spectronic)
Additional reagents and equipment for RNA quantitation (Gallagher, 2004) and
northern blot analysis (Brown et al., 2004)
Contributed by Xuehua Zhong, Asuka Itaya, and Biao Ding
Current Protocols in Microbiology (2005) 16D.4.1-16D.4.11
C 2005 by John Wiley & Sons, Inc.
Copyright
Plant RNA
Viruses
16D.4.1
NOTE: Autoclave all glassware and plasticware prior to beginning this protocol, unless
they are purchased sterile.
NOTE: Use large-orifice pipet tips to handle protoplasts. Prepare large-orifice 200- and
1000-l pipet tips by cutting tips and autoclave them. Large-orifice tips are commercially
available as well. The wider opening is necessary to avoid damaging the protoplasts.
NOTE: When testing replication of PSTVd mutants or a different type of viroid, include wild-type PSTVd as a positive control. In all experiments, use mock- (i.e., water-)
inoculated cells as a negative control.
Prepare protoplasts
1. Culture tobacco BY-2 and N. benthamiana suspension cells in 50 ml MS medium in a
250-ml flask by shaking at 250 rpm under constant ambient light at 24 C. Subculture
weekly by making a 1:25 (for tobacco) and a 1:10 dilution (for N. benthamiana).
The dilution ratio can vary based on cell growth rate and density. One useful criterion
for optimal growth rate is that, after dilution for subculturing, the cells reach the original density within 7 days. The ratios suggested above have worked best in the authors
experiments.
3. Resuspend the cells in 40 ml digestion and washing buffer with 1.5% cellulase and
0.2% macerase. Incubate the cells for 2 to 4 hr at room temperature in the dark with
slow shaking at 70 rpm.
It is good practice to microscopically examine a fraction of cells (10 l) every 30 min
after 2 hr digestion to monitor progress of digestion. Prolonged digestion will not improve
yield of protoplasts and may cause damage to the cells.
The authors have found that BY-2 cells need more time (3 to 4 hr) for digestion than N.
benthamiana cells (2 to 3 hr). Some procedures have used macerozyme as an alternative to macerase. It is reported that macerase has nearly three-fold higher activity than
macerozyme (Sinha et al., 2003). The authors also found macerase to be much more potent
than macerozyme.
4. Examine the cells under a light microscope to estimate the percentage of cells with
walls digested (i.e., protoplasts).
These cells are round-shaped with a smooth surface (see Commentary). A good yield
should be 70% to 90%.
5. Filter the protoplasts through 100-m nylon mesh over a 400-ml beaker. Refilter the
protoplasts through a 40-m nylon mesh over a 400-ml beaker.
The two-step filtration yields better quality protoplasts than a single-step filtration through
the 40-m nylon mesh. In practice, the authors place the nylon mesh over the beaker, wrap
the edge around the beaker, and fasten with a rubber band. Downward pressure is then
gently applied to the mesh to create a well to hold protoplasts. The cells will flow through
the mesh very slowly. Gentle tapping of the mesh will accelerate cell flow.
Transfecting
Protoplasts by
Electroporation
to Study Viroid
Replication
6. Transfer the protoplast suspension to a 50-ml conical tube and centrifuge 5 to 15 min
at 150 g, 4 C. Discard the supernatant and resuspend the protoplasts in 25 ml
prechilled (4 C) digestion and washing buffer.
Avoid exceeding a total volume of 30 ml (solution plus protoplasts) in the 50-ml tube;
otherwise, it will be difficult to centrifuge down the good protoplasts. The authors have
16D.4.2
Current Protocols in Microbiology
found that a 5-min centrifugation is sufficient to collect the majority of protoplasts for
infection studies, while avoiding potential damage to the protoplasts. Extending the centrifugation time to 15 min helped collect more protoplasts; however, the authors do not
recommend centrifugation beyond 15 min because longer times may not improve the yield
and may cause damage to some of the protoplasts.
7. Repeat step 6 for a second wash with prechilled (4 C) digestion and washing buffer.
Discard the supernatant and resuspend the protoplasts in prechilled (4 C) electroporation buffer to a density of 2 106 protoplasts/ml. Incubate the protoplasts 1 to 2
hr on ice before electroporation.
The authors have found that it is better to resuspend the protoplasts first in 5 to 10 ml
(i.e., 1/4 of the starting volume) of electroporation buffer, and then calculate the density
of the protoplasts by performing a 1/10 dilution on an aliquot using a hemacytometer.
Add more of electroporation buffer if the density is higher than 2 106 protoplasts/ml. Be
sure that 70% to 90% of the protoplasts are intact before proceeding to electroporation.
Perform electroporation
8. Set electroporator at 0.2 to 0.3 kV.
Make sure that all connections are in good order before electroporation. For different
electroporators, trial and error may be necessary to determine the optimal settings.
15. Isolate total RNA from the protoplasts by using RNeasy Plant Mini Kit according
to manufacturers instructions, or by the standard procedure described in Support
Protocol 2.
16. Quantify RNA by UV spectrometry at 260 nm (Gallagher, 2004) and reserve a 10-g
aliquot of RNA for northern blot analysis (Brown et al., 2004).
Plant RNA
Viruses
16D.4.3
Current Protocols in Microbiology
SUPPORT
PROTOCOL 1
Materials
Plasmid containing PSTVd cDNA with T7, T3, or SP6 promotor, suitable for in
vitro transcription (e.g., pRZ6-2; available from the authors; ding.35@osu.edu)
Restriction endonuclease (e.g., HindIII for pRZ6-2) and buffer (e.g., Invitrogen)
MEGAscript kit (Ambion)
MEGAclear kit (Ambion)
UV spectrophotometer (e.g., Thermo Spectronic)
Additional reagents and equipment for restriction digestion (Bloch and Grossmann,
1995), in vitro transcription (Tabor, 1987), and RNA quantitation (Gallagher,
2004)
1. Linearize the plasmid containing PSTVd cDNA with an appropriate restriction enzyme for 2 to 3 hr at 37 C. Terminate reaction and purify linearized plasmid according
to the manufacturers instructions for the MEGAscript kit.
2. Use linearized plasmid to synthesize PSTVd RNA by in vitro transcription using
the MEGAscript kit according to the manufacturers instructions or using a standard
procedure (e.g., Tabor, 1987).
The authors recommend using the MEGAscript kit if the laboratorys budget permits.
3. Purify RNA transcripts by using the MEGAclear kit according to the manufacturers
instructions.
4. Measure the concentration of RNA by UV spectrometry at 260 nm (Gallagher, 2004).
SUPPORT
PROTOCOL 2
Materials
Transfecting
Protoplasts by
Electroporation
to Study Viroid
Replication
16D.4.4
Current Protocols in Microbiology
Additional reagents and equipment for RNA quantitation (Gallagher, 2004) and
northern blot analysis (Brown et al., 2004)
1. Add 10 vol of TRIzol Reagent (Invitrogen) to the harvested protoplasts (e.g., 500 l
Trizol Reagent to 50 l harvested protoplasts) and mix vigorously by vortexing for
1 min.
2. Add 1 vol (i.e., 550 l) of 25:24:1 phenol:chloroform:isoamyl alcohol (25:24:1) to
the above tube and vortex thoroughly.
3. Microcentrifuge 5 min at 13,000 rpm. Carefully transfer the upper aqueous layer
to a new microcentrifuge tube and add 3 vol of 100% ethanol and 1/10 vol of 5 M
ammonium acetate to the tube. Incubate 20 min to overnight (depending on the
working schedule) at 20 C.
4. Microcentrifuge 30 min at 13,000 rpm at 4 C. Discard the supernatant and add 1 ml
of 75% ethanol. Microcentrifuge 10 min at 13,000 rpm, 4 C, discard the supernatant,
and dry the pellet at room temperature for 5 min.
5. Add 30 to 50 l DEPC-treated water and measure the RNA concentration by UV
spectrometry (Gallagher, 2004). Reserve a 10-g aliquot of RNA for northern blot
analysis (Brown et al., 2004).
Plant RNA
Viruses
16D.4.5
Current Protocols in Microbiology
Autoclave 20 min and remove from autoclave as soon as possible to avoid evaporation of H2 O and caramelization of sucrose
If not to be used immediately, store autoclaved medium up to 1 month at 4 C
Before use, add the following supplements (filter-sterilized through 0.22-m cellulose filter):
1 ml 1000 Gamborgs vitamin stock (B5 vitamins; Caisson Laboratories or Sigma;
store up to 2 years at 20 C)
100 l 10 mg/ml 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma; store up to 2 years
at 20 C)
Protoplast culturing solid medium
Supplement protoplast culturing solution (see recipe) with 1% (w/v) agar, autoclave
20 min, allow to cool to 50 C, then pour 5-mm-thick layers (4 ml) in wells of
6-well Costar tissue culture plates (Corning). Store plates up to 1 month at 4 C,
wrapped in Parafilm.
Protoplast culturing solution
4.3 g Murashige & Skoog (MS) basal salt mixture (Caisson Laboratories or Sigma)
30 g sucrose
100 mg myo-inositol (Sigma cat. no. I-5125)
10 mg KH2 PO4 [add 1 ml of 1000 (10 g/liter) stock solution]
82 g mannitol
H2 O to 1 liter
Adjust pH to 5.5 with 1 M KOH.
Autoclave 20 min and remove from autoclave as soon as possible to avoid evaporation of H2 O and caramelization of sucrose
If not to be used immediately, store autoclaved medium up to 1 month at 4 C
Before use, add the following supplements (filter-sterilized through 0.22-m cellulose filter):
1 ml 1000 Gamborgs vitamin stock (B5 vitamins; Caisson Laboratories or Sigma;
store up to 2 years at 20 C)
100 l 10 mg/ml 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma; store up to 2 years
at 20 C)
COMMENTARY
Background Information
Transfecting
Protoplasts by
Electroporation
to Study Viroid
Replication
in replication at the cellular level from defects in other functions (e.g., cell-to-cell and
long-distance transport) that are required for
systemic infection. Therefore, noninfection at
the whole-plant level cannot be definitively
interpreted as indication of nonreplication at
the cellular level. Furthermore, the long infection process, up to several weeks, often results
in new mutations or reversions of mutations
to wild-type, which complicates interpretation
of the role of specific nucleotide changes in
replication.
Analysis of viroid replication in single cells
is necessary to advance mechanistic understanding of replication. Protoplasts offer an
ideal system for such analysis. Muhlbach and
Sanger (1977) showed that protoplasts prepared from leaves of tomato (cultivar Hilda
72) supported efficient replication of Cucumber pale fruit viroid when inoculation was
16D.4.6
Current Protocols in Microbiology
Critical Parameters
Make sure the cultured cells are maintained
under good conditions. One useful measure
is the observation that after dilution for subculturing, the cells will grow to reach the
original density within 1 week. Cells with
lower growth rates will usually not grow
well after inoculation and thereby support
active viroid replication. Avoid using cells
>10 days old, because the protoplasts derived from them tend to lose competence in
supporting viroid replication. Whether this is
attributable to their recalcitrance to inoculation or to their reduced metabolic rate is
unclear.
Do not overdigest the cells. Digesting cells
over an extended period of time will not improve the yield of protoplasts and may cause
damage to the protoplasts. It is therefore important to use the shortest time possible to
achieve optimal digestion, as judged by the
percentage of protoplasts obtained (70% to
90%).
Use large-orifice pipet tips to transfer protoplasts at all steps. This is critical. The protoplasts, without cell walls, are very fragile. Use
of regular (i.e., small-orifice) tips will squeeze
and break the protoplasts. The large-orifice tips
will allow easy passage of the protoplasts without physical damage.
All pipet tips and tubes should be RNasefree. Because viroid RNA is used as the inoculum, presence of RNase in the tips and
tubes may cause degradation of the inoculum.
Furthermore, presence of RNase may also degrade the quality of RNAs extracted from the
protoplasts.
Do not use any antibiotics to maintain or
subculture cells. Because the cells are constantly maintained, use of antibiotics poses the
risk of selecting for antibiotic-resistant bacteria. If the culture cells are contaminated, simply discard them and obtain clean cell cultures.
A useful practice is to maintain several flasks
of culture cells, ideally in separate places, so
that the risk of contaminating all of them can
be minimized.
All utensils should be clean and free from
contamination by any detergents. Presence of
even trace amounts of detergents may inhibit
cell growth and damage protoplasts during or
after electroporation. One method of detecting
the presence of detergent residues on glassware
Plant RNA
Viruses
16D.4.7
Current Protocols in Microbiology
Problem
Possible cause(s)
Solution(s)
Improper temperature
Adjust temperature
Overdigestion
Troubleshooting
Some problems that may arise with the
protocols in this unit, along with their possible causes and solutions, are listed in Table
16D.4.1.
Anticipated Results
Transfecting
Protoplasts by
Electroporation
to Study Viroid
Replication
Time Considerations
With the availability of plasmids for in vitro
transcription to synthesize RNA inoculum
and hybridization probes, along with healthy,
growing cultured cells, the entire procedure
from preparation of inoculum, probes, and protoplasts, to finishing the northern blot analyses,
can be accomplished within 1 week.
Cultured cells need to be maintained constantly. Cultured cells 7 to 10 days old (starting
from subculturing) are best for preparing protoplasts. The total time for protoplast preparation, starting from cell wall digestion to having
protoplasts ready for electroporation, is 4 to
16D.4.8
Current Protocols in Microbiology
Figure 16D.4.1 Cultured cells and protoplasts of N. benthamiana. (A) Cultured cells. (B) Protoplasts prepared from cultured cells. Healthy protoplasts should account for 70% to 90% of the
total cells in a good preparation.
Figure 16D.4.2 Northern blot showing replication of PSTVd in tobacco BY2 protoplasts at increasing hours post-electroporation. (+)-cPSTVd and (+)-lPSTVd indicate monomeric circular and
monomeric linear PSTVd, respectively, produced during rolling-circle replication. 5.8S rRNA serves
as a loading control. Lane M shows in vitro RNA transcripts containing tetrameric and monomeric
PSTVd sequences and some vector sequences as size markers. See Qi and Ding (2002) for further
information. Reprinted from Qi and Ding (2002), with permission from Elsevier Publishers.
Plant RNA
Viruses
16D.4.9
Current Protocols in Microbiology
8 hr. In vitro transcription time varies depending on the methods. If the Ambion
MEGAscript kit is used for in vitro transcription, it takes 3 to 4 hr, starting with assembly of the reaction, to complete purification
of RNA transcripts using the MEGAclear kit
and measure the concentration of RNA via
UV spectrometry (see Support Protocol 1).
The time for RNA extraction from the transfected protoplasts for northern blots also varies
with the chosen method. Using the RNeasy
Plant Mini Kit or a non-kit method, it takes
2 hr.
Literature Cited
Bloch, K.D. and Grossmann, B. 1995. Digestion of
DNA with restriction endonucleases. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.1.13.1.21. John Wiley & Sons, Hoboken, N.J.
Branch, A.D. and Robertson, H.D. 1984. A replication cycle for viroids and other small infectious
RNAs. Science 223:450-454.
Brown, T. Mackey, K., and Du, T. 2004. Analysis
of RNA by northern and slot blot hybridization.
In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. 4.9.1-4.9.19. John Wiley & Sons, Hoboken,
N.J.
Faustmann, O., Kern, R., Sanger, H.L., and
Muhlbach, H.-P. 1986. Potato spindle tuber viroid (PSTV) RNA oligomers of (+) and () polarity are synthesized in potato protoplasts after
liposome-mediated infection with PSTV. Virus
Res. 4:213-227.
Feldstein, P.A., Hu, Y., and Owens, R.A. 1998. Precisely full length, circularizable, complementary
RNA: An infectious form of potato spindle tuber
viroid. Proc. Natl. Acad. Sci. U.S.A. 95:65606565.
Flores, R., Randles, J.W., Bar-Josef, M., and Diener,
T.O. 2000. Subviral agents: Viroids. In Virus
Taxonomy, Seventh Report of the International
Committee on Taxonomy of Viruses (M.H.V.
van Regenmortel, C.M. Fauquet, D.H.L. Bishop,
E.B. Carstens, M.K. Estes, S.M. Lemon, J.
Manilof, M.A. Mayo, D.J. McGeoch, C.R.
Pringle, and R.B. Wickner, eds.) pp. 1009-1024.
Academic Press, San Diego.
Gallagher, S. 2004. Quantitation of DNA and RNA
with absorption and fluorescence spectroscopy.
In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, and K. Struhl, eds.)
pp. A.3D.1-A.3D.12. John Wiley & Sons, Hoboken, N.J.
Transfecting
Protoplasts by
Electroporation
to Study Viroid
Replication
16D.4.10
Current Protocols in Microbiology
Key References
Qi and Ding, 2002. See above.
Establishes electroporation protocol to inoculate
tobacco and N. benthamiana protoplasts for studying PSTVd replication.
Internet Resources
Tabler, M. and Sanger, H.L. 1984. Cloned singleand double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated
subgenomic DNA fragments are infectious.
EMBO J. 3:3055-3062.
http://www.btxonline.com/products/
Tabler, M. and Tsagris, M. 2004. Viroids: Petite RNA pathogens with distinguished talents.
Trends Plant Sci. 9:339-348.
Plant RNA
Viruses
16D.4.11
Current Protocols in Microbiology
UNIT 16E.1
ABSTRACT
The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies
that focus on viral RNAs often involve virus infections of plant protoplasts (see
UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections,
which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes
two basic protocols for extracting viral RNAs from plant protoplasts. An approach for
preparing double-stranded viral RNA from total RNA pools is also provided. The viral
RNA prepared by using these techniques can be used for further analyses such as primer
extension, reverse transcriptionPCR, and northern blotting. Curr. Protoc. Microbiol.
C 2007 by John Wiley & Sons, Inc.
6:16E.1.1-16E.1.6.
Keywords: RNA r protoplast r isolation
INTRODUCTION
Viral RNAs can be extracted from virus-infected plant protoplasts (see UNITS 16D.1-16D-4)
using a number of different methods. In general, these protocols involve isolating and
lysing the protoplasts, removing the protein components from the lysate, precipitating
the total RNA, and resuspending the purified RNAs in an appropriate buffer. Several
variations of these basic protocols are currently used to isolate RNA from plus-strand
RNA virus-infected protoplasts. Total nucleic acids, including viral RNAs, can be extracted from protoplasts by using an EDTA-detergent lysis buffer and phenol-chloroform
extraction method (see Basic Protocol 1). By including an additional step to this protocol
(i.e., lithium chloride precipitation), double-stranded (ds) nucleic acids (including ds
forms of viral RNAs) can be separated from single-stranded (ss) nucleic acids (including
ss forms of viral RNAs). Viral RNAs can also be extracted from protoplasts by using
a guanidinium-based solution (see Basic Protocol 2). Additional methods include commercially available kits that also use guanidinium-containing solutions. These include
TRIzol Reagent (Invitrogen) and RNeasy (Qiagen). The former is similar to Basic Protocol 2, described below, while the latter utilizes a proprietary RNA binding membrane
that eliminates the phenol extraction step. The reagents and protocols for these kits are
provided by the respective suppliers and will not be discussed here.
It should be noted that the methods mentioned are largely interchangeable and most
will be effective with the types of plant protoplasts used commonly in virus research
(e.g., tobacco BY-2, Arabidopsis thaliana, Nicotiana benthamiana, barley, cucumber,
etc.). The method selected for a particular application will therefore depend largely upon
personal preference and other factors such as time and cost.
NOTE: Use sterile and RNase-free vessels and deionized, DEPC-treated H2 O (see
APPENDIX 2A) in all recipes and at all protocol steps.
Plant RNA
Viruses
Current Protocols in Microbiology 16E.1.1-16E.1.6, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16e01s6
C 2007 John Wiley & Sons, Inc.
Copyright
16E.1.1
Supplement 6
BASIC
PROTOCOL 1
Materials
Infected protoplasts (see UNITS 16.D.1-16.D.4)
Nucleic acid extraction buffer (see recipe)
25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (PCI; APPENDIX 2A)
1 M and 3M sodium acetate, pH 5.2
95% and 70% ethanol, ice-cold
Diethylpyrocarbonate (DEPC)-treated H2 O (APPENDIX 2A)
7 M lithium chloride (LiCl)
2 DNase buffer (see recipe)
RNase-free DNase I (10 U/l; bovine pancreas, Amersham Biosciences)
1.5-ml microcentrifuge tubes
Standard microcentrifuge
Tube vortexer
Additional reagents and equipment for performing agarose gel electrophoresis and
ethidium-bromide staining (Voytas, 2000) and northern blot analysis (Brown et al.,
2004)
NOTE: All reagents and vessels used should be sterile and RNase-free.
7. Remove the upper aqueous layer and transfer it to a microcentrifuge tube containing
100 l of 1 M sodium acetate and 1000 l of 95% ethanol, ice-cold.
16E.1.2
Supplement 6
16. To isolate the ds nucleic acids, carefully remove the supernatant by pipetting it
into a new microcentrifuge tube, add three volumes of 95% ethanol, and precipitate
overnight at 20 C .
17. Spin the sample in a microcentrifuge 15 min at 12,000 g, 4 C.
18. Remove the supernatant, and gently cover the nucleic acid pellet with 1 ml of ice-cold
75% ethanol and then centrifuge 15 min at 12,000 g, 4 C.
19. Remove the 70% ethanol supernatant completely and let the pellet air-dry for 10 min
and resuspend pellet in 50 l of DEPC-treated water.
20. To eliminate the DNA, add 50 l of 2 DNase buffer and 20 U DNase I (RNase-free).
21. Incubate for 30 min at 37 C, add 20 l of 3 M sodium acetate, 80 l DEPC-treated
water, and 200 l PCI.
22. Vortex on high setting for 30 sec and then centrifuge 2 min 12,000 g, ambient
temperature.
23. Remove the upper aqueous phase and transfer to a new tube, add 600 l of ice-cold
95% ethanol, vortex, and incubate 1 hr at 20 C.
24. Collect the precipitated nucleic acids by centrifuging 15 min at 12,000 g, 4 C.
Plant RNA
Viruses
16E.1.3
Current Protocols in Microbiology
Supplement 6
25. Remove the supernatant and gently cover the nucleic acid pellet with 1 ml of ice-cold
70% ethanol. Centrifuge 15 min at 12,000 g, 4 C.
26. Remove the 70% ethanol supernatant completely and let the pellet air-dry for 10 min.
27. Resuspend the dsRNA pellet in 10 to 20 l of DEPC-treated water.
BASIC
PROTOCOL 2
Materials
Infected protoplasts (see UNITS 16.D.1-16.D.4)
Guanidinium solution (see recipe)
25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (PCI; APPENDIX 2A)
2 M sodium acetate, pH 4 (APPENDIX 2A)
DEPC-treated water (APPENDIX 2A)
Isopropanol, ice-cold
70% ethanol, ice-cold
1.5-ml microcentrifuge tubes
Tube vortexer
Additional reagents and equipment for performing agarose gel electrophoresis and
ethidium-bromide staining (Voytas, 2000) and northern blot analysis (Brown et al.,
2004)
NOTE: Use sterile, RNase-free reagents and vessels.
7. Precipitate the RNA by adding 1 volume (800 l) of ice-cold isopropanol and placing
for 1 hr at 80 C or overnight at 20 C.
8. Centrifuge for 10 min at 12,000 g, 4 C.
16E.1.4
Supplement 6
9. Remove the supernatant and carefully cover the pellet with 1 ml of ice-cold 70%
ethanol.
10. Remove the 70% ethanol supernatant and let the pellet air dry for 10 min.
11. Once complete, resuspend RNA pellet in 20 to 50 l of DEPC-treated water.
2
DNase buffer
0.2 M TrisCl, pH 7 (APPENDIX 2A)
0.2 M NaCl
0.1 M MgCl2
Store up to 6 months at 4 C
Guanidinium solution
4 M guanidinium thiocyanate
25 mM sodium acetate, pH 7
20 mM EDTA (APPENDIX 2A)
50 mM 2-mercaptoethanol (APPENDIX 2A)
Store up to 1 week in dark bottle at 4 C
Nucleic acid extraction buffer
0.1 M TrisCl, pH 8.0 (APPENDIX 2A)
0.2 M sodium chloride
2 mM EDTA (APPENDIX 2A)
1% SDS
Store up to 1 week at 4 C
COMMENTARY
Background Information
These methods describe the isolation of viral RNAs associated with plus-strand RNA
plant virus infections of plant protoplasts (Section 16D). The protocols allow for the isolation of either total RNA or partially purified
RNA that also include viral RNAs. Thus, it is
important to remember that these methods do
not generate pure viral genomic RNAs, that
generally requires the prior isolation of virus
particles (from which viral genomes can be
isolated) and a customized purification scheme
for each individual virus. Instead, the protocols
described are useful for obtaining RNA preparations that contain genomic RNAs, subgenomic mRNAs, defective viral RNAs, complementary viral RNAs, and/or double-stranded
viral RNAs in association with cellular RNAs.
Though crude by some standards, these RNA
Critical Parameters
Due to the unstable nature of RNA, it
is important that extra care is taken when
performing the steps in these protocols. For
example, it is often better to be conservative
when removing the aqueous phases from organic phases and to sacrifice some sample at
Plant RNA
Viruses
16E.1.5
Current Protocols in Microbiology
Supplement 6
contains both viral and cellular RNAs. In either case, these protocols yield RNA samples
that will be stable for several years when stored
at 80 C and can be used successfully in the
many applications listed above.
Anticipated Results
Literature Cited
Time Consideration
The protocols described are moderately
time consuming but, with the exception of the
dsRNA preparation, can be completed in about
2 hr. This estimate will increase if large numbers of samples are processed simultaneously.
Chomczynski, P. and Sacchi, N. 1987. SingleStep method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal. Biochem. 162:156-159.
Morris, T.J. and Dodds, J.A. 1979. Isolation and
analysis of double-stranded RNA from virusinfected plant and fungal tissue. Phytopathology
69:854-858.
Voytas, D. 2000. Agarose gel electrophoresis. Curr.
Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.
Extracting Viral
RNAs from Plant
Protoplasts
16E.1.6
Supplement 6
UNIT 16E.3
An essential feature of viral infection is replication of the viral genome. RNA viruses
are broadly categorized into single-stranded, positive- and negative-sense RNA viruses,
and double-stranded (ds)RNA viruses. Many plant and animal viruses have plus-strand
(i.e., messenger-sense) RNA genomes in their virions. RNA genomes of positive-strand
RNA viruses share similar replication strategies despite vast differences in genome
organization, primary sequence, and secondary and tertiary structure. After release of
the genomic RNA(s) from the viral capsid, translation proceeds using host ribosomes,
leading to expression of the RNA-dependent RNA polymerase (RdRp). The RdRp,
together with known or hypothetical host proteins and additional viral proteins, forms an
active replicase that recognizes specific cis-acting elements, leading to initiation of minusstrand synthesis (Buck, 1996; Ahlquist, 2002). Minus strands, which either complex with
the plus-strand template (Khromykh and Westaway, 1997) or remain single-stranded
(Garnier et al., 1980; Axelrod et al., 1991), are transcribed into full-length plus strands
such that an asymmetric ratio of plus- and minus-strands accumulates, ranging from 10:1
to 1000:1 (Buck, 1996). A negative-sense RNA genome and dsRNA, upon entering into a
suitable host cell, must be copied into positive-sense mRNA before translation. To study
the replication of RNA viruses in tissues, cells, or protoplasts, total RNA is extracted,
the type of viral transcripts is detected, and the abundance is determined by northern
blot analysis using strand-specific probes. Though different investigators follow northern
analysis protocol with modifications based on their experimental requirements, this unit
describes the general strategies for northern analysis.
STRATEGIC PLANNING
Northern Blotting
RNA
Total RNA is extracted from tissues (see Support Protocol 1), cells, or protoplasts (Kong
et al., 1995; Wang and Simon, 1999; Qi et al., 2004; Grdzelishvili et al., 2005; Hema
et al., 2005; Herranz et al., 2005; Picard et al., 2005) and used for northern analysis. In
the total cellular RNA, ribosomal RNAs (rRNAs) are the dominant components and may
contribute >75%. Among the rRNAs, 25S (3.5 kb) and 18S (1.9 kb) are highly abundant.
Quality of RNA
Integrity of rRNA is a critical factor for successful northern blot analysis and can be
assessed by agarose gel electrophoresis and ethidium bromide staining (Fig. 16E.3.1).
Concentration of RNA
If comparing the abundance of viral RNA transcripts in different samples, it is extremely
important to load equal amounts of RNA into each lane of a gel. If equal amounts of
RNA are not loaded into each lane, it is not possible to compare and, therefore, results
in misinterpretations. Before loading the RNA onto a gel, quantitate RNA.
To quantitate RNA, dissolve the salt-free RNA in DEPC-treated water by briefly vortexing
at 4 C or gently pipetting up and down several times. Incubate 15 min on ice. Dilute
1 l RNA with 499 l DEPC-treated water and measure the absorbance at 260 nm.
A260 /A280 ratio of the extracted RNA is within the range of 1.6 to 2.0. Calculate the RNA
concentration (g/l) = O.D. 500 0.04 g/l. The yield of total RNA depends on
Plant RNA
Viruses
Contributed by Paramasivan Vijaya Palani and Na-Sheng Lin
Current Protocols in Microbiology (2007) 16E.3.1-16E.3.32
C 2007 by John Wiley & Sons, Inc.
Copyright
16E.3.1
Supplement 4
Figure 16E.3.1 Integrity and size distribution of total RNA isolated from Nicotiana benthamiana
is checked by agarose gel electrophoresis and ethidium bromide staining. A concentration of 1 g
RNA was loaded per lane. The rRNA bands should appear as sharp bands on the stained gel.
The size of 25S, 18S, and 16S rRNA is 3.5, 1.9, and 1.5 kb respectively. The 25S and 18S rRNAs
are cytoplasmic and the 16S rRNA is from the chloroplast. The large chloroplast rRNA is always
cleaved into two smaller RNAs during RNA extraction from plant tissue. The 25S ribosomal RNA
band should be present with an intensity approximately twice that of the 18S rRNA. If the rRNA
bands are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA was
degraded during preparation.
the tissue, cell, or protoplast source. For instance, the yield of total RNA is between 0.25
and 0.5 g/mg for tobacco, tomato, or potato leaf tissue (Verwoerd et al., 1989).
CAUTION: DEPC (diethylpyrocarbonate) is a potent protein denaturant and a suspected
carcinogen, and should be handled with care.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
RNA prepared from polyadenylated virusinfected samples contains viral mRNA with
poly(A) tails located at the 3 termini. In contrast, RNA prepared from nonpolyadenylated
virusinfected samples does not contain viral mRNA with poly(A) tails at the 3 termini.
The poly(A) content of the mRNA in total RNA can be compared by dot blot or slot
blot hybridization using a poly(dT) probe (Harley, 1987). Based on the signal intensities,
equivalent amounts of RNA can be loaded into lanes of a northern gel.
16E.3.2
Supplement 4
IMPORTANT NOTE: This method is applicable only when polyadenylated RNA is being
detected; it is not applicable for nonpolyadenylated RNAs.
A known amount of RNA transcript synthesized in vitro (positive control) from the
recombinant plasmid with the gene of interest is used in dot blot or slot blot hybridization
and the relative intensity of the hybridization signal is used to calibrate the expression of
the RNA.
Gels
Nondenaturing agarose gels (see Support Protocol 3 and 5) or formaldehyde agarose gels
(see Support Protocol 4) are commonly used for the separation of RNA. Nondenaturing
agarose gels are preferred because formaldehyde gels have fumes that are unpleasant and
highly toxic, are more fragile, and great care is necessary to handle them. Also, formaldehyde diffuses from the gel into the buffer during electrophoresis. RNA appear as distinct
bands in agarose gels without any denaturant, whereas RNA bands in formaldehyde gels
appear fuzzy. For resolving 0.5- to 0.8-kb RNAs, 1.5% (w/v) agarose gels are suitable
and for larger RNAs, 1% to 1.2% (w/v) agarose gels can be used.
CAUTION: Formaldehyde is highly volatile, toxic, and carcinogenic. It irritates skin,
eyes, mucous membranes, and the respiratory tract. Avoid breathing in vapors and use in
a fume hood.
Standards
To obtain quantitative results, it is essential to include positive and negative controls
during northern analysis. Positive control RNAs have structural properties similar to that
of the sample RNA and complementary to that of the probe. A positive control RNA
transcript is synthesized in vitro from DNA templates that have been engineered into a
plasmid where two different bacteriophage promoters, with opposite orientations, flank
the cloning site. A sense-strand positive control is synthesized and used for hybridization
when an antisense-strand is used as a probe. An antisense-strand positive control is
synthesized and used for hybridization when a sense-strand is used as a probe. The
negative control should consist of RNA from tissue, cells, or protoplasts that do not
express the target viral RNA.
To prepare standards, mix an in vitro transcript of sense-strand RNA with the negative
control RNA to approximately equal the mass of the sample RNA. For electrophoresis,
positive and negative control RNAs are prepared similar to that of the sample RNA.
These manipulations are necessary to reduce the presence of impurities in total RNA that
affect the intensity of the hybridization signal.
Plant RNA
Viruses
16E.3.3
Current Protocols in Microbiology
Supplement 4
Markers
The size of the viral RNA can be measured accurately by using RNA ladders or
DNA markers which can be commercially purchased (e.g., Ambion, Life Technologies, Promega). Markers are also used to detect RNase contamination or to check for
problems that may have occurred during sample preparation or electrophoresis. Glyoxylated denatured RNAs and DNAs of equal length migrate to equivalent locations on
agarose gels. Therefore, small DNAs of known size can also be used as markers in this
system. Do not use DNA standards on gels containing formaldehyde since the migration
of RNA is faster than that of the DNA of equivalent size (Wicks, 1986). Occasionally, the
contaminating vector sequence in the probe may hybridize to the RNA or DNA standards,
and may generate either a smear or discrete band where none should be.
Highly abundant rRNAs, i.e., 25S (3.5 kb) and 18S (1.9 kb), in total RNA preparations,
visualized while checking the quality of RNA in ethidium bromide agarose gels, can also
be used as markers.
In denaturing agarose gels, commonly used tracking dyes, bromophenol blue, which
migrates faster than 5S rRNA, or xylene cyanol, which migrates slower than 18S rRNA,
are also used as indicators of migration.
Gel
Prepare nondenaturing agarose gels (see Support Protocol 3 or 5) or formaldehydeagarose gels (see Support Protocol 4).
Membranes
A solid support is required to immobilize the RNA. Nitrocellulose membranes have been
the choice for several years (Thomas, 1980). The disadvantage of using the nitrocellulose
membrane is that it becomes brittle when baked under vacuum at 80 C to fix the RNA.
Also, such membranes cannot be used for reprobing. Nylon membranes (available from
Amersham Biosciences, Ambion, and PerkinElmer Life Sciences) are more durable
than nitrocellulose membranes (Reed and Mann, 1985) and therefore commonly used
for nucleic acid hybridization. A good nylon northern blot can be kept for years and
reprobed a dozen or more times with good results. Two types of nylon membranes are
available: neutral/uncharged and positively charged.
Neutral/uncharged nylon membranes
When using a neutral/uncharged nylon membrane, use of a neutral or nonalkaline buffer
over a wide pH range is preferred for the transfer of RNA. Nucleic acid retention capacity of a neutral/uncharged nylon membrane is 200 to 300 g/cm2 and nucleic acids of
>50 bp bind efficiently. Nucleic acids are immobilized either by baking or UV irradiation at 254 nm. Commonly used neutral nylon membranes are Hybond-N (Amersham
Biosciences) and Gene-screen (PerkinElmer Life Sciences). The disadvantage of an uncharged membrane is that it has a lesser capacity to bind nucleic acids compared to a
charged membrane.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.4
Supplement 4
agents in the prehybridization and hybridization solutions, and also by decreasing the
transfer time of RNA from gel to membrane. Nucleic acids in charged membranes are
immobilized by either baking or UV cross-linking. Commonly used, charged membranes
include Hybond-N+ (Amersham Biosciences) and Gene-screen Plus (PerkinElmer Life
Sciences). For both neutral and charged nylon membranes, buffers of low ionic strength
over a wide pH range can be used (Sambrook and Russel, 2001).
DNA/RNA probes
DNA or RNA probes are prepared (see Support Protocols 8 and 9) and used for hybridization. The working probe must be single-stranded. RNA or DNA polymerase is
used to produce labeled RNA or DNA, respectively, copies from the starting DNA.
Probes are labeled by an in vitro synthesis reaction or by PCR. One of the four nucleotide
precursors carries a labeled group and is incorporated into the newly synthesized RNA
or DNA. Double-stranded probes have secondary structure and are not ideal for use,
therefore, to improve the efficiency of hybridization, denature the double-stranded probe
by heating 5 min at 100 C. Double-stranded DNA probes are about three times less
sensitive than single-stranded probes. High sensitivity in northern blotting is obtained
from single-stranded DNA or RNA probes radiolabeled in vitro by 32 P. When preparing
the radiolabeled probe, consider the half-life, emission type, energy, and specific activity
of the radioactive material. The probe concentration in the hybridization solution should
be 10 ng/ml if the specific activity is 108 dpm/g, or 2 ng/ml if the specific activity is
109 dpm/g.
CAUTION: When using radiolabeled dNTPs, order only the required amount of isotope
for probe preparations. Always wear a laboratory coat and protective shield when preparing and purifying probes. Prepare the probe as soon as the isotope arrives. Follow the
general safety guidelines for use and disposal of radioactive materials. See UNIT 1A.4 for
more information.
BASIC
PROTOCOL
The following methods are most commonly used to separate RNA: (1) electrophoresis of
glyoxal/DMSO-denatured RNA on a nondenaturing agarose gel (see Support Protocol 3;
McMaster and Carmichael, 1977; Cheng and Tsai, 1999; Cheng et al., 2002; Annamalai
et al., 2003; Yeh et al., 2004; Hema et al., 2005; Hsu et al., 2006); (2) electrophoresis
of formamide/formaldehyde-denatured RNA on an agarose gel containing formaldehyde
(see Support Protocol 4; Boedtker, 1971; Lehrach et al., 1977; Seeley et al., 1992); and
(3) electrophoresis of formamide-denatured RNA on a nondenaturing agarose gel and
post-treatment of the gel with formaldehyde (see Support Protocol 5). This method is
preferred to overcome the problems with formaldehyde during electrophoresis (Kong
et al., 1997; Guan et al., 2000; Sun et al., 2005).
CAUTION: Formamide and formaldehyde are toxic, carcinogenic, and can irritate skin,
eyes, mucous membranes, and the respiratory tract. Avoid inhalation or direct contact.
Plant RNA
Viruses
16E.3.5
Current Protocols in Microbiology
Supplement 4
Materials
Sample RNA (Support Protocol 1 or 2)
Methylene blue solution (see recipe)
0.2 SSC (APPENDIX 2A)
1% (w/v) SDS
Prehybridization solution (see recipe)
Low-stringency wash buffer (see recipe)
High-stringency wash buffer (see recipe)
Strip buffer (see recipe), 70 to 75 C
Blotting paper
UV crosslinker (Stratagene), optional
80 C vacuum oven, optional
Microwave oven, optional
Glass tray
Camera with yellow filter, optional
Hybridization tube
65 C hybridization oven with rotation
Plastic wrap
X-ray film (e.g., Kodak Biomax MS film) and cassette (e.g., Hypercasette,
Amersham Biosciences), optional
Densitometer (e.g., GS-700, BioRad), optional
PhosphorImager (Molecular Dynamics, Amersham Pharmacia Biotech)
Additional reagents and equipment for electrophoresis (see Support Protocols 3 to
5); membrane transfer (see Support Protocols 6 and 7); DNA/RNA probe
preparation (see Support Protocols 8 and 9)
Electrophorese and transfer RNA
1. Perform electrophoresis of sample RNA as described in Support Protocols 3 to 5.
2. Cut and prepare the membrane and subsequently transfer the RNA from the gel to
the membrane by the upward or downward capillary system (see Support Protocols 6
and 7).
IMPORTANT NOTE: Always use gloves when handling the membrane.
Transfer of denatured RNA from the agarose gel to the surface of a membrane is the
critical step in northern blot analysis. Capillary transfer by the upward (see Support
Protocol 6, Fig. 16E.3.2) or downward (see Support Protocol 7, Fig. 16E.3.3) system is
usually the method of transfer.
Crosslinking by UV irradiation is highly preferred since fixing results are best and
quickly accomplished in a commercial UV crosslinker.
3b. Baking under vacuum: Air dry the membrane, place between two pieces of blotting
paper, and bake 2 hr under vacuum in a 80 C vacuum oven.
16E.3.6
Supplement 4
3c. Baking in a microwave oven: Place the damp membrane on a dry piece of blotting
paper and heat for 2 to 3 min at full-power (750 to 900 W) in a microwave oven.
Any membrane that is not immediately used for hybridization should be dried thoroughly,
wrapped in blotting paper, placed in a sealable plastic bag, and can be stored for several
months at room temperature, preferably under vacuum.
Hybridize membrane
6. Prehybridize the membrane by placing the membrane in a hybridization tube with
the RNA-side facing in and add 0.2 ml prehybridization solution/cm2 membrane.
Incubate 1 hr in a 65 C hybridization oven with rotation (about six to eight
rotations/min).
Alternatively, resealable plastic bags can be used on a shaker or rocker in a suitable
incubator.
7. Prepare DNA or RNA probe (see Support Protocols 8 and 9). If the probe is already
prepared and stored at 70 C, thaw the probe on ice before use.
Strand-specific oligonucleotide probes are commonly used for hybridization of nonpolyadenylated viral RNA (for preparation see Support Protocol 8).
Double-stranded probes have secondary structure, therefore, to improve the efficiency
of hybridization, denature the double-stranded probe by heating 5 min at 100 C. Chill
the probe in ice immediately and use it.
Single-stranded probes usually do not require denaturation.
8. Pipet the 32 P-labeled probe directly into the prehybridization solution and continue
to incubate with rotation (about six to eight rotations/min) overnight at 42 C (DNA
probe) or 65 C (RNA probe).
Use 10 ng/ml probe if the specific activity is 108 dpm/g or 2 ng/ml if the specific activity
is 109 dpm/g.
If resealable bags are used, the probe can be added by inserting a syringe to the opened
plastic bag and the bag should be resealed with great care.
10. Remove the probe not homologous to the target mRNA by the following stringent
washes:
a. Low-stringency wash: Wash the membrane in a large volume (50 ml) of
low stringency wash buffer for 1 to 2 hr at 37 C with rotation (six to eight
rotations/min).
b. High-stringency wash: Discard the buffer and continue washing two times with
50 ml high-stringency wash buffer for 1 hr at 65 C.
c. Remove the membrane from the wash solution. Blot excess liquid and cover the
membrane in plastic wrap as quickly as possible.
Plant RNA
Viruses
If the blot is to be reused, do not allow membrane to dry until the blot has been stripped.
16E.3.7
Current Protocols in Microbiology
Supplement 4
Materials
Infected plant tissues
Liquid nitrogen
RNA extraction buffer (see recipe), 80 C
24:1 (v/v) chloroform/isoamyl alcohol mixture, 4 C
4 M lithium chloride (LiCl)
DEPC-treated water (APPENDIX 2A)
3 M sodium acetate, pH 5.2
Isopropanol/100% ethanol, ice cold
70% ethanol
10 U/l RNase-free DNase I (Roche)
0.1% to 0.5% SDS in TE buffer, pH 7.6 or 0.1 mM EDTA, pH 7.5
Deionized formamide
Sterile mortar and pestle
15-ml propylene snap-cap tube
Centrifuge
Micropipettor
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
Extract RNA
1. Isolate infected plant tissues by dissection, cut into small pieces, and place immediately in liquid nitrogen.
Frozen tissues can be used immediately or stored at 70 C until use.
16E.3.8
Supplement 4
2. Transfer 0.5 g frozen tissue to a mortar containing liquid nitrogen and pulverize
tissue to a fine powder using a pestle.
3. Immediately transfer the powdered tissue to a 15-ml polypropylene snap-cap tube
with a precooled spatula, add 2.5 ml hot (80 C) RNA extraction buffer, and mix
the contents vigorously by inverting 30 sec.
4. Add 0.5 ml of 24:1 chloroform/isoamyl alcohol mixture and mix thoroughly. Centrifuge 5 min at 12,000 g, 4 C.
Precipitate RNA
5. Collect upper aqueous phase and add an equal volume of 4 M LiCl. Allow the RNA
to precipitate overnight at 20 C or 2 hr at 70 C.
6. After incubation, vortex or thaw the RNA precipitate on ice and centrifuge 15 min
at 12,000 g, 4 C.
7. Remove and discard the supernatant with a micropipettor. Dissolve the pellet in
700 l DEPC-treated water and vortex. Briefly spin down to remove large debris.
8. Add 1/10 vol of 3 M sodium acetate, pH 5.2, and 2.5 vol ice-cold 100% ethanol or
1 vol ice-cold isopropanol. Mix well and precipitate RNA overnight at 20 C or 2
hr at 70 C.
9. Collect the precipitated RNA by centrifuging 15 min at 12,000 g, 4 C. Carefully
remove and discared the ethanol/isopropanol with a micropipettor.
Do not decant the supernatant as the pellet may be lost.
10. Wash the pellet with 1 ml of 70% ethanol, centrifuge 5 min at 12,000 g, 4 C, and
remove any remaining ethanol with a micropipettor.
11. Keep the tube open for 10 to 20 min on ice to allow ethanol to evaporate. Do not
overdry the pellet as it affects solubilization.
12. Resuspend the RNA pellet in 250 l DEPC-treated water and store the RNA solution
at 70 C until use.
Store RNA
14a. Suspend RNA precipitates in 100% ethanol and store indefinitely at 20 C.
14b. Dissolve RNA precipitates in aqueous buffers (0.1% to 0.5% SDS in TE buffer, pH
7.6 or 0.1 mM EDTA, pH 7.5) and store indefinitely at 20 C. Remove SDS by
chloroform extraction and ethanol precipitation.
14c. Dissolve the RNA precipitate in deionized formamide and store indefinitely at
20 C.
Formamide provides stability and protection against degradation by RNases. Formamide
can be removed by ethanol precipitation.
Plant RNA
Viruses
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Current Protocols in Microbiology
Supplement 4
SUPPORT
PROTOCOL 2
Materials
Oligo(dT) cellulose (Ambion, Invitrogen)
10 N sodium hydroxide (NaOH)
40 U/l RNasin (Promega)
DEPC-treated water (APPENDIX 2A)
1 column loading buffer (see recipe)
Total RNA (Support Protocol 1)
Elution buffer (see recipe)
100% ethanol
Platform rocker or rotator
Disposable poly-prep columns (Bio-Rad)
1. Suspend 0.5 to 1.0 g of oligo(dT) cellulose in 0.5 to 1 ml of 0.1 N NaOH in a 1.5-ml
microcentrifuge tube and allow to swell for 2 min. Add 1 l RNasin in column
buffer and elution buffer before use.
The oligo(dT) cellulose should disperse readily (1 g poly(A) RNA binds to 1 mg
oligo(dT) cellulose).
2. Wash the oligo(dT) cellulose three times with DEPC-treated water by centrifuging
15 sec at 3000 g, room temperature. Remove the supernatant with a micropipettor
after centrifugation.
3. Wash the oligo(dT) cellulose with an equal volume of 1 column loading buffer
and centrifuge 15 sec or until the pH is >8.0 (use pH paper for measurement) at
3000 g, room temperature.
4. Denature the total RNA 5 min at 65 C. Cool the solution to room temperature and
add an equal volume of 2 column loading buffer.
5. Add the total RNA solution to the oligo(dT) cellulose and rock the tube gently for
30 to 60 min at room temperature.
Rocking or rotating increases the efficiency of mRNA binding to the oligo(dT) cellulose.
7. To remove poly(A) RNA from the column, wash the column with 5 column
volumes of 1 column loading buffer or until the OD260 of the final wash fraction
(diluted to 1:20) is <0.05 or 0 absorbance.
8. Elute poly(A) RNA from the column with 2 column volumes of elution buffer.
Collect the fractions, dilute each fraction 100-fold, and measure A260 . Pool the
fractions having an absorbance >0.05 and precipitate with 100% ethanol.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
After the removal of poly(A) from the matrix, low salt buffer is used to destabilize the
double-stranded structure of poly(A) RNA and to elute the poly(A) RNA from the matrix.
16E.3.10
Supplement 4
SUPPORT
PROTOCOL 3
Glyoxal is a commonly used denaturant that eliminates the secondary structure of singlestranded RNA. The two aldehyde groups of glyoxal react slightly under acid conditions
with the imino groups of guanosine to form a cyclic compound that prevents the formation
of intrastrand Watson-Crick bonds (Shapiro and Hachmann, 1966; Nakaya et al., 1968).
Glyoxalated RNA is unable to form stable secondary structures and it migrates on
agarose gels at a rate that is approximately proportional to the log10 of its size (McMaster
and Carmichael, 1977). Integrity of the sample RNA should be checked before adding
glyoxal because glyoxal reacts with ethidium bromide. Agarose gel electrophoresis of
glyoxalated RNA must be carried out at low ionic strength to prevent super renaturation
of the RNA and the commonly used buffers are 10 mM sodium phosphate (pH 6.3) or
40 mM 3-N-morpholinopropanesulfonic acid (MOPS), pH 7.0.
Materials
Agarose (SeaKem)
1 sodium phosphate buffer, pH 6.3
Sample RNA, salt-free (Support Protocol 1 or 2)
DEPC-treated water (APPENDIX 2A)
Standards
Markers
Glyoxal reaction mix (see recipe)
Nondenaturing gel loading buffer (see recipe)
10 SSC (APPENDIX 2A)
55 C water bath
2-mm comb
Horizontal electrophoresis apparatus for 20 10cm gels (built-in recirculation
system, optional)
Peristaltic pump, optional
1.5-ml microcentrifuge tubes
Glass plates
Prepare gel
1. To prepare 150 ml of a 1% agarose gel (20 10cm), add 1.5 g SeaKem agarose
to 150 ml of 1 sodium phosphate buffer, pH 6.3, and microwave until agarose is
completely dissolved.
2. Cool agarose to 55 C in a 55 C water bath and cast the gel using a 2-mm comb
with a sufficient number of teeth for all samples to be analyzed, including a few
extra wells for running standards and markers. Allow the gel to solidify for at least
1 hr at room temperature.
The gel should be 3- to 10-mm thick. Thin gels (<3-mm thick) do not support efficient
transfer of RNA to the membrane and also tend to deform during RNA transfer. When
preparing low concentration (i.e., 0.5%) agarose gels, first prepare a supporting gel
(i.e., 1%) without wells. After the gel hardens, pour the lower percentage gel directly on
top of the supporting gel. Stacked gels avoid fracturing during handling. It is important
that both gels be prepared from the same batch of 1 running buffer.
3. After the gel solidifies, remove the comb, place the gel in the electrophoresis tank,
and add 1 sodium phosphate buffer to cover the gel to a depth of 1 mm.
Set up a peristaltic pump if the electrophoresis system does not have a built-in recirculation system.
Plant RNA
Viruses
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Supplement 4
Prepare RNA
4. Dissolve salt-free sample RNA in DEPC-treated water and prepare the denaturation
mix in a 0.2-ml microcentrifuge tube as follows (prepare standards and markers in
the same manner):
5 l
10 l
RNA (1 to 10 g)
glyoxal reaction mix
Loading of >10 g RNA per lane causes smearing of the RNA during electrophoresis.
5. Incubate RNA solutions 1 hr at 55 C. Cool the mixture 10 min on ice and briefly
centrifuge at 7000 g, 4 C.
Perform electrophoresis
6. To the glyoxylated RNA mix, add 3 l nondenaturing gel loading buffer, mix well,
and immediately load into the wells of the gel. Load standards and markers on the
extreme wells of the gel to enable easy excision of marker lanes.
7. Perform electrophoresis at 4 to 5 V/cm until the bromophenol blue has migrated
to 8 cm, circulating the buffer during the run. As the gel is running, cut the
membrane, blotting papers, and paper towels (see Support Protocols 6 and 7).
The major drawback of using glyoxal as a denaturing agent is the upward shift in pH
of the electrophoresis buffer that occurs in the cathodic chamber during elecrophoresis.
If this shift occurs, a steep pH gradient will form as small ions in the buffer migrate
rapidly along the gel from the cathode, resulting in removal of the glyoxal from the RNA
(Nakaya et al., 1968). Therefore, it is necessary to recirculate the buffer mechanically.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.12
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electrophoresis. However, diffusion can be overcome by running gels for shorter periods
of time at a higher voltage (7 to 10 V/cm). Agarose-formaldehyde gels are preferred
for northern analysis because formaldehyde can be easily removed from the gel after
electrophoresis.
Materials
Agarose (SeaKem)
DEPC-treated water (APPENDIX 2A)
1 and 10 MOPS buffer, pH 8.0 (see recipe)
Formladehyde (deionized; see recipe)
Sample RNA, salt-free (Support Protocol 1 or 2)
Standards
Markers
Formamide, stored indefinitely at 20 C
Formaldehyde gel loading buffer (see reicipe)
55 C water bath
Horizontal electrophoresis apparatus with 20 10cm gel capacity (built-in
recirculation system, optional)
2-mm comb
Peristaltic pump, optional
1.5-ml microcentrifuge tubes
Glass plate
Prepare gel
1. Prepare 150 ml of 1% agarose gel (20 10cm) containing 2.2 M formaldehyde
by adding 1.5 g SeaKem agarose to 115 ml DEPC-treated water and microwaving
until agarose completely dissolves. Cool to 55 C and add 15 ml of 10 MOPS
buffer and 27 ml deionized formaldehyde.
When preparing low concentration (0.5%) agarose gels, first prepare a supporting gel
(1%) without wells. After the gel hardens, pour the lower percentage gel directly on top
of the supporting gel. Stacked gels avoid fracturing during handling. It is important that
both gels be prepared from the same batch of 1 running buffer.
2. Cool to 55 C and cast the gel using a 2-mm comb with a sufficient number of teeth
for all samples to be analyzed, including a few extra wells for running standards
and markers. Allow the gel to solidify for at least for 1 hr at room temperature.
3. Remove the comb, place the gel in the electrophoresis tank, and add 1 MOPS
buffer to cover the gel to a depth of 1 mm.
Set up a peristaltic pump if the electrophoresis system does not have a built-in recirculation system.
Prepare RNA
4. Prepare denaturation mix as follows (prepare standards and markers in the same
manner) in a 1.5-ml microcentrifuge tube:
1.0 l
1.0 l
2.0 l
5.0 l
RNA (1 to 20 g)
10 MOPS buffer
formaldehyde (deionized)
formamide
Loading of >20 g RNA per lane causes smearing of the RNA during electrophoresis.
CAUTION: Formamide is an irritant and destroys skin, mucous membrane, and the
respiratory tract. Avoid direct contact.
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5. Incubate the mixture 1 hr at 55 C. Cool the mixture on ice and briefly microcentrifuge to collect the mixture at the bottom of the tubes.
Perform electrophoresis
6. Add 1 l formaldehyde gel loading buffer to each sample, mix well, and place tubes
on ice. Load standards and markers in the extreme wells of the gel to enable easy
excision of marker lane.
7. To prevent diffusion of formaldehyde from the gel into the buffer, electrophorese
at 7 to 10 V/cm. To avoid change of pH in the electrophoresis buffer, circulate the
buffer. As the gel is running, cut membrane, blotting paper, and paper towels (see
Support Protocols 6 and 7).
Materials
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
Agarose (SeaKem)
1 sodium phosphate buffer, pH 6.3 (APPENDIX 2A)
Sample RNA, salt-free (Support Protocol 1 or 2)
DEPC-treated water (APPENDIX 2A)
50% to 70% formamide, stored indefinitely at 20 C
Standards
16E.3.14
Supplement 4
Markers
Nondenaturing gel loading buffer (see recipe)
6% formladehyde (deionized; see recipe)
10 SSC (APPENDIX 2A)
55 C water bath
Horizontal electrophoresis apparatus with 20 10cm gel capacity (built-in
recirculation system, optional)
2-mm comb
Peristaltic pump, optional
1.5-ml microcentrifuge tubes
Glass plate
Prepare gel
1. To prepare 150 ml of a 1.5% agarose gel (20 10cm), add 2.25 g SeaKem agarose
to 150 ml of 1 sodium phosphate buffer, pH 6.3, and microwave until completely
dissolved.
2. Cool to 55 C and cast the gel using a 2-mm comb with a sufficient number of teeth
for all samples to be analyzed, including a few extra wells for running standards
and markers. Allow the gel to solidify for at least 1 hr at room temperature.
The gel should be 3- to 10-mm thick. Thin gels (<3-mm thick) do not support efficient
transfer of RNA to the membrane and also tend to deform during RNA transfer. When
preparing low concentration (0.5%) agarose gels, first prepare a supporting gel (1%)
without wells. After the gel hardens, pour the lower percentage gel directly on top of the
supporting gel. Stacked gels avoid fracturing during handling. It is important that both
gels are prepared from the same batch of buffer.
3. Remove the comb, place the gel in the electrophoresis tank, and add 1 sodium
phosphate buffer, pH 6.3, to cover the gel to a depth of 1 mm.
Set up a peristaltic pump if the electrophoresis system does not have a built-in recirculation system.
Prepare RNA
4. Dissolve salt-free RNA in appropriate volume of DEPC-treated water in a 1.5-ml
microcentrifuge tube. Denature total RNA by heating 1 hr at 55 C in 50% to 70%
formamide. Cool the RNA 10 min on ice and collect the mixture at the bottom of
the tube by briefly centrifuging at 7000 g, 4 C. Prepare standards and markers
in the same manner.
CAUTION: Formamide is an irritant and destroys skin, the mucous membrane, and the
respiratory tract. Avoid direct contact.
Perform electrophoresis
5. To the denatured RNA mix, add 3 l of nondenaturing gel loading buffer, mix well,
and immediately load into the wells of the gel. Load standards and markers on the
extreme wells of the gel to enable easy excision of marker lanes.
6. To avoid change of the electrophoresis buffer pH, recirculate the buffer. Run the gel
at 4 to 5 V/cm until the bromophenol has migrated to 8 cm. As the gel is running,
cut membrane, blotting paper, and paper towels (see Support Protocols 6 and 7).
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Current Protocols in Microbiology
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well. Plot the log10 of the size fragments of RNA against the distance migrated.
Using the resulting curve, calculate the size of the viral mRNA after hybridization.
CAUTION: UV radiation is dangerous and can damage the retina of the eyes. It is also
mutagenic and carcinogenic. Use safety glasses or a UV protection shield.
Materials
2 and 20 SSC (APPENDIX 2A)
Gel (Support Protocols 3 to 5)
Nylon membrane
Glass plates
Plastic wrap
Blotting paper (Schleicher & Schuell GB002)
Thick blotting paper (Whatman 3 MM, Schleicher & Schuell GB004)
Glass rod or pipet
Paper towels and 500-g weight
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
Figure 16E.3.2 Upward capillary transfer. Capillary transfer of RNAs from the agarose gel to
the membrane is achieved by an upward ow of surrounding buffer through the gel to the stack of
paper towels.
16E.3.16
Supplement 4
1. On a glass plate, spread out a piece of plastic wrap large enough to surround the
gel.
This barrier is necessary to prevent buffer flow directly from the reservoir to the paper
towels stacked on top of the gel.
2. Cut a piece of blotting paper that is longer and wider than the gel, soak in 2 SSC,
and place in the center of the plastic wrap.
3. Cut three thick pieces of blotting paper similar to the size of the gel, soak in 2
SSC, and place in the center of the presoaked larger blotting paper.
4. Place the gel on the wet thick blotting paper. Remove air bubbles between the gel
and the blotting paper by rolling a glass rod/pipet over them.
5. Cut a piece of membrane 5 mm larger than the gel, soak in 2 SSC for 2 min.
Float the membrane on the surface of the buffer and then immerse the membrane into
the buffer. If the membrane is not saturated after floating for 2 min, then use a new
membrane as the unsaturated membrane affect the transfer of RNA.
6. Wet the top of the gel with 2 SSC and place the wet membrane on top of the gel
so that the corners are aligned. Avoid trapping air bubbles under the membrane.
Never move the membrane once it has been placed on top of the gel.
7. Cut three thick blotting papers similar to the size of the gel, soak in 2 SSC, and
place on top of the membrane without any bubbles between the membrane and
blotting paper.
Do not disturb the gel once the membrane has been placed on top of it.
8. Pour sufficient 20 SSC (transfer buffer) on the plastic wrap. Surround the gel with
the plastic wrap but do not cover the gel.
9. Cut a stack of paper towels (5- to 10-cm high), the size of the gel, and place the
stack on top of the wet thick blotting papers.
Do not soak the paper towels in buffer. Precisely pack the paper towels and avoid extra
over the edge of the gel, which will affect the efficiency of RNA trasfer.
10. Place a thin glass plate on top of the paper towels to prevent evaporation.
11. Over the glass plate, place an 500-g weight on top. Ensure that the setup is
balanced. Transfer RNA overnight. Do not disturb the setup.
12. After the transfer, disassemble the capillary system. Mark the positions of the wells
on the membrane in a corner with a pencil. Remove the membrane, drain the buffer,
and place the membrane RNA-side up on a piece of dry blotting paper.
SUPPORT
PROTOCOL 7
Materials
Nylon membrane
Gel (see Support Protocols 3 to 5)
20 SSC (APPENDIX 2A)
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Figure 16E.3.3 Downward capillary transfer. Capillary transfer of RNAs from the agarose gel to the membrane is achieved by a downward ow of buffer from the reservoir through the gel into the stack of paper
towels.
5. Place the gel on top of the membrane so that the cut corners are aligned. Ensure
that air bubbles are not present between the membrane and the gel. If air bubbles
are present, remove by rolling a glass rod or pipet over the gel. Do not disturb the
gel once it has been placed on top of the membrane.
6. Wet the top surface of the gel with 20 SSC and cover with three thick blotting
papers the size of the gel and soaked in 20 SSC.
7. Connect the stack to the reservoir troughs containing 20 SSC with two large
pieces of wet thick blotting papers larger than the gel as wicks.
8. Place a thin glass plate on top of the stack to prevent evaporation.
9. Allow downward transfer of RNA to occur for 4 hr.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
During transfer, check whether adequate buffer is present in the troughs. If necessary,
gently add buffer directly to the troughs without disturbing the capillary system.
16E.3.18
Supplement 4
10. After the transfer, disassemble the capillary system. Mark the position of the wells
in a corner on the membrane with a pencil. Remove the membrane, drain the buffer,
and place membrane, RNA-side up, on a piece of dry blotting paper.
5
SUPPORT
PROTOCOL 8
CAUTION: Perform the reaction and purify the oligonucleotide probe behind a plexiglass
screen as the mixture contains [-32 P]ATP. See UNIT 1A.4 for more information.
Materials
100 pmol/l synthetic oligonucleotides
Bacteriophage T4 polynucleotide kinase and 10 bacteriophage T4 polynucleotide
kinase buffer (New England BioLabs)
>5000 Ci/mmol [-32 P]ATP
1 M TrisCl, pH 8.0 (APPENDIX 2A)
5 M ammonium acetate
70% and 100% ethanol, ice cold
TE buffer, pH 7.6 (APPENDIX 2A)
1.5-ml microcentrifuge tubes
37 and 68 C water baths
Additional reagents and equipment to measure specific activity (see Support
Protocol 9)
1. Set up the following reaction mix in a 1.5-ml microcentrifuge tube:
1 l
2 l
15 l
1.5 l
5. Add 40 l water to the tube containing the radiolabeled oligonucleotide and mix
well.
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9. Wash the pellet with 0.5 ml of ice-cold 70% ethanol and centrifuge 5 min at 12,000
g, 4 C.
10. Remove the ethanol using a micropipettor. Keep the tube open on ice until the
ethanol evaporates.
11. Dissolve the radiolabeled oligonucleotide probe in 100 l TE buffer, pH 7.6.
The probe can be used immediately or stored at 70 C until the half-life of the radiolabeled compound.
SUPPORT
PROTOCOL 9
Materials
Plasmid DNA
Restriction enzymes
DEPC-treated water (APPENDIX 2A)
5 mM ATP, GTP, and UTP (APPENDIX 2A)
100 M CTP
10 transcription buffer
20 mCi/l or 3000 Ci/mM [-32 P] rCTP
40 U/l RNasin (Promega)
15 U/l T7/SP6 DNA-dependent RNA polymerase
10 U/l RNase-free DNase I (Roche)
Scintillation fluid
37 C (for T7/T3 polymerase) or 40 C (for SP6 polymerase) water bath
Nuc-Trap push column (Stratagene), optional
Scintillation counter
Whatman filter paper disk (DE81 filter, Whatman)
Additional reagents and equipment for restriction digestion and purifying DNA
(Moore and Dowhan, 2002)
1. Digest plasmid DNA with a restriction enzyme that cuts downstream of the desired
probe sequence. Extract digested DNA with phenol/chloroform, precipitate with
ethanol, and redissolve DNA in DEPC-treated water to a final concentration of
1 g/l (Moore and Dowhan, 2002).
Northern Analysis
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Minus-Strand
RNAs
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2. Prepare the transcription reaction mix behind a plexiglass screen in the order given:
1.0 l
10.0 l
5.0 l
2.5 l
5.0 l
Linearized DNA (1 g)
DEPC-treated H2 O
5 mM ATP, GTP, and UTP + 100 M CTP
10 transcription buffer
[-32 P] rCTP (20 mCi/l or 3000 Ci/mM)
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Current Protocols in Microbiology
Supplement 4
Mix the components at room temperature by gently tapping the tube, then add:
0.5 l
1.0 l
3. Briefly centrifuge to bring the contents to the bottom of the tube and incubate 1 hr
at 37 C (T7/T3 DNA-dependent RNA polymerase) or 40 C (SP6 DNA-dependent
RNA polymerase).
4. Terminate the reaction by adding 1 l RNase-free DNase I to the reaction tube.
Gently tap the tube and incubate 15 min at 37 C.
5. Purify the riboprobe by ethanol precipitation, column chromatography (e.g., NucTrap push column), or elution after polyacrylamide gel electrophoresis.
Purification by column chromatography is safer and facilitates the removal of unincorporated isotopes.
7. Calculate the specific activity of the radiolabeled probe (Sambrook and Russel,
2001) using the following equation:
L (2.2 109 ) ( PI )
m + [(1.3 103 ) ( PI ) ( L / S )]
where, L = input radioactive label (Ci), PI = proportion of the precursor that has been
incorporated (cpm in washed filter/cpm in unwashed filter), m = mass of template DNA
(ng), and S = specific activity of input label (Ci/nmol).
The numerator of this equation is the product of three terms: the total dpm in this
reaction [L (2.2 106 cpm/(Ci)], the proportion of this dpm which was incorporated
(PI), and a factor to convert the final value for specific activity from cpm/ng to cpm/g
(103 ).
The denominator represents: the total mass of RNA (ng) at the end of the reaction,
equal to the starting mass of DNA (ng) synthesized during the reaction. The latter is
calculated from the number of nanomoles of rNMP incorporated [(PI)(L/S)] multiplied
by four times the average molecular mass of the four rNMPs (4 325 ng/nmol = 1.3
103 /ng/nmol).
8. Use the probe immediately for hybridization or store at 70 C until the half-life of
[-32 P] CTP.
SUPPORT
PROTOCOL 10
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.22
Supplement 4
inactivated and the protected RNA is incubated with the riboprobe, if it has not been
previously incubated with the riboprobe, and then used for gel analysis.
Materials
Sample RNA (Support Protocol 1)
32
P-labeled riboprobe (see Support Protocol 9)
Annealing buffer A (see recipe)
RNase A
Annealing buffer B (see recipe)
Proteinase K
SDS
Viral RNA
Annealing buffer C (see recipe)
30 , 37 , 45 , 60 , 80 , and 100 C water baths
Additional reagents and equipment for purifying DNA (Moore and Dowhan, 2002)
To prepare sample RNA for detecting low abundant plus-strand RNAs:
1a. Incubate the DNase I-treated sample RNA with 0.2 pmol of 32 P-labeled riboprobe
in 20 l of annealing buffer A 5 min at 100 C and then 20 min at 45 C.
2a. Remove the single-stranded RNAs (ssRNA) in the mixture by digesting with 20 g
RNase A in 200 l annealing buffer B 30 min at 30 C.
3a. Terminate RNase digestion with 30 g of proteinase K and SDS (0.4% final concentration) 10 min at 37 C.
4a. Recover the protected dsRNA by phenol-chloroform extraction and ethanol precipitation (Moore and Dowhan, 2002).
5a. Separate the RNA in an agarose gel, transfer the RNA to the membrane (see Support
Protocols 6 and 7), and detect the plus-strand RNAs by autoradiography.
To prepare sample RNA for detecting low abundant minus-strand RNAs:
1b. Incubate DNase I-treated sample RNA with an excess amount (0.3 g) of viral
RNA in 30 l annealing buffer C with the following sequential incubations: 5 min
at 80 C, 10 min at 60 C, and 10 min at 37 C.
2b. Remove ssRNAs with 1 g/ml RNase A for 30 min at 30 C.
3b. Terminate RNase digestion with 30 g of proteinase K and SDS (0.4% final concentration) for 10 min at 37 C.
4b. Recover the protected dsRNAs by phenol-chloroform extraction and ethanol precipitation (Moore and Dowhan, 2002).
5b. Mix dsRNAs with 0.07 pmol of 32 P-labeled riboprobe in 20 l of annealing buffer B
and incubate the mixture 5 min at 100 C to denature the dsRNAs and then incubate
1 hr at 45 C to anneal the minus-strand RNA with the probe.
6b. Separate the RNA on an agarose gel, transfer the RNA to a membrane (see Support
Protocols 6 and 7), and detect the minus-strand RNAs by autoradiography.
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Annealing buffer A
40 mM piperazine-N,N -bis(2-ethanesulfonic acid) (PIPES), pH 6.4
0.4 M NaCl
1 mM EDTA
80% formamide
Prepare fresh
Annealing buffer B
0.2 M NaCl
0.1 M LiCl
10 mM TrisCl, pH 7.5 (APPENDIX 2A)
1 mM EDTA
Prepare fresh
Annealing buffer C
0.3 M KCl
50 mM TrisCl, pH 8.0 (APPENDIX 2A)
1 mM EDTA
Prepare fresh
Bakers yeast RNA, 20 mg/ml
1 g bakers yeast RNA (Sigma)
50 ml TE buffer, pH 8.0 (APPENDIX 2A)
Store in 25-ml aliquots indefinitely at 20 C
Column loading buffer, 2
40 mM TrisCl, pH 7.6 (APPENDIX 2A)
1 mM NaCl
2 mM EDTA
0.2% SDS
Prepare fresh stock solutions of TrisCl, NaCl, and EDTA, and autoclave. Dilute
with sterile DEPC-treated water and cool to 65 C, then add SDS from 10% stock
solution.
Denatured salmon DNA, 10 mg/ml
0.5 g salmon sperm DNA (Sigma)
50 ml TE buffer, pH 8.0 (APPENDIX 2A)
Shear in syringe with 18-G needle
Stir 2 to 4 hr at room temperature and store in 25-ml aliquots indefinitely at 20 C
Before use, boil 5 min and chill quickly
Elution buffer
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.24
Supplement 4
Formaldehyde
Purchase 37% to 40% (w/v) or 12.3 M formaldehyde solution (Bio-Rad). Formaldehyde readily oxidizes when exposed to air and becomes yellow (pH < 4.0). Use
fresh stock solution.
P-labeled probe (10 ng/ml if the specific activity is 108 dpm/g or 2 ng/ml if the
specific activity is 109 dpm/g)
10 ml prehybridization buffer (see recipe)
Prepare fresh
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MOPS buffer, 10
0.2 M MOPS (free acid; adjust to pH 7.0 with 2 N NaOH)
20 mM sodium acetate
10 mM EDTA
Sterilize through a 0.45-m Millipore filter and store until solution turns yellow at
room temperature in the dark
Nondenaturing gel loading buffer, 6
50% glycerol
10 mM sodium phosphate buffer, pH 6.3 (see recipe)
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
Store in 0.1-ml aliquots indefinitely at 20 C
Prehybridization buffer
250 ml formamide
25 ml 1 M sodium phosphate buffer, pH 6.3 (see recipe)
80 ml 5 M NaCl
5 ml 0.1 M EDTA
50 ml 100 Denhardts solution (APPENDIX 2A)
12.5 ml 10 mg/ml denatured salmon DNA (see recipe)
12.5 ml 20 mg/ml bakers yeast RNA (see recipe)
25 ml 10% SDS
40 ml H2 O
Store in 10-ml aliquots indefinitely at 20 C
RNA extraction buffer
51 ml DEPC-treated H2 O
5 ml 4 M lithium chloride
20 ml 1 M TrisCl, pH 8.0 (APPENDIX 2A)
4 ml 0.5 M EDTA
20 ml 10% SDS
Prepare fresh, mix with buffered phenol (APPENDIX 2A) in a 1:1 ratio, and heat at
80 C before use
Sodium phosphate buffer, 1 M (100, pH 6.3)
31.95 g Na2 HPO4
106.95 g NaH2 PO4 H2 O
Make up to 1 liter with water
Store up to 3 months at room temperature
Strip buffer
10 mM TrisCl, pH 7.4 (APPENDIX 2A)
0.2% SDS
Prepare fresh stock solution of TrisCl and autoclave. Dilute with sterile DEPCtreated water and after cooling to 65 C, add SDS from 10% stock solution. Store
up to 3 months at room temperature.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.26
Supplement 4
COMMENTARY
Background Information
Extraction of total RNA
For intact RNA isolation, guanidium hydrochloride and guanidium thiocyanate are
two strong denaturants used to disrupt cells,
solubilize their components, and denature
endogenous RNases (Ullrich et al., 1977;
Chirgwin et al., 1979; Han et al., 1987). However, these methods are unsuitable for handling
numerous samples. Some investigators, when
handling many samples, follow a single-step
technique for RNA extraction, which generally
does not yield high-purity RNA. For RNA extraction, commercial reagents, such as Trizol
(Life Technologies), Isogen (Nippon Gene),
and Tri reagent (Molecular Research Center),
can also be used following the manufacturers
instructions.
Separation of poly(A) RNA from total RNA
Viral mRNA may or may not have a poly(A)
tail at their 3 termini. Based on the presence or
absence of the poly(A) tail at their 3 termini,
viruses are classified as polyadenylated or
nonpolyadenylated, respectively. If the RNA
is from a polyadenylated virus-infected sample, then DNA contamination can be avoided
by separating poly(A) RNA from total RNA
by affinity chromatography or batch elution.
Affinity chromatography is generally the preferred method to purify >25 g poly(A) RNA
from total RNA. In this method, mRNA form
stable RNA-DNA hybrids with short chains
of oligo(dT) (generally 18 to 30 nucleotides in
length) linked to a supporting cellulose matrix.
A percentage of 1% to 10% of total RNA is
recovered as poly(A) RNA. Batch chromatography on oligo (dT)-cellulose is a technique
of choice when working with a small amount
of total RNA (<25 g). Poly(A) RNA can be
separated from total RNA by using commercial kits (Invitrogen and Ambion).
Transfer of RNA from gel to membrane
Many methods, including capillary blotting, vacuum blotting, electroblotting, and
semi-dry blotting, have been used to transfer RNA from gel to membrane. The capillary system is the original simple technique
devised by Southern (1975) for the transfer
of nucleic acids from gel to membrane using an ascending flow of buffer (Fig. 16E.3.2).
Capillary transfer is commonly used because
it does not need any special equipment. This
system has two types, the upward or downward capillary system. In an upward capillary
system, the transfer of RNA from gel to membrane is by ascending flow of buffer and this
system is more suitable for transferring small
RNA species (<1 kb). In a downward capillary system, transfer of RNA from agarose gel
to membrane is by descending flow of buffer
(Fig. 16E.3.3). Downward capillary systems
result in faster transfer and increased efficiency
of transfer for longer RNA (Chomczynski and
Mackey, 1994).
DNA or RNA probes
For northern analysis, a DNA or RNA probe
is used. In vitro synthesis of a DNA probe is
accomplished by the following methods.
Random-primed labeling
In random-primed labeling, template
DNA is denatured and then cooled slowly
in the presence of all possible combinations
of hexanucleotides, which can bind to suitable complementary sequences within the
DNA strands. The reaction mix contains four
dNTPs, at least one of which has a labeled
group, and a Klenow subunit of DNA polymerase I. Primer binds to the template in a
random manner and the probe is of high specific activity.
Nick translation
In nick translation, a single-strand break
is introduced resulting in a 3 -hydroxyl termini and 5 -phosphate termini using DNase I.
The exposed nick becomes the starting point
for introducing newly labeled nucleotide using DNA polymerase while the existing nucleotides are removed from the other side of
the nick by exonuclease activity of the same
enzyme. The synthesis allows for the incorporation of labeled nucleotides in place of the
previously unlabeled ones.
End-labeling
For details on end-labeling, see Support
Protocol 8.
Labeling by PCR
Double-stranded DNA or single-stranded
DNA probes are labeled by PCR in the presence of two or one primer, respectively. Probe
labeling by PCR eliminates the need of cloning
a desired fragment of DNA into a vector containing bacteriophage promoters. A very small
amount of DNA (1 to 10 ng) is required. The
probe DNA fragment can be directly synthesized and labeled independently in the presence of an isotope or nonisotope such as biotinylated or DIG-labeled dNTPs by PCR.
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Radiolabeling
For radiolabeling the riboprobe, [32
P]UTP is the best choice, as it is specific to RNA. Alternatively, [-32 P]GTP, [32
P]CTP, [-32 P]CTP, or [-32 P]ATP can
be used. 32 P-labeling of riboprobes afford a
high sensitivity of detection (Koev and Miller,
2000; Cheng et al., 2002; Annamalai et al.,
2003; Choi et al., 2004; Komoda et al., 2004;
Grzelishvili et al., 2005). RNA can also be
transcribed in vitro using commercial kits
(Ambion and Promega).
Nonisotopic labeling
Nonisotopic labeling involves chemical
coupling of a modified reporter molecule to a
nucleotide precursor. Nonisotopic probes have
become increasingly popular in recent years
and have replaced radioactive probes in northern analysis. Advantages of these techniques
are that the technologies are safe, probes can
be stored for at least 1 year, and hybridization solutions can be reused several times. Two
common nonisotopic systems include biotinstreptavidin and digoxigenin. Hybridization
buffers and conditions vary in these systems.
In both systems, the probe is detected with either chromogenic, fluorescent, or chemiluminescent substrate. Colorimetric detection uses
the substrates NBT and BCIP to generate purple/brown precipitate directly on the membrane. Biotin- or digoxigenin-labeled probes,
upon coupling to the alkaline phosphatase substrate (CSPD or CDP-Star), produces a light
signal that can be detected by exposing the
membrane to an X-ray film.
RNase contamination
RNase contamination is the most serious
problem in northern analysis. RNase contamination cannot be removed by autoclaving, but
it can be avoided by taking careful precautionary steps.
Biotin-streptavidin system
The biotin-streptavidin system utilizes extremely high-affinity ligands: biotin (a naturally occurring vitamin) and streptavidin (a
bacterial protein). Biotin is incorporated in
the probe by using biotinylated dNTPs during
probe synthesis.
Digoxigenin system
Digoxigenin (DIG; a plant steroid) containing nucleotides is incorporated into the probe
and detected using a DIG-specific antibody.
DIG-probe synthesis incorporates DIG-11dUTP by PCR, random primer labeling, T7or SP6-mediated transcription, end-labeling,
nick translation, etc. (Vlot et al., 2001;
Haasnoot et al., 2003).
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
Preparation of riboprobes
Rioprobes are prepared by in vitro transcription using either linearized plasmid or
PCR-amplified DNA fragments encoding a
Critical Parameters
Gloves
Wear gloves at all times and use new gloves
while handling equipment when contamination is suspected. Pipets and pipet tips are another source of RNase contamination. Therefore, use a special set of pipets and tips for
RNA work. Glassware, spatulas, and stir bars
should be decontaminated by baking 4 hr at
300 C. Reagents and plasticware should be
from fresh packages set aside for RNA work.
Plasticware can be treated with commercial
RNase inactivators like RNasezap (Ambion).
Prepare all solutions and reagents in RNasefree water and chemicals reserved for RNA
work.
Dedicated reagents
Use separate stocks of reagents for RNA
work. It is better to store solutions in small
aliquots and to discard previously used solutions or those that are suspected to have
contamination. The growth of microorganisms in buffers and reagents may cause RNase
contamination. Therefore, simply discard the
contaminated solutions and prepare fresh with
RNase inhibitors.
DEPC treatment
DEPC, a highly reactive alkylating agent, is
the most common and potent RNase inhibitor.
Store small aliquots of DEPC in a dry environment. Glassware and plasticware should be
washed with 0.1% DEPC-treated water and allow it to stand for 1 hr to overnight at room
temperature, then rinse with DEPC-treated
water several times and autoclave. In aqueous
solutions, DEPC hydrolyzes rapidly to CO2
and ethanol, and hydrolyzation is accelerated
16E.3.28
Supplement 4
by Tris and amines. Therefore, DEPC is incompatible with buffers like Tris.
Dedicated apparatus
Use a specific horizontal electrophoresis
apparatus and combs for resolving RNA and
never run DNA in the RNA unit. Wash gel
box, electrophoresis tank, and combs with detergent, rinse with water, and fill with 3% H2 O2
for 30 min at room temperature. Rinse the tank
and comb thoroughly with DEPC-treated water before use.
Choice of membrane
Use of positively charged nylon membranes
is always better than neutral or uncharged
membranes since the former retain nucleic
acids by covalent bonding.
Preparation of riboprobes
When in vitro transcription is performed,
it is essential to consider the disposition of
Problem
Cause
Possible solution
Prehybridize for longer periods
Improper washing
RNA is degraded
High background on
the lanes
No signal
Plant RNA
Viruses
16E.3.29
Current Protocols in Microbiology
Supplement 4
Specific activity
(dpm/g)
Type of probe
Method of synthesis
Size of probe
Double-stranded
DNA
Random priming
400600 nt
using DNA templates
2 109
Single-stranded
DNA
Random priming
using RNA templates
400600 nt
1 109
Double-stranded
DNA
Nick translation
400 nt
5 108 1 109
Single- or
double-stranded
DNA
PCR
Generates radiolabeled
DNAs of defined length,
depending on the spacing
of primers
1 109 2 109
Single-stranded
RNA
In vitro transcription
of double-stranded
DNA templates
Generates radiolabeled
RNAs of defined length
1 109
Time
Procedure
Day 1
Day 2
Day 3
Troubleshooting
Table 16E.3.1 presents some of the problems commonly encountered when performing these techniques, as well as their possible
causes and solutions.
Anticipated Results
The specific activity of different probes
varies (Table 16E.3.2). Riboprobes offer high
sensitivity of detection than DNA probes and
32
P-labeled riboprobes are more sensitive.
Time Considerations
A northern blot can be completed in 3 days
as shown in Table 16E.3.3.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
Literature Cited
Ahlquist, P. 2002. RNA-dependent RNA polymerase, viruses, and RNA silencing. Science
296:1270-1273.
16E.3.30
Supplement 4
Qi, Y., Zhong, X., Itaya, A., and Ding, B. 2004. Dissecting RNA silencing in protoplasts uncovers
novel effects of viral suppressors on the silencing pathway at the cellular level. Nucl. Acids
Res. 32:e179.
Herrin, D.L. and Schmidt, G.W. 1988. Rapid reversible staining of northern blots prior to hybridization. BioTechniques 6:196-200.
Hsu, Y.H., Chen, H.S., Cheng, J., Annamalai, P.,
Lin, B.Y., Wu, C.T., Yeh, W.B., and Lin, N.S.
2006. Crucial role of the conserved structure
of bamboo mosaic virus satellite RNA in down
regulation of helper viral RNA replication. J.
Virol. 80:2566-2574.
Ishikawa, M., Meshi, T., Ohno, T., and Oakada, Y.
1991. Specific cessation of minus-strand RNA
accumulation at an early stage of Tobacco mosaic virus infection. J. Virol. 65:861-868.
Khromykh, A.A. and Westaway, E.G. 1997. Subgenomic replicons of the flavivirus Kunjin: Construction and applications. J. Virol. 71:14971505.
Koev, G. and Miller, W.A. 2000. A positive-strand
RNA virus with three very different subgenomic
RNA promoters. J. Virol. 74:5988-5996.
Plant RNA
Viruses
16E.3.31
Current Protocols in Microbiology
Supplement 4
Wang, J. and Simon, A.E. 1999. Symptom attenuation by a satellite RNA in vivo is dependent
on reduced levels of virus coat protein. Virology
259:234-245.
Wicks, R.J. 1986. RNA molecular weight determination by agarose gel elctrophoresis using
formaldehyde as denaturant: Comparison of
RNA and DNA molecular weight markers. Int.
J. Biochem. 18:277-278.
Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, A., Rutter, W., and Goodman, H. 1977.
Rat insulin genes: Construction of plasmids containing the coding sequences. Science 196:13131319.
Verwoerd, T.C., Dekker, B.M., and Hoekema, A.
1989. A small scale procedure for the rapid isolation of plant RNAs. Nucl. Acids Res. 17:2362.
Vlot, A.C., Neeleman, L., Linthorst, H.J.M., and
Bol, J.F. 2001. Role of the 3 -untranslated regions of Alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in
plants and in the assembly of virions. J. Virol.
75:6440-6449.
Voytas, D. 2000. Agarose gel electrophoresis.
In Current Protocols in Molecular Biology
(F.M. Ausubel, R. Brent, R.E. Kingston, D.D.
Moore, J.G. Seidman, J.A. Smith, and K. Struhl,
eds.) pp. 2.5A.1-2.5A.9. John Wiley & Sons,
Hoboken, N.J.
Northern Analysis
of Viral Plus- and
Minus-Strand
RNAs
16E.3.32
Supplement 4
UNIT 16F.1
ABSTRACT
Full-length, biologically active cDNA clones of the positive-strand RNA plant viruses are
indispensable for investigating the functions of viral genes and control elements as well as
generating virus-derived gene expression and silencing vectors. Even though engineering
of such clones for 4- to 10-kb viral RNAs has become routine, it remains a challenging
task for 15- to 20-kb RNA genomes of the monopartite viruses in a family Closteroviridae.
This unit describes strategic considerations and techniques used to generate an infectious
cDNA clone of a closterovirus. The use of agroinfection to improve specific infectivity
C
of the resulting clone is also explained. Curr. Protoc. Microbiol. 7:16F.1.1-16F.1.26.
2007 by John Wiley & Sons, Inc.
Keywords: RNA virus r full-length clone r cDNA r agroinfection
INTRODUCTION
Application of reverse genetics to RNA viruses requires generation of their infectious
cDNA clones, which are amenable to genetic manipulations using a vast array of DNAspecific enzymes and established protocols for DNA amplification, cloning, and mutagenesis. For the viruses with large genomes, conversion of the RNAs into faithful cDNA
clones is a challenging task. Such conversion involves putting together multiple cDNA
fragments for which reliable consensus nucleotide sequences must be obtained. In addition, large RNA transcripts of the viral cDNA clones are prone to degradation and
therefore exhibit relatively low specific infectivity. In this unit, the authors present strategic considerations and techniques designed to facilitate the generation of biologically
active cDNA clones for large RNA viruses.
It is also important to develop a reliable procedure for conversion of the clones back to
RNA that can be used to initiate infection. In plants this can be accomplished by using in
vitro transcription followed by mechanical inoculation (e.g., see UNIT 16B.3) of the resulting
RNA. However, mechanical inoculation is inefficient for many closteroviruses that are
normally limited to phloem and hence are recalcitrant to this approach. This limitation can
be overcome by using Agrobacterium-mediated delivery of viral cDNAs (agroinfection).
The efficiency of agroinfection can be drastically increased by co-expression of the plant
virus-derived suppressors of RNA silencing (Chiba et al., 2006; Basic Protocol 9).
This unit provides protocols that were used to engineer an infectious molecular clone of
Beet yellow virus (BYV). This virus belongs to the genus Closterovirus (family Closteroviridae) and possesses a one-component, 15.5-kb, positive-strand, RNA genome
(Dolja, 2003). Methods are provided for isolation of single-stranded (ss) and doublestranded (ds) genome-size viral RNAs (Basic Protocols 1 and 2) and for determining
the exact 3 - and 5 -terminal sequences of these RNAs (Basic Protocol 3 and Alternate
Protocol 1; Basic Protocol 4 and Alternate Protocol 2). Procedures are described for
molecular cloning of these sequences and introducing plant transcriptional control elements upstream and downstream from the viral cDNAs (Strategic Planning and Basic
Plant RNA
Viruses
Current Protocols in Microbiology 16F.1.1-16F.1.26, November 2007
Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16f01s7
C 2007 John Wiley & Sons, Inc.
Copyright
16F.1.1
Supplement 7
Protocols 5 and 6). Procedures are also provided also for cloning long cDNA fragments
using reverse transcription PCR (RT-PCR; Basic Protocol 7) as well as traditional cDNA
synthesis (Basic Protocol 8) for the generation of a full-length DNA copy of the viral
genome. Finally, testing of biological activity of the resulting clones using agroinfection
(Basic Protocol 9 and Support Protocols 1 & 2; also see UNIT 16B.2) of host plants is
explained.
CAUTION: Plant viruses are subject to regulation by USDA Animal and Plant Health
Inspection Service (APHIS). An APHIS permit must be obtained for virus propagation
in plants. Researchers also need to conform to local and institutional policies for working
with infectious agents. See Chapter 1, Section A and other appropriate resources (e.g.,
APPENDIX 1B) for further information.
STRATEGIC PLANNING
Availability of high-quality viral RNA for which a nucleotide sequence is known is a
prerequisite for obtaining a cDNA clone. As a starting point, this unit uses the published sequence of a European isolate of BYV (GenBank accession no. X73476). A
restriction map of corresponding cDNA was generated using online NEBcutter program from New England Biolabs (http://tools.neb.com/NEBcutter2/index.php). ClustalW
(http://www.ebi.ac.uk/clustalw) is a convenient tool for comparing nucleotide or amino
acid sequences of the clones using multiple sequence alignments. The GenBank sequences are only a starting point for obtaining a cDNA clone because they often do not
result in complete biological activity of an engineered virus.
Using low-copy plasmids for all crucial cloning steps (e.g., manipulations with very long
cDNA fragments or final assembly of the resulting clone) is recommended. This helps
avoid excessive accumulation of the foreign DNA and reduces potential toxicity of the
viral cDNA to bacteria used for cloning. Such toxicity is a common problem with viruses
having large genomes (Lai, 2000). For constructing a cDNA clone of BYV (Prokhnevsky
et al., 2002) the authors used pCB301 mini-binary vector provided by Dr. D.J. Oliver
(see Xiang et al., 1999). This plasmid has a relatively small size of 3.5 kb, accumulates
to a low level in E. coli, carries a convenient polylinker site (Fig. 16F.1.1, step 1), and
can be used for agroinfection. A NOS terminator sequence (required for transcription
termination; see Fig. 16F.1.2) should be first added to the pCB301 by regular cloning
using unique sites S and K (Fig. 16F.1.1, step 3).
The first steps in generating a full-length cDNA clone are identification of exact terminal
sequences of the viral RNA genome and cloning of the corresponding cDNA fragments
(Fig. 16F.1.4, steps 4 and 5). Because authentic genome termini are critical for infectivity,
at least two different methods should be used for determining these RNA regions. The
3 -terminal nucleotide sequences of the viral RNAs, which lack poly(A) tails, can be
determined by using two simple techniques that yield equally good results. The authors
recommend using both of them to ensure correct identification of the very terminal
nucleotides that are critical for virus viability. Both nucleotide sequencing and cDNA
cloning are performed using reverse transcription and either conventional synthesis of
the dsDNA or PCR amplification. The deoxyoligonucleotide primers for these reactions
should be of 24 to 30 residues in length with the GC content close to 50%. Designing
primers that overlap naturally occurring restriction sites in the viral genome is suggested
to simplify future manipulations.
Cloning of Large
Positive-Strand
RNA Viruses
The next steps include insertion of a ribozyme downstream from the 3 -terminal nucleotide of a viral cDNA (Fig. 16F.1.1, step 4) and RNA polymerase promoter upstream
from the 5 -terminal nucleotide of viral cDNA (Fig. 16F.1.1, step 5). These control elements are needed to ensure transcription and processing of exact copies of viral RNAs
upon agroinoculation.
16F.1.2
Supplement 7
Figure 16F.1.1 Diagram showing consecutive steps (and their associated Protocols) for generating a full-length cDNA clone for beet yellow virus (BYV) in a mini-binary plasmid. Dotted boxes
represent viral cDNA. Restriction sites used for cloning: A, SacI; B, BglII; X, XbaI; N, SnaBI; T,
BstEII; S, SmaI; K, KpnI. Other abbreviations: 5 and 3 , 5 and 3 termini of BYV cDNA, respectively; 35S, cauliower mosaic virus (CaMV) 35S promoter; NOS, nopaline synthase terminator;
RB and LB, transferred DNA (T-DNA) right border and left border, respectively; Rz, ribozyme
sequence.
Figure 16F.1.2
Plant RNA
Viruses
16F.1.3
Current Protocols in Microbiology
Supplement 7
Finally, internal cDNA fragments are added to complete assembly of the full-length clone
(Fig. 16F.1.1, steps 6 and 7; Basic Protocols 7 and 8). Each of the cDNA fragments must
be sequenced in at least three independent clones to determine a consensus nucleotide
sequence and exclude incorporation of incidental mutations that are often detrimental for
virus infectivity. The final steps involve transformation of the Agrobacterium with the
full-length cDNA clone and infectivity tests. For these purposes the authors use plants of
Nicotiana benthamiana, which is an experimental host for many viruses including BYV
and is amenable to infiltration with agrobacterial culture.
Note that parameters for the polymerase chain reactions described in this unit were
optimized for the PTC-100 thermocycler (MJ Research, distributed by Bio-Rad); they
have not been tested on other machines.
BASIC
PROTOCOL 1
Materials
5 mg/ml virus suspension of interest
10% (w/v) SDS (DNase and RNase free; Bio-Rad)
Neutral saturated phenol (EMD Chemicals)
Neutral phenol (EMD Chemicals)/chloroform/isoamyl alcohol mixture (25:24:1)
Chloroform/isoamyl alcohol (24:1)
7.5 M ammonium acetate
100% and 70% (v/v) ethanol (cold)
Sterile nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
1.5-ml microcentrifuge tube
Refrigerated microcentrifuge
Vacuum evaporator (e.g., SpeedVac concentrator, Thermo Scientific)
Additional reagents and equipment for performing agarose gel electrophoresis
(Voytas, 2000) and spectrophotometrically quantifying nucleic acids (Gallagher,
2004)
Extract ssRNA from virus particles
1. To 0.7 ml of 5 mg/ml virus suspension, add 70l of 10% (w/v) SDS and vortex
briefly.
2. Immediately add an equal volume (0.77 ml) of neutral phenol and shake 1 minute.
3. Centrifuge 1 min at maximum speed, room temperature in a tabletop microcentrifuge.
4. Transfer upper aqueous phase (avoiding the interphase) to a fresh tube and extract
twice with 0.8 ml of neutral phenol/chloroform/isoamyl alcohol mixture (25:24:1)
by shaking 1 min and centrifuging as in step 3. After each centrifugation transfer the
upper phase to a fresh tube.
5. Extract twice with 0.8 ml of chloroform/isoamyl alcohol (24:1) as in step 4, transferring the upper phase after each extraction to a fresh tube.
Cloning of Large
Positive-Strand
RNA Viruses
6. Adjust supernatant to 2.5 M with 7.5 M ammonium acetate and precipitate the RNA
with 2.5 volumes 100% cold ethanol.
7. Incubate a few hours at 20 C.
16F.1.4
Supplement 7
BASIC
PROTOCOL 2
The dsRNA purification method described in (Valverde, 1990) recommends two rounds
of chromatography on cellulose sorbent. The authors have found that one round is
sufficient to obtain dsRNA preparation suitable for applications discussed in this unit.
Precipitating the nucleic acids after the phenol extraction and before loading them onto
cellulose sorbent improves the recovery and quality of dsRNA. This method can be easily
scaled up if more dsRNA is needed for a particular protocol; it is also applicable to small
RNA viruses.
Materials
1 STE buffer (see recipe)
SDS, DNase- and RNase-free (Bio-Rad)
Phenol equilibrated with 1 STE buffer (see recipe for buffer)
7.5 molar ammonium acetate
95% (v/v) ethanol
CF-11 cellulose (Whatman)
1 STE(see recipe)/15% ethanol
Nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
Mortar and pestle, chilled in ice
50-ml conical, polypropylene centrifuge tubes
5-ml polypropylene columns (Qiagen)
Additional reagents and equipment for performing agarose gel electrophoresis
(Voytas, 2000) and spectrophotometrically quantifying nucleic acids (Gallagher,
2004)
Extract dsRNA from virus particles
1. Grind 5 g of plant tissue in 10 ml of ice-cold 1 STE buffer using mortar and pestle
chilled in ice. Transfer the homogenate into a centrifuge tube.
2. Add 1.5 ml of 10% SDS and extract (by quickly inverting the tubes 10 times) twice
with equal volume (16.5 ml) of phenol equilibrated with 1 STE buffer. Centrifuge
5 min at 5000 g, room temperature. Transfer supernatants into a fresh tube after
each centrifugation.
3. Adjust the combined supernatants to 2.5 M ammonium acetate, using 7.5 M ammonium acetate. Pellet nucleic acids as described in Basic Protocol 1, steps 6 to 8.
Plant RNA
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Current Protocols in Microbiology
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4. Resuspend the pellet in 10 ml 1 STE buffer, add 2 ml of 95% ethanol, and mix
well.
5. Add 1 g of dry CF-11 cellulose powder to the tube, vortex well, and incubate 30 min
at room temperature, with occasional mixing.
6. Load the cellulose slurry into an empty 5-ml polypropylene column and allow to
drain.
7. Wash column with 1 STE/15% ethanol and elute the retained dsRNA with 4
aliquots (1 ml each) of 1 STE without ethanol. Combine eluates and pellet the
dsRNA as described in Basic Protocol 1, steps 6 to 8.
8. Resuspend pellet in 1 ml of nuclease-free water and clarify by microcentrifuging
1 min at maximum speed, room temperature, to remove residual cellulose. Transfer
the supernatant into fresh tube and precipitate the dsRNA.
Materials
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.6
Supplement 7
Primer
Sequence (5 to 3 )
C-dG-PCR
GGATCCGAGCTCGAATTCGC
dG-PCR
GCGAATTCGAGCTCGGATCC
dT-Sac
GGTGAGCTCTTTTTTTTTTTTTTTTTT
5-p20-Nde
GCCATATGACTAGCTCTGTCGAAC
C-BYV-BglII
TACGAGAGATCTCGTAAGCTTCAT
5-35S
TAGAGCTCAACATGGTGGAGCAC
3-35S-vir
GGATGGTTAAAAACTGCTCTCCAAATGAAATGAACTTCC
5-vir-35S
CATTTCATTTGGAGAGCAGTTTTTAACCATCCTTC
3BYV-Rib
TACCCGGGCCGTTTCGTCCTCACGGACTCATCAGAAGACAT
GTGAATCATGTCTTGACGGCCCTTATTTTTTCTTC
BYV-BstEII
GTGACGGAGGTGACCTACCTGCC
Plant RNA
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Supplement 7
5. Incubate 2 hr at 30 C.
6. Extract once with neutral phenol/chloroform/isoamyl alcohol mixture and once with
chlorophorm/isoamyl alcohol as described in Basic Protocol 1, steps 4 and 5.
7. Precipitate the product by adding 25 l of 7.5 M ammonium acetate and 200 l of
ethanol. Microcentrifuge 10 min at maximum speed, 4 C.
26 to 30 cycles
1 min
1 min
1 min
93 C
56 C
72 C
(denaturation)
(annealing)
(extension).
16F.1.8
Supplement 7
14. Digest the product with appropriate restriction endonucleases (see Bloch and Grossman, 1995).
Appropriate restriction endonucleases are those with restriction sites that were included in
the sequences of the PCR primers (in this case, BamHI present in dG-PCR and C-dG-PCR
primers, and NdeI present in 5-p20-Nde primer).
15. Clone the released fragments into a suitable plasmid cloning vector.
Any commonly available high-copy plasmid such as pGEM plasmid series (Promega) can
be used at this step since products of the described RT-PCR reaction are of relatively
small sizes and unlikely to cause any problem associated with the toxicity of viral cDNA
in E. coli.
16. Analyze the clones by restriction digestion and select those that release restriction
fragments of expected sizes or bigger. The bigger than expected products can result
if the previous work underestimated viral RNA length or mismapped the 3 terminus.
17. Select at least ten clones and sequence them to obtain a consensus sequence of a
putative 3 end of the viral cDNA.
ALTERNATE
PROTOCOL 1
This method is based on the ability of yeast poly(A) polymerase to add adenosine
residues to the 3 end of RNA. This enzyme works more efficiently than the E. coli poly(A)
polymerase. The resulting polyadenylated viral RNA can be used as a template for cDNA
synthesis using oligo-(dT) primer to initiate the reaction. A fragment that corresponds to
the 3 end of the viral RNA is then PCR amplified and cloned for sequencing and analysis.
Both ssRNA and dsRNA preparations can be used as a starting material, although the
authors have found that ssRNA yields higher amounts of the resulting PCR product.
This procedure is best suited for nonpolyadenylated viral RNAs. Presence of extensive
stretches of adenosine residues near the 3 end of RNA may lead to appearance of truncated RT-PCR products due to annealing of the oligo(dT) primer to those regions. This
method does not discriminate between naturally occurring and artificially added 3 terminal adenosine residues; correctly determining the identity of very terminal nucleotide(s)
calls for an independent approach such as the primer-ligation method (Basic Protocol 3).
Although primer ligation (Basic Protocol 3) and polyadenylation methods (this protocol)
each can produce reliable results, using both methods increases confidence in terminal
sequence determinations. In addition, depending on their structural features, RNAs of
different viruses may be better substrates for either T4 RNA ligase or yeast poly(A)
polymerase used in the alternative methods.
Materials
1 g/ml viral ssRNA (Basic Protocol 1)
5 yeast poly(A) polymerase buffer (USB)
10 mM ATP: prepared using 100 l 100 mM ATP (Fermentas) and 900 l sterile
nuclease-free water; store in 50-l aliquots up to a few months at 20 C
600 U/l yeast poly(A) polymerase (USB)
Neutral phenol (EMD Chemicals)/chloroform/isoamyl alcohol mixture (25:24:1)
Plant RNA
Viruses
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Current Protocols in Microbiology
Supplement 7
2. Incubate 20 min at 30 C.
One should not exceed the recommended incubation time for the poly(A) tailing reaction
because it can result in formation of needlessly long oligo(A) tails which may hamper
future sequencing of the cloned fragments.
16F.1.10
Supplement 7
1 min
30 sec
30 sec
30 sec
1 min
93 C
46 C
52 C
56 C
72 C
(denaturation)
(annealing)
(annealing)
(annealing)
(extension).
13. Analyze clones by restriction digestion and select those that release restriction fragments of expected sizes or bigger.
14. Select at least 10 clones and sequence them to obtain consensus sequence of a
putative 3 end of the viral cDNA.
To map a end of a capped, positive-strand viral RNA, the authors suggest using the
RLM-RACE kit (FirstChoice RLM-RACE kit, Ambion; or equivalent) with modifications
described below. This kit has been used in the authors laboratory for determining 5
ends of both viral and cellular capped RNAs and has proven to be a reliable tool.
Standard protocols for reverse transcription and PCR amplification used in the authors
BASIC
PROTOCOL 4
Plant RNA
Viruses
16F.1.11
Current Protocols in Microbiology
Supplement 7
laboratory yield superior results compared to the protocols provided by the supplier
of the kit. This maybe attributed to use of the SuperScript II RT enzyme, which lacks
RNaseH activity, rather than the M-MLV reverse transcriptase recommended by the
kits manufacturer. In addition, a blend of a regular Taq polymerase and Taq extender
PCR additive increased the amount of the PCR product. While an original RLM-RACE
protocol suggests two consecutive rounds of PCR using two sets of nested primers,
one round of PCR amplification yields enough PCR product for further cloning, due to
a substantial concentration of the viral RNA. (The original RLM-RACE method was
designed to amplify 5 ends of low-abundance mRNAs.)
Materials
1 g/l viral ssRNA (Basic Protocol 1)
Nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
FirstChoice RLM-RACE kit (Ambion) including:
10 calf intestine alkaline phosphatase buffer
calf intestine alkaline phosphatase
10 tobacco acid pyrophosphatase buffer
tobacco acid pyrophosphatase
5 RACE adapter
Random primer
10 RNA ligation buffer
T4 RNA ligase
5 RACE outer primer
Neutral phenol (EMD Chemicals)/chloroform/isoamyl alcohol mixture (25:24:1)
Chloroform/isoamyl alcohol mixture (24:1)
7.5 M ammonium acetate
Isopropanol
10 Taq extender buffer (Stratagene)
4 mM (each) dNTP mixture: prepared using 8 l of each of four 100 mM dNTPs
(Promega) and 168 l sterile RNase-free water; store up to a few months at
20 C
100 mM dithiothreitol (DTT)
200 U/l SuperScript II RT (Invitrogen)
Sequence-specific primer carrying a convenient restriction site at its 5 end (e.g.,
C-BYV-BglII; see Table 16F.1.1)
Taq extender PCR additive (Stratagene)
5 U/l Taq DNA Polymerase
High-copy plasmid cloning vector (e.g., pGEM plasmid series; Promega)
0.6-ml microcentrifuge tubes
PCR tubes
Thermal cycler (e.g., PTC-100 thermocycler; Bio-Rad)
Additional reagents and equipment for performing agarose gel electrophoresis
(Voytas, 2000), digestion of DNA with restriction endonucleases (Bloch and
Grossman, 1995), and cloning DNA (Sambrook and Russell, 2001)
Treat RNA with alkaline phosphatase
1. Mix in a 0.6-ml microcentrifuge tube:
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.12
Supplement 7
2. Incubate 1 hr at 37 C.
Incubations may be performed in the thermal cycler or other heating devices.
3. Extract once with neutral phenol/chloroform/isoamyl alcohol mixture and once with
chloroform/isoamyl alcohol.
4. Add 10 l of 7.5 M ammonium acetate and 30 l of isopropanol to precipitate the
RNA.
5. Microcentrifuge 10 min at maximum speed, 4 C.
Remove cap
6. Discard the supernatant and resuspend the RNA pellet in 7 l nuclease-free water.
7. Add 1 l of 10 tobacco acid pyrophosphatase buffer and 2 l tobacco acid pyrophosphatase.
8. Incubate 1 hr at 37 C.
Plant RNA
Viruses
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Current Protocols in Microbiology
Supplement 7
30 sec
1 min
2 min
93 C
52 C
72 C
(denaturation)
(annealing)
(extension).
BASIC
PROTOCOL 5
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.14
Supplement 7
Materials
10 Taq extender buffer (Stratagene)
4 mM (each) dNTP mixture: commercially prepared using 8 l of each of four 100
mM dNTPs (Promega) and 168 l sterile RNase-free water; store up to a few
months at 20 C
100 pmol/l oligonucleotide PCR primers, in sterile water: 5 sequence-specific
primer BYV-BstEII, ribozyme-encoding primer 3BYV-Rib (see Table 16F.1.1)
Taq extender PCR additive (Stratagene)
Taq DNA polymerase
Nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
Plasmid vector containing viral cDNA (see Basic Protocol 3, step 17 or Alternate
Protocol 1, step 14)
Restriction endonucleases (SmaI or XmaI and BstEII)
Modified plasmid vector (see Fig. 16F.1.1, steps 2 and 3): e.g., pCB301 mini-binary
vector provided by Dr. D. J. Oliver (see Xiang et al., 1999) with added NOS
terminator sequence and modified polylinker (see Strategic Planning)
PCR tubes
Thermal cycler (e.g., PTC-100 thermocycler; Bio-Rad)
Additional reagents and equipment for performing agarose gel electrophoresis
(Voytas, 2000), digestion of DNA with restriction endonucleases (Bloch and
Grossman, 1995), and cloning DNA (Sambrook and Russell, 2001)
1. In a PCR tube, mix the following:
Plant RNA
Viruses
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Current Protocols in Microbiology
Supplement 7
1 min
1 min
2 min
93 C
56 C
72 C
(denaturation)
(annealing)
(extension).
3. Digest the PCR product with SmaI (or XmaI) and BstEII and clone the fragments
into a modified plasmid vector pCB301 (Fig. 16F.1.1, Step 4).
BASIC
PROTOCOL 6
Figure 16F.1.3 Diagram showing consecutive steps of the PCR-assisted generation of the fusion
between 35S RNA polymerase promoter and 5 -terminal region of the viral cDNA. The dotted boxes
represent viral cDNA, while the cross-hatched arrows correspond to the 35S promoter.
16F.1.16
Supplement 7
C-BYV-BglII in Table 16F.1.1) and generate products with overlapping ends (3 -end of
35S promoter and 5 end of viral cDNA). As a template for the latter PCR, one can use
a clone carrying the correct sequence of BYV obtained in Alternate Protocol 2. This
PCR amplifies the 5 terminus of the BYV cDNA and adds a few anchoring nucleotides
complementary to the 3 end of CaMV 35S promoter. After the two first rounds of PCR,
the fragments are joined together by mixing products of both reactions and performing a
third PCR using primers complementary to the 5 and 3 ends of the desired full-length
product (primers 5-35S and C-BYV-BglII; see Fig. 16F.1.3). Overlap in 20 nucleotides
is usually sufficient for annealing of the two fragments during the annealing step of a
second PCR. Gel purification of the products of the first PCR reactions significantly
improves the yield of a full-length product.
BASIC
PROTOCOL 7
The procedures described up to this point generate a cassette that contains the 35S
promoter, 5 - and 3 -terminal regions of the viral cDNA, ribozyme, and NOS terminator
in a low-copy plasmid (Fig. 16F.1.1, step 5). This cassette is used for assembly of the fulllength clone by stepwise addition of large internal cDNA fragments using appropriate
restriction sites naturally present in viral cDNA. It is critical to ensure that viral cDNA
fragments represent a consensus sequence. Due to the inherent low fidelity of PCR,
amplification of large viral cDNAs results in accumulation of incidental mutations that
render the full-length clone nonviable with virtual certainty. Use of high-fidelity enzymes
(e.g., PfuTurbo DNA polymerase; Stratagene) may help alleviate the problem but in the
authors experience has never solved it entirely. One way to circumvent this problem is to
screen several independent PCR-derived clones, and reassemble a full-length clone from
mutation-free variants. Although this approach was successfully used for animal viruses
(Yount et al., 2003; Lee et al., 2005), it requires extensive sequencing and subcloning
efforts.
An alternative strategy is to use an RT-PCR-generated "draft" clone as a convenient
structural platform for replacing defective fragments with those obtained by conventional
cDNA synthesis. The protocol below details the generation of sequences by RT-PCR for
cloning into an appropriate low-copy plasmid (the draft clone). Basic Protocol 8 describes
conventional cDNA synthesis of viral sequences and their incorporation into the draft
clone, where they replace the RT-PCR-generated sequences.
High-quality viral RNA in microgram quantities allows RT-PCR amplification of cDNA
fragments up to 10 kb long. Generating fragments of 4 to 6 kb is recommended; they
provide an optimal balance between the size of the product (the longer the PCR product
the fewer cloning steps are needed) and the amount of the output DNA. A blend of Taq
polymerase and Taq extender PCR additive significantly improves the yield and quality
of the PCR product. It is also worth noting that the 10 Taq extender buffer supplied with
the enzyme already has an optimal concentration of Mg2+ ions for the enzyme mixture
and does not require additional fine tuning of the reaction conditions.
Materials
1 g/l viral ssRNA (Basic Protocol 1)
Nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
100 pmol/l oligonucleotide primers (see Table 16F.1.1): 3 and 5
sequence-specific primers
First-Strand synthesis buffer (Invitrogen)
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Viruses
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Current Protocols in Microbiology
Supplement 7
26 cycles
1 min
1 min
4 min
93 C
56 C
72 C
(denaturation)
(annealing)
(extension).
16F.1.18
Supplement 7
BASIC
PROTOCOL 8
This protocol describes conventional cDNA synthesis of viral sequences and their incorporation into the draft clone (see Basic Protocol 7), where they replace the RTPCR-generated sequences. It is recommended as an optimal way to generate a faithful,
biologically active, full-length cDNA clone of viral genome.
Even the traditional cDNA synthesis using E. coli DNA Polymerase I may result in a
population of fragments, some of which can carry unwanted mutations. This could be
due to the natural variability of the virus population as well as the lack of proofreading
activity of the M-MLV (e.g., SuperScript II) reverse transcriptase. However, this approach
provides a dramatic improvement in the quality of the cDNA compared to RT-PCR.
Sequencing of clones obtained by conventional cDNA cloning has revealed that virtually
all of them have a consensus sequence. More specifically, a 12,800-nucleotide fragment
of the BYV that was cloned and assembled using this method had only three nonlethal
mutations, compared to on average one lethal mutation introduced by RT-PCR per 1000
bases of a product.
The process of conventional synthesis and cloning of the cDNA fragments is no more
difficult than the RT-PCR approach. With the availability of viral RNA of sufficient
quality one can expect to see a discrete band after digestion of a cDNA with the restriction
enzymes that flank the desired region. Using microgram quantities of the input RNA,
the authors have been able to generate cDNAs of up to 7 kb in concentrations sufficient
for routine cloning manipulations. For practical purposes, using restriction endonuclease
sites that produce fragments of 4 kb is recommended.
Materials
Nuclease-free water (see APPENDIX 2A for DEPC-treated solutions or purchase
commercially)
1 g/l viral ssRNA (Basic Protocol 1)
Sequence-specific primer
First-Strand synthesis buffer (Invitrogen)
4 mM (each) dNTP mixture: prepared using 8 l of each of four 100 mM dNTPs
(Promega) and 168 l sterile RNase-free water; store up to a few months at
20 C
100 mM dithiothreitol DTT
200 U/l SuperScript II RT (Invitrogen)
10 E. coli DNA ligase buffer (Invitrogen)
3 U/l RNase H (Invitrogen)
10 U/l E. coli DNA ligase (Invitrogen)
10 U/l E. coli DNA polymerase (Invitrogen)
Neutral phenol (EMD Chemicals)/chloroform/isoamyl alcohol (25:24:1)
Chloroform/isoamyl alcohol (24:1)
7.5 M ammonium acetate
100% and 70% (v/v) ethanol
0.6-ml microcentrifuge tubes
Vacuum source
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Viruses
16F.1.19
Current Protocols in Microbiology
Supplement 7
Second-strand synthesis
5. Add the following to the tube from the step 4:
131 l nuclease-free water
16 l 10 E. coli DNA ligase buffer
6 l 4 mM dNTP mixture
0.7 l RNAse H
1.5 l E. coli DNA ligase
5 l E. coli DNA polymerase I.
6. Incubate 8 hr at 16 C.
7. Extract once with neutral phenol/chloroform/isoamyl alcohol mixture and once with
chloroform/isoamyl alcohol.
8. Precipitate by adding 100 l of 7.5 M ammonium acetate and 750 l of 100%
ethanol. Microcentrifuge 10 min at maximum speed, 4 C.
9. Discard the supernatant and wash the pellet with 70% ethanol. Dry under vacuum.
Cloning of Large
Positive-Strand
RNA Viruses
At this step the vector is retained and the released "draft" insert is discarded because
it originates from the error-prone RT-PCR and most likely carries multiple detrimental
mutations.
16F.1.20
Supplement 7
12. Clone the cDNA fragment released in step 10 into the plasmid released in step 11.
Repeat steps 10 to 12 as many times as necessary to replace all draft clone DNA
with conventionally synthesized cDNA.
As a result of this cloning step, the RT-PCR-derived cDNA region (which tends to contain
multiple mutations introduced by PCR amplification of large DNA stretches) is replaced
by a much more faithful cDNA obtained via conventional cDNA synthesis.
13. Sequence the entire cDNA to confirm that it does not carry any detrimental mutation.
14. Prepare a glycerol stock for each plasmid-carrying strain after the first passage in
a liquid culture by mixing 850 l of a liquid culture and 150 l of sterile 100%
glycerol in an microcentrifuge tube. Store indefinitely at 80 C.
BASIC
PROTOCOL 9
A. tumefaciens C58 GV2260 cultures carrying a full-size cDNA copy of a large virus may
grow very slowly in the medium containing three antibiotics (kanamycin, rifampicin and
streptomycin). If this rate of growth becomes impractical for research progression, one
may consider propagating the bacterial culture in the presence of 50 g/ml kanamycin
alone. However, this can lead to gradual disappearance of the Ti plasmid and loss of
virulence of the bacterial strain. Because of such potential events, it is imperative to
make a glycerol stock of each plasmid-carrying strain after the first passage in a liquid
culture (see Basic Protocol 8, step 12) to ensure preservation of the clone. Agrobacterium
cultures carrying large binary plasmids should never be allowed to overgrow because
this may lead to aberrant rearrangement of the plasmid. Each culture grows to its own
maximum density, after which it begins to lose its infectivity. As a rough guideline,
do not exceed a density of 1.0 at OD600 . For reliable and reproducible inoculation of
plants, always start the experiment from a frozen glycerol stock cultures and grow for
the shortest possible time required to attain the desired cell density. The efficiency of the
viral infection in agroinoculated leaves can be increased to a few thousand-fold by using
plant virus-derived suppressors of RNA silencing, such as BYV p21 or potyviral HC-Pro
(Chiba et al., 2006) as described below.
Materials
A. tumefaciens C58 GV2260 carrying viral cDNA cultured on agar plates (Support
Protocol 1)
LB medium (APPENDIX 4A) supplemented with 25 g/ml kanamycin, 12.5 g/ml
rifampicin and 25 g/ml streptomycin (see APPENDIX 4A)
LB medium (APPENDIX 4A) supplemented with 25 g/ml kanamycin, 12.5 g/ml
rifampicin and 25 g/ml streptomycin (see APPENDIX 4A), 10 mM MES, and 20
M acetosyringone (see recipes for stock solutions)
Agrobacterium induction solution (see recipe)
Plant RNA
Viruses
16F.1.21
Current Protocols in Microbiology
Supplement 7
2. Transfer the entire contents of the tube into 100 ml of LB medium containing the
same antibiotics, 10 mM MES, and 20 M acetosyringone. Incubate overnight at
28 C, with shaking at 250 rpm.
3. Centrifuge culture 10 min at 5000 g, room temperature.
4. Decant the supernatant and resuspend the pellet in 10 ml Agrobacterium induction
solution.
5. Adjust the culture to an OD600 of 1 with Agrobacterium induction solution. Incubate
2 hr at room temperature.
6. Infiltrate 3 to 4 week old (6 to 8 leaf stage) N. benthamiana plants with a 3-ml syringe
without a needle as follows:
a. Fill the syringe with 3 ml of the bacterial culture from step 4.
b. Press the syringe gently against the lower surface of the leaf, placing the finger of
the other hand underneath the infiltration point.
c. Slowly inject the bacterial suspension into the leaf blade.
The leaf blade darkens gradually in the area where the liquid fills intercellular space.
Wearing lab coat, gloves, and face shield is strongly recommended to avoid contamination
of the skin and clothing. Infiltrate the maximum number of young leaves.
The first systemic symptoms of BYV infection can be detected 14 to 24 days after inoculation. These symptoms include leaf vein clearing and, later, deformation and curling of
the leaves. In the next week or two the plants wilt and die.
SUPPORT
PROTOCOL 1
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.22
Supplement 7
1. Remove a tube with the electrocompetent A. tumefaciens C58 GV2260 cells from
80 C freezer and thaw on ice.
2. Add 1 l of a binary vector carrying viral cDNA to the tube and mix by gently
pipetting.
3. Transfer the suspension into an ice-cold, standard, 2 mm electrode gap, electroporation cuvette.
4. Set parameters of pulse as follows: 2,000 V, 500 Ohm, 50 F.
The authors use the BTX ECM 630 electroporator.
SUPPORT
PROTOCOL 2
5. Transfer cells into a sterile centrifuge bottle and centrifuge 10 min at 5000 g, 4 C.
Carry out all procedures on ice from this point on.
6. Decant the supernatant and carefully resuspend the pellet in 100 ml of ice-cold,
sterile distilled water. Centrifuge 10 min at 5000 g, 4 C.
Plant RNA
Viruses
16F.1.23
Current Protocols in Microbiology
Supplement 7
Acetosyringone, 150 mM
Dissolve 300 mg of acetosyringone (4 -hydroxy-3 ,5 -dimethoxyacetophenone) in
10 ml DMSO. Store up to 1 month at 20 C in 1 ml aliquots.
STE buffer, 1
50 mM Tris
100 mM NaCl
1 mM Na2 EDTA
Adjust pH to 6.8 with HCl
Autoclave
Store up to 1 month at 4 C
COMMENTARY
Background Information
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.24
Supplement 7
Anticipated Results
Starting with 0.2 to 0.5 mg of the highquality virion RNA and 0.05 to 0.1 mg of the
viral dsRNA, it should be possible to identify terminal nucleotide sequences of the viral
genome and generate enough RT/PCR products and cDNA fragments to engineer biologically active cDNA clone of a closterovirus.
However, this task could be very challenging for viruses that accumulate to low levels
and/or are restricted to inconvenient hosts such
as woody plants.
Time Considerations
A realistic estimate for the completion of
infectious cDNA clone of a closterovirus is one
year. Availability of a consensus nucleotide sequence of the entire viral genome and presence of the useful restriction endonuclease
sites could facilitate the cloning. On the other
hand, lack of a convenient experimental host
or incidental toxicity of viral cDNA could play
a negative role and slow the progress down.
Literature Cited
Almazan, F., Gonzalez, J.M., Penzes, Z., Izeta,
A., Calvo, E., Plana-Duran, J., and Enjuanes,
L. 2000. Engineering the largest RNA virus
genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. U.S.A. 97:55165521.
Ayllon, M.A., Gowda, S., Satyanarayana, T., Karasev, A.V., Adkins, S., Mawassi, M., Guerri, J.,
Moreno, P., and Dawson, W.O. 2003. Effects of
Plant RNA
Viruses
16F.1.25
Current Protocols in Microbiology
Supplement 7
Key References
Peremyslovet al., 1998. See above.
This paper describes generation of the fully biologically active cDNA clone of BYV and its tagging
via insertion of the reporter gene encoding green
fluorescent protein.
Prokhnevsky et al., 2002. See above.
This paper describes generation of the binary vector designed to launch infectious viral genome via
agroinfection that dramatically improved systemic
infectivity of the cDNA clone.
Chiba et al., 2006. et al., 2006. See above.
This paper describes the use of viral RNA silencing
suppressors to drastically increase the infectivity of
virus launched by agroinfection.
Cloning of Large
Positive-Strand
RNA Viruses
16F.1.26
Supplement 7
UNIT 16F.3
16F.3.1
Supplement 4
Materials
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
16F.3.2
Supplement 4
Polymerase
Commentsa
Supplier
Vent polymerase
Pfu polymerase
Promega
Tli polymerase
Promega
Pfu polymerase
Stratagene
Stratagene
rTth polymerase XL
Applied Biosystems
Pwo polymerase
Roche
ExTaq polymerase
Panvera
LA Taq polymerase
Panvera
Novagen
KOD XL polymerase
Novagen
Invitrogen
Invitrogen
Elongase Amplification
System
Complicated to use
Invitrogen
Pfu polymerase
Fermentas
a Comments indicate the authors experience with selected polymerases, under specific conditions. In some cases the
authors did not attempt to optimize the reactions, thus the properties indicated may not apply in all conditions.
Plant RNA
Viruses
16F.3.3
Current Protocols in Microbiology
Supplement 4
2. Incubate the sample 5 min at 65 C, then transfer the reaction tube quickly onto ice.
This step reduces long-distance secondary structure that could interfere with the reverse
transcription reaction.
12 min at 37 C
12 min at 42 C
12 min at 47 C
12 min at 51 C
12 min at 55 C.
The initial low temperature favors annealing of the downstream primer to the RNA template. As the primer is extended by the reverse transcriptase the thermal stability of the
RNA/ssDNA duplex rises. Then the incubation temperature can be raised to levels which
melt secondary structure without disrupting binding of the nascent ssDNA strand with the
RNA template. If a primer with a high Tm is used, it may be possible to incubate immediately at higher temperatures. Alternatively, an initial 10 min incubation at 37 C followed
by incubations at higher temperatures for the remainder of the reaction may work well.
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
16F.3.4
Supplement 4
1 cycle:
1 min
1 min
6 min
10 min
94 C
52 C
72 C
72 C
(denaturation)
(annealing)
(extension)
(final extension).
The extension time is determined by the length of the fragment being amplified. The rule
of thumb is to allow 1 min/kilobase. Additionally the annealing temperature may need to
be raised if there is nonspecific product generated, or it may need to be lowered if using
oligonucleotide primers with a low Tm . An extension temperature of 72 C is standard
for most thermostable DNA polymerases. Although the product literature for Platinum
Pfx DNA polymerase calls for extension at 68 C, this laboratory has found 72 C to
be adequate. See Kramer and Coen (2001), for complete optimization guidelines and
resources for calculating the Tm .
16F.3.5
Current Protocols in Microbiology
Supplement 4
Here the protocol makes use of the TOPO cloning technology developed by Invitrogen.
Their Zero Blunt TOPO PCR cloning system allows for very rapid cloning of blunt-ended
PCR products. Other TOPO systems allow for T/A cloning or directional cloning. The
salt solution described above is a proprietary solution provided with the cloning kit.
The vector comes precut with topoisomerase I covalently bound to the 3 end of each
DNA strand. The activity of this enzyme is used to efficiently clone the PCR product.
Blunt-end fragments clone with the highest efficiency. Therefore, the Zero Blunt TOPO
PCR cloning system containing the vector pCR-Blunt II-TOPO is preferred in this
laboratory.
A
purify viral RNA
anneal downstream
primer to anchor oligo
reverse transcribe to
create first strand
anneal upstream
primer to first strand
extend primer to
create second strand
Key:
RNA strand
DNA strand
anchor
downstream primer
upstream primer
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
Figure 16F.3.1 Use of ligation-anchored PCR (LA-PCR) to determine the sequence of the 3
end region of an unknown RNA virus (A).
16F.3.6
Supplement 4
13. Transfer 2 l of the TOPO reaction to a vial of One Shot TOP10 Chemically
Competent E. coli cells. Incubate 5 to 30 min on ice.
14. Heat shock 30 sec at 42 C, then place back on ice.
15. Add 250 l room temperature SOC medium to a vial of transformed cells. Cap and
incubate horizontally with shaking at 37 C for 1 hr.
16. Plate on LB containing selective antibiotic and incubate overnight at 37 C.
17. Pick 12 to 24 colonies for screening and analysis.
B
purify viral RNA
anneal downstream
primer to viral genome
reverse transcribe to
create first strand
ligate anchor to 3
end of first strand
anneal upstream
primer to first strand
extend primer to
create second strand
Key:
RNA strand
DNA strand
anchor
downstream primer
upstream primer
Figure 16F.3.1 (continued) Use of LA-PCR to determine the 5 end region sequence (B).
Plant RNA
Viruses
16F.3.7
Current Protocols in Microbiology
Supplement 4
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
16F.3.8
Supplement 4
4. Incubate the mixture 5 min at 65 C. Then transfer the reaction tube quickly onto
ice.
5. Add the following to the reaction tube (20 l final volume):
12 min at 37 C
12 min at 42 C
12 min at 47 C
12 min at 51 C
12 min at 55 C.
7. Heat to 85 C to terminate the reaction.
8. Store first-strand reaction at 80 C.
1 cycle:
1 min
1 min
6 min
10 min
94 C
52 C
72 C
72 C
(denaturation)
(annealing)
(extension)
(final extension).
Plant RNA
Viruses
16F.3.9
Current Protocols in Microbiology
Supplement 4
11. Check PCR products by electrophoresis on a 0.8% agarose gel (Voytas, 2000).
Cloning of the 3 end fragment is done just as in the Basic Protocol. Bear in mind that
the goal of this Support Protocol is to determine the 3 end sequence of an unknown
genome. Once the sequence of the 3 terminal region is known, a downstream primer
complementary to this region needs to be designed as described in the Basic Protocol.
Then the full-length genome can be amplified using this downstream primer.
SUPPORT
PROTOCOL 2
12 min at 37 C
12 min at 42 C
12 min at 47 C
12 min at 51 C
12 min at 55 C.
5. Add 1 l of 1.5 M sodium hydroxide. Mix and place in a boiling water bath for
5 min.
Treatment with NaOH hydrolyzes the RNA, leaving the ssDNA first-strand free for amplification without interference from the original RNA template. An alternative procedure
is to treat with the RNA/DNA hybrid with RNase H. However, this leaves RNA fragments
with phosphorylated 3 ends that will compete with the ssDNA for ligation to the anchor
oligomer.
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
6. Increase volume to 100 l with nuclease-free water. Exchange the buffer using a
YM-30 column. Resuspend the ssDNA first strand in 10 l nuclease-free water.
16F.3.10
Supplement 4
1 cycle:
1 min
1 min
6 min
10 min
94 C
52 C
72 C
72 C
(denaturation)
(annealing)
(extension)
(final extension).
11. Check PCR products by electrophoresis on a 0.8% agarose gel (Voytas, 2000).
Cloning of the 5 end fragment is done just as in the Basic Protocol. Remember that the
goal of this Support Protocol is to determine the 5 end sequence of an unknown genome.
Once the sequence of the 5 terminal region is known, a upstream primer homologous to
this region needs to be designed as described in the Basic Protocol. Then the full-length
genome can be amplified using this upstream primer.
SUPPORT
PROTOCOL 3
When dealing with difficult or unknown templates, newly designed primer pairs, or
variable first-strand concentrations, it is critical to use the optimal annealing temperature
during PCR. Advances in thermocycler technology allow one to compare twelve different
annealing temperatures at one time using a single gradient PCR block. The authors
laboratory uses the DNA Engine with gradient block from Bio-Rad. Most suppliers of
thermocyclers sell a unit with this capability. Application of this simple protocol can
save weeks of time when adjustment of the annealing parameters is the bottleneck in the
cloning pipeline.
Plant RNA
Viruses
16F.3.11
Current Protocols in Microbiology
Supplement 4
65 l 10 PCR buffer
13 l 50 mM MgSO4
78 l 2.5 mM dNTPs (2.5 mM each of dATP, dCTP, dGTP, dTTP)
13 l (325 pmol) upstream primer
13 l (325 pmol) downstream primer
13 l ssDNA first-strand template
13 l (32.5 U) Platinum Pfx DNA polymerase
442 l sterile water.
2. Dispense 50-l aliquots into every tube of a 12-tube strip (0.2-ml tube volume)
3. PCR amplify as follows:
30 cycles:
1 cycle:
1 min
1 min
6 min
10 min
94 C
40 to 60 C gradient
72 C
72 C
(denaturation)
(annealing)
(extension)
(final extension).
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
2. Place the gel on a UV light box and use a clean razor blade to remove a gel plug
containing the desired band.
Keep the UV light on for minimal period of time possible, to limit UV radiation damage
to the DNA and the user.
16F.3.12
Supplement 4
4. Add 1 vol buffer-saturated phenol. Vortex briefly and microcentrifuge for 2 min at
maximum speed, 4 C.
5. Remove the upper aqueous phase and transfer to a new tube.
6. Add 1 vol phenol/chloroform/isoamyl alcohol (25:24:1). Vortex briefly and centrifuge for 2 min at maximum speed, 4 C.
7. Remove the upper aqueous phase and transfer to a new tube.
8. Add 1/10 vol of 3 M sodium acetate (pH 5.5) and 2 vol of 95% ethanol at 20 C.
Mix and place at 80 C for 1 hr.
9. Microcentrifuge 30 min at maximum speed, 4 C.
10. Wash the pellet with 1 ml of 70% ethanol at 20 C. Microcentrifuge 10 min at
maximum speed, 4 C.
11. Dry the pellet under vacuum for 5 min.
12. Dissolve the pellet in 15 l nuclease-free water.
COMMENTARY
Background Information
The study of RNA viruses was revolutionized in the 1980s by two major methodological breakthroughs. The first was construction of full-length infectious clones of viral
genomes (Taniguchi et al., 1978; Racaniello
and Baltimore, 1981; Ahlquist et al., 1987).
This, combined with rapid developments in
site-directed mutagenesis, allowed viral reverse genetics to become the norm. The second major breakthrough was the polymerase
chain reaction. This allows very simple sitedirected mutagenesis and the ability to work
with extremely small amounts of starting genetic material (Kowalik et al., 1990). Perhaps
the most extreme example of the application
of PCR and in vitro transcription to character-
Plant RNA
Viruses
16F.3.13
Current Protocols in Microbiology
Supplement 4
in the quasispecies may contain sets of polymorphisms that must occur together on the
same genome to allow replication. Hence, assembly of a clone from separately cloned fragments derived from different viable genomes
may not produce an infectious transcript.
Nonpolyadenylated RNAs require special
attention for cloning of the complete 3 end.
For host mRNAs and many viral genomes
an oligo(dT) primer complementary to the
poly(A) tail will suffice for first-strand synthesis. However, unlike mRNAs, the genomes
of many positive-strand RNA viruses are
not polyadenylated. Examples include the
bromoviruses, tobamoviruses, tombusviruses,
luteoviruses, cucumoviruses, and alfamoviruses of plants; nodaviruses of insects and
fish; and the pestiviruses, flaviviruses, and hepaciviruses of mammals. The methods for fulllength RT-PCR cloning detailed in this unit
have been applied to produce seven independent full-length clones of cereal yellow dwarf
virus-RPV (CYDV-RPV) in about one week.
The clones differed in infectivity. Some were
not infectious. This variation in infectivity reflects the slight variations in sequence between
each RNA, even though all were cloned from
a highly homogeneous virus isolate. Extensive previous attempts to assemble separately
cloned fragments into an infectious clone were
unsuccessful. Thus, this unit should be valuable to researchers attempting to construct one
or many full-length infectious clones of any
RNA virus whether or not it harbors a poly(A)
tail.
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
RNA purification
RNA purification methods vary from system to system and from virus to virus (see
Section 16E). To obtain full-length clones it
is essential to start with high-quality, fulllength RNA. A number of standard precautions should be followed. Wear gloves to avoid
contaminating the virus sample with fingertip
nucleases. Use nuclease-free pipet tips and microcentrifuge tubes. Prepare reagents in baked
glassware using nuclease-free water (Ambion
or Sigma) and chemicals dedicated to RNA
work. Ideally, use freshly extracted viral RNA
which has been stored at 80 C and has
gone through a minimum of freeze/thaw cycles. Before proceeding to subsequent steps,
visually check the viral RNA by agarose gel
electrophoresis. For electrophoresis, the E-gel
precast agarose gel system (Invitrogen) works
well for quick assessment of viral RNA quality. The E-gel system eliminates the need for
laborious treatments of gel rigs and running
buffers with diethylpyrocarbonate (DEPC) or
other RNase deactivators. Presence of ribonuclease in a gel system can degrade RNA in the
gel leading to the erroneous conclusion that
the whole batch is degraded.
Design of primer pairs
If the 5 and 3 terminal sequences of an
RNA virus are known, then an effective pair of
upstream and downstream primers can be designed. The downstream primer will be used
first and will come into direct contact with
the RNA genome of the virus. Thus, ensure
that this primer is nuclease free. Many commercial suppliers of oligomers offer RNasefree synthesis. At minimum, dissolve the
newly synthesized primer in nuclease-free water (Ambion) and handle it as if working with
RNA. The downstream primer must be complementary to the 3 end region of the viral
RNA genome. Design the 5 prime extension
of the primer with a unique restriction enzyme
site that incorporates the terminal bases of the
genome. Ideally, create a blunt-ended restriction site which is not found in the viral genome
and which will leave a perfect viral 3 end when
the eventual full-length clone is cut at this site.
For example, the genomes of barley yellow
dwarf virus and the Tombusviridae terminate
in CCC. By using the 5 prime extension of
the downstream primer to add GGG, a unique
SmaI site is created. When the full-length clone
is linearized with SmaI, the resulting transcript
terminates with a perfect CCC 3 end. If it is
not possible to create a unique restriction site
16F.3.14
Supplement 4
Plant RNA
Viruses
16F.3.15
Current Protocols in Microbiology
Supplement 4
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
PCR amplification
Successful PCR amplification of any DNA
molecule requires several variables to work
in harmony. Annealing temperature, extension
time, primer concentration, primer Tm , magnesium concentration, and polymerase quality all need to be optimal. A number of reviews address general optimization of PCR
conditions (Kramer and Coen, 2001). Here,
the matters found most critical to a successful reverse transcriptase PCR (RT-PCR) or
a ligation-anchored PCR (LA-RT-PCR) are
addressed.
DNA polymerase: The polymerase must
have a proofreading function in order to avoid
introducing base changes that could render
the full-length clone noninfectious. The DNA
polymerase must also be processive enough
to amplify the entire genome. By amplifying
the entire genome at once, the time-consuming
process of piecing together fragments of the
genome from subclones is avoided. This also
obviates the possibility of introducing lethal
mutations at subclone junctions. A trade-off
comes at this point, however. In general, the
commercially available polymerases claiming
16F.3.16
Supplement 4
kits (Qiagen) and then used immediately without further preparation of the ends. Nonproofreading DNA polymerases add a single dATP
residue to the 3 end of each strand of the PCR
product. The proofreading DNA polymerase
used in this protocol polishes the ends by
removing the overhanging adenosine residue.
Thus, a population of cDNA molecules is created during PCR where the majority have true
blunt ends, ideally compatible with the Zero
Blunt TOPO vectors. The topoisomerase is
very efficient: a 6-kb fragment, representing
a full-length viral genome, which was extremely difficult to clone using traditional ligation methods, was readily cloned in this laboratory using the TOPO system on the first try.
The PCR amplified genome was inserted into
a TOPO vector and transformed into E. coli
within 3 hr.
Anticipated Results
Time Considerations
The Basic Protocol, resulting in full-length
clones ready for screening, can be executed in
1 to 2 days. First-strand synthesis requires 1
to 2 hr. Amplification takes 2 to 5 hr, depending on the size of the viral genome. TOPOmediated insertion of the amplified fragment
into the vector takes 1 to 4 hr depending on
if gel purification is needed. If LA-PCR is required to determine the ends sequences of the
genome, the 5 and 3 ends should be cloned
simultaneously. Clones of the end fragments
can be obtained in 3 days.
Acknowledgements
This work was supported by USDA National Research Initiative grant no. 200435600-14227, the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa,
and by Hatch Act and State of Iowa Funds.
Literature Cited
Ahlquist, P., French, R., and Bujarski, J.J. 1987.
Molecular studies of brome mosaic virus using
infectious transcripts from cloned cDNA. Adv.
Virus Res. 32:215-242.
Plant RNA
Viruses
16F.3.17
Current Protocols in Microbiology
Supplement 4
Boyer, J.C. and Haenni, A.L. 1994. Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415-426.
Bujarski, J.J. and Miller, W.A. 1992. Use of in vitro
transcription to study gene expression and replication of spherical, positive sense RNA plant
viruses. CRC Press, Boca Raton, Fla.
Carninci, P., Nishiyama, Y., Westover, A., Itoh,
M., Nagaoka, S., Sasaki, N., Okazaki, Y.,
Muramatsu, M., and Hayashizaki, Y. 1998.
Thermostabilization and thermoactivation of
thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA.
Proc. Natl. Acad. Sci. U.S.A. 952:520-524.
Chester, N. and Marshak, D.R. 1993. Dimethyl
sulfoxide-mediated primer Tm reduction: A
method for analyzing the role of renaturation
temperature in the polymerase chain reaction.
Anal. Biochem. 209:284-290.
Das, M., Harvey, I., Chu, L.L., Sinha, M., and
Pelletier, J. 2001. Full-length cDNAs: More than
just reaching the ends. Physiol. Genomics 6:5780.
Domingo, E. 2003. Quasispecies and the development of new antiviral strategies. Prog. Drug Res.
60:133-158.
Troutt,
A.B.,
McHeyzer-Williams,
M.G.,
Pulendran, B., and Nossal, G.J. 1992.
Ligation-anchored PCR: A simple amplification technique with single-sided specificity.
Proc. Natl. Acad. Sci. U.S.A. 89:9823-9825.
Hawkins, P.R., Jin, P., and Fu, G.K. 2003. Fulllength cDNA synthesis for long-distance RTPCR of large mRNA transcripts. Biotechniques
34:768-770, 722-773.
Kowalik, T.F., Yang, Y.Y., and Li, J.K. 1990. Molecular cloning and comparative sequence analyses
of bluetongue virus S1 segments by selective
synthesis of specific full-length DNA copies of
dsRNA genes. Virology 177:820-823.
Kramer, M.E. and Coen, D.M. 2001. Enzymatic
amplification of DNA by PCR: Standard procedures and optimization. In Current Protocols
in Molecular Biology (F.M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J.G. Seidman, J.A.
Rapid Full-Length
Cloning of Nonpolyadenylated
RNA Virus
Genomes
16F.3.18
Supplement 4
UNIT 16G.1
Robert A. Owens1
1
ABSTRACT
Two-dimensional PAGE involving an initial fractionation under nondenaturing conditions
followed by a second electrophoresis under denaturing conditions provides a powerful
means to detect viroids and other small circular RNAs. This unit describes a method
known as R(eturn) PAGE in which denaturation is achieved by simultaneously raising
the temperature and lowering the ionic strength during the second electrophoresis. Under
denaturing conditions, circular RNAs migrate more slowly than their corresponding
linear forms. Following fractionation, RNAs are visualized by staining with ethidium
bromide, SYBR Gold, or silver nitrate. Unlike nucleic acid hybridization or RT-PCR,
viroid identification by R-PAGE requires no nucleotide sequence information. Curr.
C 2008 by John Wiley & Sons, Inc.
Protoc. Microbiol. 10:16G.1.1-16G.1.9.
Keywords: viroids r polyacrylamide gel electrophoresis (PAGE) r circular RNAs r
(R)eturn PAGE
INTRODUCTION
Viroids are the smallest autonomously replicating pathogens yet described. These small,
unencapsidated, circular RNA molecules lack mRNA activity yet are able to induce a wide
range of disease symptoms in susceptible plant hosts. Although molecular hybridization
or polymerase chain reaction (PCR) techniques have now largely replaced polyacrylamide gel electrophoresis (PAGE) for routine detection of well characterized viroids,
electrophoresis remains an essential tool for the detection of viroid-like molecules of
unknown sequence. Unlike these other techniques, PAGE requires no nucleotide sequence
information.
The migration rate of a macromolecule during PAGE is determined by three factors:
charge, size, and shape. Two-dimensional PAGE (initial fractionation under nondenaturing conditions followed by electrophoresis under denaturing conditions) provides a
powerful means to detect viroids and other small circular RNAs because they migrate
more slowly than linear RNAs under denaturing conditions. This unit describes a method
known as R(eturn) PAGE in which denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength during the second electrophoresis.
Following fractionation, RNAs are visualized by staining with ethidium bromide, SYBR
Gold, or silver nitrate.
STRATEGIC PLANNING
Although viroids have been isolated from a wide range of plant species, no animal
or human infections have been reported. Thus, Biosafety Level 1 (BSL-1) precautions
are adequate for most laboratory manipulations. In the United States, laboratory and
greenhouse studies require a permit from the Animal and Plant Health Inspection Service
(PPQ Form 526). Experimental protocols involving viroids should also be reviewed by
the local Institutional Biosafety Committee (IBC).
Plant RNA
Viruses
Current Protocols in Microbiology 16G.1.1-16G.1.9, August 2008
Published online August 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16g01s10
C 2008 John Wiley & Sons, Inc.
Copyright
16G.1.1
Supplement 10
BASIC
PROTOCOL 1
RNA ISOLATION
No single RNA extraction protocol works well with every possible viroid/host combination. As described by Hanold et al. (2003), the challenge is to prepare undegraded viroid
RNA in concentrations sufficient to be visible on the gel, yet containing a sufficiently low
level of impurities so as not to interfere with R-PAGE. Viroid concentrations are usually
highest in young, symptomatic tissue. Small amounts of leaf tissue (0.1 to 1.0 g) can be
conveniently powdered in liquid N2 using a mortar and pestle and extracted with TRIzol
reagent as described by the manufacturer (Invitrogen). Total RNA yields usually range
between 250 to 1000 g/g tissue fresh weight. Ribosomal and other large single-stranded
RNAs are insoluble in 2 M LiCl; thus, LiCl precipitation is often used to enrich an RNA
preparation for viroids prior to R-PAGE.
Materials
White quartz sand (50 to 70 mesh; Sigma, cat. no. 14808-60-7 or equivalent)
Liquid nitrogen
Plant tissue (fresh or frozen)
TRIzol reagent (Invitrogen)
Chloroform (molecular-biology-grade)
Isopropanol (molecular-biology-grade)
70% (v/v) ethanol/0.1 M sodium acetate (see recipe)
RNase-free H2 O
4 M LiCl
Absolute ethanol
Mortar and pestle (autoclaved)
Spectrophotometer
Additional reagents and equipment for measuring A260 using a spectrophotometer
(Gallagher and Desjardins, 2006)
1. Add an appropriate amount of sand (0.2 g/g tissue) to an autoclaved mortar, carefully
fill the mortar with liquid N2 , add the plant tissue (fresh or frozen), and grind until
the tissue has been reduced to a fine powder and the liquid N2 has evaporated.
Although not essential, addition of sand facilitates the grinding process and helps ensure
that the tissue is finely powdered.
2. Add the appropriate amount of TRIzol reagent (1 ml/100 mg tissue), quickly mix
the reagent with the powdered tissue to form a thick paste, and allow to thaw.
CAUTION: Use gloves and eye protection when working with TRIzol reagent. Avoid
contact with skin or clothing and use in a chemical fume hood. Avoid breathing vapor.
4. Wash RNA pellets with 70% (v/v) ethanol/0.1 M sodium acetate, dissolve in RNasefree H2 0, and measure A260 using a spectrophotometer (see Gallagher and Desjardins,
2006).
16G.1.2
Supplement 10
5. Assuming that an A260 of 1.0 indicates 40 g/ml single-stranded RNA, adjust RNA
to a final concentration of 1 to 2 mg/ml, add an equal volume of 4 M LiCl, and
incubate overnight at 0 C to 4 C.
IMPORTANT NOTE: Chill RNA on ice before starting LiCl fractionation and add the
4 M LiCl solution dropwise with constant mixing. A flocculent precipitate containing
primarily rRNA and large mRNA should appear only SLOWLY (after 1 to 2 hr). Too rapid
precipitation may nonspecifically trap viroid RNA.
R-PAGE ANALYSIS
As described by Schumacher et al. (1986) and Singh and Boucher (1987), R-PAGE
was originally performed using a precursor to the current Bio-Rad PROTEAN II xi
vertical electrophoresis cell and required large volumes of running buffer. The procedure
described below has been adapted for use in a minigel format and requires only 300 ml
of buffer.
BASIC
PROTOCOL 2
Materials
GlassPlus or equivalent glass cleaner
95% ethanol
Gel solution (see Table 16G.1.1)
1 TBE
Isolated RNA (Basic Protocol 1)
6 gel loading buffer (see recipe)
0.5 g/ml ethidium bromide in water
Compatible plates, spacers, and combs designed for Bio-Rads Mini PROTEAN 3
apparatus (available from Bio-Rad Laboratories)
JGC-3 Joey Gel Casting System (Owl Separation Systems), optional
P8DS Emperor Penguin Vertical Electrophoresis System (Owl Separation Systems)
Micropipet equipped with a drawn-out plastic tips
Haake Model DC10-P5/U circulating baths (available from Fisher Scientific) or
any circulating bath capable of maintaining temperatures between 25 and
100 C is also acceptable
Cast the gel
1. Clean glass plates as necessary with GlassPlus or equivalent glass cleaner followed
by 95% ethanol.
2. Assemble glass plates and spacers.
Although not required, use of the Mini PROTEAN 3 casting frame and stand (Bio-Rad
Laboratories) or the JGC-3 Joey Gel Casting System (Owl Separation Systems) eliminates
many potential problems with leakage during polymerization.
For many purposes, a 10-well, 0.75- or 1.0-mm comb, and spacers work well.
Plant RNA
Viruses
16G.1.3
Current Protocols in Microbiology
Supplement 10
Table 16G.1.1 Volumes of Reagents Used to Cast Polyacrylamide Gels for R-PAGE
Component
40% (w/v) acrylamide stock (see recipe)
5%
7.5%
10%
3.1 ml
4.7 ml
6.25 ml
2.5 ml
2.5 ml
2.5 ml
H2 O
19.3 ml
17.7 ml
16.1 ml
TEMEDa
12.5 l
12.5 l
12.5 l
125 l
125 l
125 l
Total
25 ml
25 ml
25 ml
3. Calculate the volume of gel solution required and prepare according to the recipes
given in Table 16G.1.1, taking care to add the ammonium persulfate last. Quickly
mix.
CAUTION: Acrylamide is a neurotoxin. See UNIT 1A.3 for guidelines on handling and
contact your local safety officer concerning guidelines for disposal. It is not necessary to
deaerate the acrylamide solution before adding ammonium persulfate. Deaeration may
lead to incomplete polymerization around the comb.
4. Tilt the glass plate-spacer assembly slightly, fill with gel solution, and insert the comb.
Avoid trapping air bubbles under the teeth of the comb or against the glass plates.
5. Allow polymerization to proceed for 30 to 60 min at room temperature.
When polymerization is complete, a refractive line will appear 1 to 2 mm below the
surface of the acrylamide between the teeth of the comb.
6. Carefully remove the comb, first wetting it with a small amount of 1 TBE to avoid
disturbing the wells.
If the gel is not to be used immediately, place a laboratory tissue soaked in 1 TBE over
the comb, wrap the entire gel cassette in plastic wrap, and store at 4 C.
10. Connect electrodes to a power supply, set the voltage at 100 V, and run until the blue
xylene cyanol FF marker dye reaches the bottom of the gel.
Identification of
Viroids by Gel
Electrophoresis
11. Turn off power and disconnect leads from power supply.
16G.1.4
Supplement 10
14. Reconnect electrodes making sure to REVERSE THE POLARITY and wait 5 min
for RNA denaturation to occur before resuming electrophoresis at 100 V.
During electrophoresis under denaturing conditions, RNAs migrate from the bottom of
the gel toward the top. Thus, it is essential to REVERSE THE LEADS, connecting red (+)
to black () and black () to red (+).
15. Turn off power and disconnect the leads when the xylene cyanol marker dye reaches
the top of the gel.
Running time should be 45 min (constant voltage).
16. Discard upper and lower reservoir buffer and remove gel assembly.
BASIC
PROTOCOL 3
Materials
Electrophoresed gel (Basic Protocol 2)
Staining solution (0.5 g/ml ethidium bromide in water)
Scalpel or single-edged razor blade
Plastic or glass dish for staining
Platform shaker
Plastic wrap
UV transilluminator
Additional reagents and equipment for photographing the gel (Voytas, 2000)
1. Lay the gel assembly on the benchtop, remove the spacers (if necessary), and carefully
separate the glass plates using a scalpel or single-edged razor blade.
In most cases, the gel remains attached to the lower plate.
2. Transfer the gel to an appropriate staining solution, using either a plastic (ethidium
bromide or SYBR Gold) or glass (silver stain) dish.
Use only dishes with smooth bottoms to avoid scratching the gel.
3. For ethidium bromide, use sufficient staining solution (0.5 g/ml ethidium bromide
in water) to just float the gel and gently agitate for 15 to 30 min on a platform shaker.
CAUTION: Ethidium bromide is a potent mutagen; see UNIT 1A.3 for guidelines on handling and disposal.
Plant RNA
Viruses
16G.1.5
Current Protocols in Microbiology
Supplement 10
COMMENTARY
Background Information
Identification of
Viroids by Gel
Electrophoresis
The use of denaturing PAGE to identify viroids exploits the unusual circular nature of
these small (246 to 400 nucleotides), highly
structured RNA molecules. Under denaturing
conditions, circular RNAs migrate much more
slowly than the corresponding linear forms.
Either high temperature or chemicals such as
urea and/or formamide can be used as the
denaturant. Sambrook and Russell (2001) provides an excellent introduction to the principles of nucleic acid electrophoresis, espe-
16G.1.6
Supplement 10
Viroid concentration
Under favorable growing conditions, concentrations of Potato spindle tuber viroid
(PSTVd) in diseased tissue are high enough
that the viroid can be easily detected by ethidium bromide staining after analysis of total cellular RNA. Where R-PAGE of a total
RNA preparation fails to reveal an obvious
band in the viroid window, the analysis should
be repeated using low-molecular-weight RNA.
Removing rRNA and other high-molecularweight RNAs by LiCl precipitation allows one
to load the RNA from several-fold more tissue
onto a single lane, thereby increasing the sensitivity of the analysis (Fig 16G.1.1C, compare
lanes 4 and 6). Staining with SYBR Gold or
silver rather than ethidium bromide can yield
an additional increase in sensitivity.
Critical Parameters
Running time
It is obviously important that the molecule
of interest not run off the gel during the first
electrophoresis. Perhaps not quite so obvious
is the need to run as many of the small linear cellular RNAs as possible off the bottom
of the gel so that they do not later clutter the
viroid window. Their strong secondary structure causes viroids to behave anomalously during electrophoresis under nondenaturing conditions; thus, choosing an appropriate set of
RNA markers is critical.
If available, low-molecular-weight RNA
preparations containing one or more viroids of
known size (i.e., 246 to 403 nucleotides) are
ideal. Where the existence of circular RNAs
<250 nt (i.e., Avocado sunblotch viroid) or
>600 nt (i.e., the slow-slow dimer of Coconut cadang-cadang viroid) is suspected, it
may be wise to construct a series of selfcircularizing RNAs of precisely known size,
derived from the less abundant form of tobacco
ringspot virus satellite RNA (Feldstein et al.,
1997). At the present time, circular RNA markers are not commercially available.
Troubleshooting
Optimizing R-PAGE for viroids of different
size
R-PAGE analysis using a 5% gel works well
for viroids that are similar to PSTVd in size
and/or structural properties. Provided that appropriate circular RNA markers are available,
changing the gel concentration or run times to
improve the separation is straightforward. One
potential source of confusion stems from the
6
XC
C
L
L
H
C
C+L
XC
BPB
denaturing conditions (1 TBE, 8 M urea, 55 C), (B) first (i.e., nondenaturing) R-PAGE electrophoresis, and (C) second R-PAGE electrophoresis. RNAs were visualized by ethidium bromide
staining after electrophoresis, and the positions of circular PSTVd (C), linear PSTVd (L), and
a linear host RNA (H) of unknown size are marked. Lane 1, precisely-full-length (i.e., 359 nt)
PSTVd RNA synthesized in vitro; lane 2, purified PSTVd marker containing both circular and
linear molecules; lane 3, 2 g total RNA from uninfected tomato; lane 4, 2 g total RNA from
PSTVd-infected tomato; lane 5, 3 g low-molecular-weight RNA from uninfected tomato; lane 6,
3 g low-molecular-weight RNA from PSTVd-infected tomato. Positions of the bromphenol blue
(BPB) and xylene cyanol (XC) tracking dyes are marked.
Plant RNA
Viruses
16G.1.7
Current Protocols in Microbiology
Supplement 10
failure of the particular combination of temperature and low ionic strength used during the
second electrophoresis to completely denature
an unknown viroid RNA. On the positive side,
Singh and Boucher (1987) have exploited this
situation to separate closely related sequence
variants of PSTVd. Different banding patterns
may reflect differences in either nucleotide
number or nucleotide sequence. For general
PAGE troubleshooting, please see Sambrook
and Russell (2001) and Gallagher (2006).
Viroids with branched secondary structures
Viroids with branched structures are insoluble in 2 M LiCl. Three of the 29 viroid
species currently known have branched secondary structures, and titers of at least one of
these branched viroids (i.e., Chrysanthemum
chlorotic mottle viroid) in infected tissue are
low. Where such a situation is suspected, readers should consult Navarro and Flores (1997)
for additional guidance.
Anticipated Results
Identification of
Viroids by Gel
Electrophoresis
Time Considerations
Isolation of total RNA using TRIzol reagent
is rapid and convenient. Provided that the number of samples to be processed is limited (i.e.,
<10), preparations of total RNA can be ready
for electrophoresis within 2 to 3 hr. LiCl fractionation involves an overnight incubation at
0 to 4 C; nevertheless, preparations of lowmolecular-weight RNA should be ready for
electrophoresis before noon on the second day.
When carried out in a minigel format as described in Basic Protocol 2, R-PAGE requires
only 3 to 3.5 hr from sample application to
photography of the ethidium- or SYBR Goldstained gel.
Literature Cited
Brown, T., Mackey, K., and Du, T. 2004. Analysis
of RNA by northern and slot blot hybridization.
Curr. Protoc. Mol. Biol. 67:4.9.1-4.9.19.
Feldstein, P.A., Levy, L., Randles, J.W., and Owens,
R.A. 1997. Synthesis and two-dimensional electrophoretic analysis of mixed populations of
circular and linear RNAs. Nucleic Acids Res.
25:4850-4854.
Gallagher, S.R. 2006. One-dimensional SDS gel
electrophoresis of proteins. Curr. Protoc. Mol.
Biol. 75:10.2A.1-10.2A.37.
Gallagher, S.R and Dejardins, P.R. 2006. Quantitation of DNA and RNA with absorption and
fluorescence spectroscopy. Curr. Protoc. Mol.
Biol. 76:A.3D.1-A.3D.21.
Hanold, D., Semancik, J.S., and Owens, R.A. 2003.
Polyacrylamide gel electrophoresis. In Viroids
(A. Hadidi, R. Flores, J.W. Randles, and J.S.
Semancik, eds.) pp. 95-102. CSIRO Publishing,
Collingwood, Australia.
Kingston, R.E., Chomczynski, P., and Sacchi,
N. 1996. Guanidine methods for total RNA
preparation. Curr. Protoc. Mol. Biol. 36:4.2.14.2.9.
Navarro, B. and Flores, R. 1997. Chrysanthemum
chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with
hammerhead ribozymes. Proc. Natl. Acad. Sci.
U.S.A. 94:11622-11267.
16G.1.8
Supplement 10
Key References
Flores, R. and Owens, R.A. 2008. Viroids. In
Encyclopedia of Virology, Third Edition (B.W.J.
Mahy and M.H.V. Van Regenmortel, eds.). Elsevier/Academic Press, London, in press.
The largest single reference source of current virological knowledge for both specialist and general
readers.
Steger, G. and Riesner, D. 2003. Molecular characteristics. In Viroids (A. Hadidi, R. Flores, J.W.
Randles, and J.S. Semancik, eds.) pp. 15-29.
CSIRO Publishing, Collingwood, Australia.
A concise summary of the structural properties of
viroids.
Singh, R.P. 2007. Molecular detection of plant
viroids. In Biotechnology and Plant Disease
Management (Z.K. Punja, S.H. De Boer, and
H. Sanfacon, eds.), pp. 277-300. CAB International, Wallingford, U.K.
R-PAGE and other electrophoretic methods are
compared with other detection techniques such as
molecular hybridization and RT-PCR.
Internet Resources
http://subviral.med.uottawa.ca/
Subviral RNA Database. Maintained by Martin
Pelchat [RNA Group/Groupe ARN, Departement de
biochimie, Universite de Sherbrooke, Sherbrooke
(Quebec) J1H 5N4, Canada], this website contains
the sequences of more than 2400 viroids and related
RNAs.
http://www.ictvonline.org
The Universal Database of the International Committee on the Taxonomy of Viruses is a convenient
source of current information on viroid (and virus)
taxonomy.
http://www.probes.invitrogen.com/handbook/
Invitrogen Detection Technologies: Contains an informative discussion of nucleic acid staining using
SYBR Gold, SYBR Green I, and SYBR Green II.
Plant RNA
Viruses
16G.1.9
Current Protocols in Microbiology
Supplement 10
UNIT 16H.2
16H.2.1
Figure 16H.2.1 Schematic diagram of small-RNA isolation and cloning steps in the Basic Protocol and
Alternate Protocol.
Isolation and
Cloning of Small
RNAs from
Virus-Infected
Plants
protocols for small-RNA cloning are described. The Basic Protocol uses unmodified
small RNAs, taking advantage of the fact that small RNAs processed by Dicer have both
a 5 -phosphate and 3 -hydroxyl terminus. In the Alternate Protocol, the small RNAs are
dephosphorylated prior to ligation of the 3 adapter. Dephosphorylation can enhance the
efficiency of ligation reaction by preventing the circularization of small RNAs. However,
this step also increases the chance of cloning RNA degradation products generated by
endogenous RNases. The Alternate Protocol may be used to clone small RNAs from
high quality RNA extracts of plants infected with low abundance viruses.
16H.2.2
Current Protocols in Microbiology
BASIC
PROTOCOL
In this protocol, gel-purified small RNAs are ligated directly to a nonphosphorylated 5 adapter oligonucleotide using T4 RNA ligase. The ligation products are separated from
the excess of 5 adapter on a 15% denaturing polyacrylamide gel and are subsequently
ligated to a 5 -phosphorylated 3 -adapter oligonucleotide with a blocked 3 -hydroxyl
terminus. The final ligation products are separated from the excess of 3 adapter and are
subjected to reverse transcription and PCR amplification. The gel-purified PCR products
are digested with EcoRI and NcoI restriction enzymes and subsequently concatamerized
using T4 DNA ligase. The concatamers are ligated into a cloning vector digested with
EcoRI and NcoI, and then TOP10 cells are transformed with the recombinant plasmids.
Individual colonies are screened for the size of concatamer inserts by PCR, and selected
PCR fragments are purified and submitted for sequencing. The small-RNA sequences
are extracted from the sequence manually or automatically, using software tools (e.g.,
Staden Package, http://staden.sourceforge.net; or software developed in-house).
Materials
Virus-infected leaf tissue
Liquid nitrogen
Extraction buffer (see recipe)
Buffered phenol, pH 8.0 (APPENDIX 2A)
25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (APPENDIX 2A)
24:1 (v/v) chloroform/isoamyl alcohol
4 M sodium acetate, pH 5.2
Absolute ethanol
80% ethanol
70% ethanol in a wash bottle
RNase-free H2 O (APPENDIX 2A) or double-distilled autoclaved H2 O
15% denaturing polyacrylamide gels, fifteen well, 20 16 0.15 cm (see recipe;
also see Ellington and Pollard, 2001)
0.5 TBE buffer (APPENDIX 2A): sterilize by autoclaving
Gel-loading solution (see recipe)
10-nucleotide DNA oligo ladder (see recipe)
10 mg/ml ethidium bromide (APPENDIX 2A)
0.3 M NaCl, RNase-free
20 mg/ml glycogen (Roche)
100 M 5 adapter (see Table 16H.2.1)
Dimethyl sulfoxide (DMSO)
10 PAN ligation buffer (see recipe)
31 U/l RNAguard (Amersham Pharmacia)
40 U/l T4 RNA ligase (Amersham Pharmacia)
100 M 3 adapter (Table 16H.2.1)
100 M PCR 3 primer (Table 16H.2.1)
0.1 M dithiothreitol (DTT)
5 first-strand buffer (Invitrogen)
2 mM deoxyribonucleoside triphosphates (dNTPs: dATP, dCTP, dGTP, dTTP,
2 mM each; APPENDIX 2A)
200 U/l reverse transcriptase (Superscript II, RNase H , Invitrogen)
150 mM KOH/20mM Tris base
150 mM HCl
1 and 10 TE buffer (see APPENDIX 2A for 1)
10 PCR buffer (Roche)
PCR 5 primer (Table 16H.2.1)
Plant RNA
Viruses
16H.2.3
Current Protocols in Microbiology
Oligonucleotide
Sequencea
5 adapter
5 TGGGAATTCCTCACTrArArA 3
3 adapter
5
PCR 5 primer
5 CATGGGAATTCCTCACTAAA 3
PCR 3 primer
5 TACAGTCCATGGATAGAAA 3
M13 F primer
5 TTCCCAGTCACGACGTT 3
M13 R primer
5 CAGGAAACAGCTATGAC 3
5 GCGCTCTTGACTCGTTGTGC 3
5 ACGTGTCGACATCACGCTGGAAATGATACA 3
5 GATAATACGACTCACTATAGGGCCAGGGCGCAGATTGAGC 3
5 ACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTA
CTTTCTCTTA 3
5 ATGGATCCCTCGAGGTCGACCCTAGGTGGTCTCATCCTCA
GTTCGAGAAGTAACCCGGGAT 3
5 ATGACTAGTAGTAGGCTCTCTCTGTCCTTGAGGATGATGC
TCAAGTCTAGCTCTAAGCTGCTCAAGCTCTC 3
5 AGTCTTCTCATCCATAGAAGCAGTAGTAGGAATATCGTAA
TCAAGAGCACCAGATGAAGAGATCTTCTTGAATCTGTTAG 3
rUrUrUCTATCCATGGACTGTidT 3
P
a A, C, G, T, DNA residues; rA, rU, RNA residues; P , 5 phosphate; idT, 3 -inverted deoxythymidine.
Isolation and
Cloning of Small
RNAs from
Virus-Infected
Plants
16H.2.4
Current Protocols in Microbiology
3. Transfer the aqueous upper phase to a new tube, avoiding precipitated material from
the interface. Add 6 ml of 25:24:1 phenol/chloroform/isoamyl alcohol, vortex 15
sec, and centrifuge 5 min at 3500 g, 4 C.
CAUTION: Phenol is extremely toxic and highly corrosive, and can cause severe burns
(UNIT 1A.3). Wear appropriate gloves, goggles, and protective clothing. Always use in a
chemical fume hood.
The interface contains precipitated proteins. If a large precipitate forms after the first
phenol/chloroform/isoamyl alcohol extraction, additional extraction may be required to
remove all proteins including endogenous RNases.
4. Transfer the aqueous upper phase to a new tube. Add 6 ml of 24:1 chloroform/isoamyl
alcohol. Vortex 15 sec and centrifuge 5 min at 3500 g, 4 C.
5. Again, transfer the aqueous upper phase to a new tube and precipitate the total
nucleic acid by adding 1/20 vol. of 4 M sodium acetate, pH 5.2, and 3 vol absolute
ethanol. Mix by inverting the tube, and incubate 10 to 15 min on ice or store at
20 C overnight.
6. Recover the total nucleic acid (TNA) by centrifuging 30 min at 3500 g, 4 C.
Remove the supernatant by aspiration.
7. To remove residual salts from the pellet, slowly add an equal volume of 80% ethanol
down the side of the tube, and centrifuge immediately 5 min at 3500 g, 4 C.
Avoid resuspending the TNA pellet in 80% aqueous ethanol, because short RNAs are
soluble to some extent in 80% ethanol in the absence of salt.
Plant RNA
Viruses
16H.2.5
Current Protocols in Microbiology
8. Remove the ethanol by aspiration without disturbing the pellet, and collect the
residual ethanol at the bottom of the tube by an additional 10-sec centrifugation.
Remove the residual liquid completely using a small pipet tip and air dry the pellet
5 to 10 min at room temperature.
9. Place the tube on ice and dissolve the pellet in 200 l RNase-free water. Dilute 2 l
TNA in 1 ml water to estimate the concentration of the total RNA by measuring the
absorbance (A260 ) in a 1-cm quartz cuvette (Gallagher, 2004). Transfer the remaining
TNA extract to a siliconized microcentrifuge tube and store up to 1 year at 70 C.
Regular reaction tubes may absorb the low-molecular-weight nucleic acids to some extent.
Use siliconized reaction tubes where possible.
Quality of RNA can be checked by denaturing 2 to 5 l sample with 1 vol gel-loading
solution at 65 C for 5 min and analyzing on a 1% agarose gel.
The authors have found that the phenol/chloroform/isoamyl alcohol extraction provides
the highest yield, although other RNA isolation methods (e.g., Trizol reagent) may also be
used. The relatively small amount of genomic DNA in TNA will not interfere with further
processes. Avoid commercial RNA isolation kits with silica-gel-membrane purification
because the small-RNA fraction does not bind to the column. LiCl precipitation must also
be avoided because small RNAs do not coprecipitate with long RNA transcripts.
12. Mix 800 to 1000 g TNA with an equal volume of gel-loading solution. Denature
the nucleic acid solution, as well as 40 l of 10-nucleotide DNA oligo ladder, by
incubating the tube for 5 to 10 min at 65 C.
13. Load the sample in the center wells of the gel. Load 40 l denatured 10-nucleotide
DNA oligo ladder. Leave one well empty between the sample and the size marker to
avoid contamination.
It is important to apply the 10-nucleotide DNA oligo ladder in a similar volume as the
TNA sample to ensure similar gel-running behavior.
14. Separate the small RNAs by running the gel 2.5 hr using at 350 V (7 W) using
0.5 TBE until the bromphenol blue dye in the gel-loading solution reaches the
bottom of the gel.
15. Dismantle the electrophoresis apparatus and stain the gel by soaking in 100 ml of
0.5 TBE buffer containing 0.25 g/ml ethidium bromide (dilute from 10 mg/ml
stock) for 10 min. Visualize the nucleic acids in the gel using a 360-nm UV transilluminator. To protect the gel from RNase contamination, cover the transilluminator
with plastic wrap.
CAUTION: Ethidium bromide is a powerful mutagen and is toxic (UNIT 1A.3). Wear
appropriate gloves when working with solutions that contain ethidium bromide.
Isolation and
Cloning of Small
RNAs from
Virus-Infected
Plants
CAUTION: UV light is mutagenic and carcinogenic, and can damage the retina of the
eyes. Wear UV-protective glasses or face shield, and appropriate gloves.
16H.2.6
Current Protocols in Microbiology
A small portion of nucleic acids can diffuse into the solution during staining. To reduce
the loss of small RNAs and subsequent adapter ligation products, the DNA size markers
may be visualized separately, by slicing off the marker lane, staining it, and returning it
to its original position.
16. Excise the gel slice encompassing 18- to 28-nucleotide small RNAs defined by the
mobility of the 20- and 30-nucleotide DNA size markers.
RNA migrates slightly more slowly than DNA of the same sequence and length, necessitating the adjustment of size estimates when excising the small RNAs. Avoid unnecessarily
long UV exposure, which will damage the nucleic acids.
17. Cut the gel slice into 1 1mm squares, transfer to a preweighed 15-ml polypropylene conical tube, and weigh the gel slices. Elute the small RNAs from the gel by
adding 3 vol RNase-free 0.3 M NaCl and incubate the tube overnight at 4 C under
constant agitation (i.e., using rotary shaker or rocker).
18. Transfer the supernatant to a fresh tube (keep at 4 C) and repeat the elution of the
gel with 2 vol RNase-free 0.3 M NaCl for 6 to 8 hr at 4 C.
The more elution buffer, the higher the efficiency of recovery.
19. Combine the supernatants by pipetting and pellet residual polyacrylamide by centrifuging 10 min at 3500 g, 4 C. Transfer the supernatant to a new tube, avoiding
the polyacrylamide from the bottom.
A 3-ml volume of supernatant can be precipitated in a 15-ml conical tube. Large volumes
of gel may require greater amounts of extraction buffer for efficient elution, which can
either be precipitated in a larger tube or split into multiple 15-ml tubes.
20. Precipitate the RNA from the supernatant by adding glycogen solution to a final
concentration of 2 g/ml and 3 vol absolute ethanol. Incubate samples overnight at
20 C.
16H.2.7
Current Protocols in Microbiology
29. Excise the gel slice encompassing 36- to 46-nucleotide ligated small RNAs defined
by the mobility of the 30- and 60-nucleotide DNA size markers.
30. Elute the ligation products from the gel slice with RNase-free 0.3 M NaCl as described in steps 17 and 18.
31. Combine the supernatants in one tube and pellet residual polyacrylamide by centrifuging 10 min at 3500 g, 4 C. Transfer the supernatant to a fresh tube(s),
avoiding the polyacrylamide from the bottom.
32. Precipitate the RNA from the supernatant by adding 20 mg/ml glycogen solution
to a final concentration of 2 g/ml and 3 volumes absolute ethanol, and incubate at
20 C overnight.
45. Transfer the RNA to a siliconized microcentrifuge tube and place on ice. Denature
the nucleic acids by incubating the tube for 1 min at 90 C. Place the tube immediately
on ice for 1 min.
16H.2.8
Current Protocols in Microbiology
50 l 10 PCR buffer
50 l 2 mM dNTPs
5 l 100 M PCR 5 primer
5 l 100 M PCR 3 primer
330 l H2 O
10 l 5 U/l Taq DNA polymerase.
56. Divide the reaction mixture among five 200-l PCR tubes and run a polymerase
chain reaction (PCR; also see Kramer and Coen, 2001) using the parameters listed
below:
1 cycle:
25 cycles:
1 cycle:
2 min
1 min
1 min
1 min
5 min
94 C
94 C
50 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
57. Recombine the PCR reactions in one 1.7-ml microcentrifuge tube, and perform one
500-l 25:24:1 phenol/chloroform/isoamyl alcohol extraction and one 500-l 24:1
chloroform/isoamyl alcohol extraction, as described in steps 49 and 50.
58. Transfer the aqueous upper phase to a fresh siliconized tube and precipitate the DNA
as described in step 51.
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Viruses
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Current Protocols in Microbiology
61. Add 10 l of 6 DNA loading solution and load the sample in two to four center
wells of a 15%, fifteen-well native polyacrylamide gel (also see Chory and Pollard,
1999).
The volume that can be loaded is determined by the size of the wells. The wells should
not be fully loaded, and more wells may be needed to load the total volume.
A native polyacrylamide gel is used to separate the cDNAs in nondenaturing conditions,
addition of formamide to the sample, denaturation by heating and prerunning of the gel
are not required.
62. Load 1 g of 20-bp PCR low DNA ladder in a similar volume as the cDNA sample
to ensure similar gel running behavior. Leave one well empty between the sample
and the size marker to avoid contamination.
63. Separate the cDNAs by running the gel 3 hr at 350 V (8 W) using 0.5 TBE
buffer until the bromophenol blue dye of the gel-loading solution reaches the bottom
of the gel.
64. Dismantle the gel-running apparatus and stain the gel by soaking in 100 ml of 0.5
TBE buffer containing 0.25 g/ml ethidium bromide for 10 min. Visualize the nucleic
acids in the gel using a 360-nm UV transilluminator.
A double band, 40 and 60 bp, is often visible in the gel. The 40-bp band corresponds
to amplified ligation product of directly joined 5 - and 3 -adapter oligonucleotides without
a small-RNA insert. Avoid contamination by this product in gel slices.
65. Excise the gel slice encompassing 59- to 65-bp cDNAs defined by the mobility of
the DNA size markers.
66. Elute the DNA from the gel slice with 0.3 M NaCl as described in step 17 and 18.
67. Combine the supernatants and pellet residual polyacrylamide by centrifuging 10
min at 3500 g, 4 C. Transfer the supernatant to a fresh tube(s), avoiding the
polyacrylamide from the bottom.
68. Precipitate the DNA from the supernatant by adding 1/20 vol of 4 M sodium acetate,
pH 5.2, 20 mg/ml glycogen solution to a final concentration of 2 g/ml, and 3 vol
absolute ethanol. Incubate at 20 C overnight.
16H.2.10
The undigested PCR product should be 60 bp, the digested PCR product should be
50 bp.
Plant RNA
Viruses
16H.2.11
Current Protocols in Microbiology
84. Pick individual white colonies from the agar plate using a pipet tip and restreak on
a master plate. Transfer the same pipet tips to the wells of the microtiter plate filled
with the PCR mixture and resuspend the remaining colony by pipetting up and down.
The cells in the colonies will be lysed during the first PCR cycle. W-1 is a detergent.
85. Run a PCR (also see Kramer and Coen, 2001) using the following parameters:
1 cycle:
30 cycles:
1 cycle:
2 min
30 sec
30 sec
1 min
5 min
94 C
94 C
55 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
86. Separate 5 l of the PCR products on a 2% standard agarose gel (Voytas, 1992) using
a 100-bp DNA ladder as a size marker.
PCR products equivalent or longer than 400 bp contain at least three small-RNA sequences. The expected size of the PCR product from the empty vector is 249 bp.
87. Purify the remaining PCR products with the QIAquick PCR purification kit as
described by the manufacturer. Sequence or submit the purified PCR product for
automated sequencing using M13 F or M13 R primers.
Instead of using QIAquick column SAP/Exo I reaction may be used to prepare PCR
products for sequencing.
ALTERNATE
PROTOCOL
16H.2.12
Current Protocols in Microbiology
Plant RNA
Viruses
16H.2.13
Current Protocols in Microbiology
Extraction buffer
1 EB (see recipe)
2% SDS
Prepare just before use
Gel-loading solution
10 ml deionized formamide
200 l 0.5 M EDTA, pH 8.0
1 mg xylene cyanol FF
1 mg bromphenol blue
Store up to 1 year at 4 C
CAUTION: Formamide is teratogenic. Wear appropriate gloves and safety glasses.
Isolation and
Cloning of Small
RNAs from
Virus-Infected
Plants
16H.2.14
Current Protocols in Microbiology
This protocol is isotope free, utilizes unmodified small RNAs and is routinely used to
characterize miRNAs and siRNAs from various plant tissues. The TNA isolation is adapted
from White and Kaper (1989). The small-RNA
cloning protocol results from modifications of
protocols originally published by the Tuschl,
Bartel, and Carrington groups (Elbashir et al.,
2001; Lau et al., 2001; Llave et al., 2002; Pfeffer et al., 2003).
Anticipated Results
Depending on the size of the concatamerized DNA, every recombinant plasmid should
yield at least three small-RNA sequences. Sequence analyses of a typical clone are shown
in Figure 16H.2.2.
Time Considerations
The small-RNA cloning is a technically demanding multistep procedure. If one step fails,
the small-RNA clones will never be obtained.
The protocol can be safely interrupted at
every ethanol precipitation step. The precipitated nucleic acid can be stored in ethanol at
20 C for several days prior to collecting the
pellet by centrifugation.
The cloning procedure can be performed
within two weeks. Isolation and quality analyses of TNA extract require 1 day. Gel purification of small RNA with repeated elution
from the gel requires 2 days. Ligation of the
5 adapter to small RNA and purification of
the first ligation product with repeated elution
from the gel require 2 days. Ligation of the
3 adapter to the 5 -adapter-small-RNA hybrid
and purification of the final ligation product
with repeated elution from the gel requires
2 days. Reverse transcription and PCR amplification of the cDNA require 1 day. Gel
purification of the cDNA corresponding to
the 5 -adapter-small-RNA-3 -adapter ligation
product with repeated elution from the gel requires 2 days. EcoRI and NcoI restriction digestion of the PCR product and concatamerization of EcoRI and NcoI restriction-digested
DNA require 1 day. Cloning of concatamers
into EcoRI and NcoI restriction-digested vector requires 1 day. PCR screening of colonies
and sequencing of concatamer inserts require
1 day.
Acknowledgments
The authors thank Gyorgy Szittya and Alan
Herr for critically reading the manuscript.
Attila Molnar is a European Molecular
Plant RNA
Viruses
16H.2.15
Current Protocols in Microbiology
Figure 16H.2.2 Example of a concatamer sequence after cloning small RNAs from viral infected
Nicotiana benthamiana leaf tissue. The lowercase letters represent the vector sequence while the
uppercase letters represent the concatamer sequence. Adapter sequences are underlined and
the EcoRI and NcoI restriction sites are emboldened. Small-RNA sequences are highlighted (dark
gray).
Literature Cited
Chory, J. and Pollard, J.D. 1999. Separation of
small DNA fragments by conventional gel electrophoresis. In Current Protocols in Molecular
Biology (F.M. Ausubel, R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K.
Struhl, eds.) pp. 2.7.1-2.7.8. John Wiley & Sons,
Hoboken, N.J.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001.
RNA interference is mediated by 21- and 22nucleotide RNAs. Genes Dev. 15:188-200.
Ellington, A. and Pollard, J.D. 1998. Purification
of oligonucleotides using denaturing polyacrylamide gel electrophoresis. In Current Protocols
in Molecular Biology (F.M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J.G. Seidman, J.A.
Smith, and K. Struhl, eds.) pp. 2.12.1-2.12.7.
John Wiley & Sons, Hoboken, N.J.
Isolation and
Cloning of Small
RNAs from
Virus-Infected
Plants
16H.2.16
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Plant RNA
Viruses
16H.2.17
Current Protocols in Microbiology
UNIT 16I.1
Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, United
Kingdom
2
Institute of Molecular Plant Sciences, The University of Edinburgh,
Edinburgh, United Kingdom
ABSTRACT
Potato virus Xbased vectors are a well established system for rapid in planta studies. The vectors can be used for expression of proteins in plants and to down-regulate
genes through virus-induced gene silencing. The development of binary-based vectors
for Agrobacterium delivery makes this system well suited to high-throughput studies.
Protocols are given for establishing infections to achieve expression and VIGS through
in vitro transcription and Agrobacterium delivery to glasshouse and in vitrogrown plant
C 2008 by John Wiley & Sons,
material. Curr. Protoc. Microbiol. 8:16I.1.1-16I.1.13.
Inc.
Keywords: potato virus X r virus vector r gene silencing r Agrobacterium
tumefasciens r solanaceous plants
INTRODUCTION
Vectors based on potato virus X (PVX) have been the most widely used plant virusbased
vectors. This is in part due to the fact that they have been made freely available through
The Sainsbury Laboratory (http://www.ayeaye.tsl.ac.uk/), where PVX-based vectors were
originally constructed. PVX-based vectors have proven valuable experimental tools. They
can be used for virus-induced gene silencing (VIGS) through the insertion of partial
cDNA sequences, or for the expression of proteins through the insertion of full-length
cDNAs from plants, of cDNAs encoding therapeutic proteins, or of cDNAs for other
foreign proteins (e.g., derived from other plant pathogens).
Various strategies have been tested for the production of vectors from PVX. The most
successful strategy is that originally developed: expression of the foreign gene is achieved
by its insertion between a duplication of the subgenomic promoter sequence that, in the
wild-type RNA virus, directs the production of a subgenomic mRNA from which the
viral coat protein is translated. This unit describes procedures for infecting plants with
PVX-based vectors that use this strategy to achieve protein expression or VIGS.
In early PVX-based vectors, such as P2C2S (Figure 16I.1.1), full-length cDNA copies of
PVX were cloned into plasmids under the control of a T7 RNA polymerase promoter sequence. The vector P2C2S differs from later binary-based vectors (pGR106 and pGR107
described further) in that it contains a shorter promoter sequence to drive expression of
the coat protein gene. Such vectors give higher levels of foreign protein expression (S.
Santa Cruz, unpub. observ.), possibly due to reduced promoter competition. In Basic
Protocol 1, a method is given in which such a plasmid is linearized, the linear DNA
transcribed in vitro, and the run-off transcripts produced used for manual inoculation of
plants. Subsequently PVX-based vectors, e.g., pGR106 and pGR107 (Figure 16I.1.1),
were produced in which genetically engineered, full-length, cDNA copies of PVX were
Current Protocols in Microbiology 16I.1.1-16I.1.13, February 2008
Published online February 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16i01s8
C 2008 John Wiley & Sons, Inc.
Copyright
Plant RNA
Viruses
16I.1.1
Supplement 8
Figure 16I.1.1 Schematic representations of PVX and PVX-based vectors. Genome organization
of wild-type PVX (A). The ve major ORFs in the 6.4-kb genome are indicated by boxes. The
subgenomic mRNAs from which the 5 distal ORFs are expressed are indicated by lines below,
with the sizes of the mRNAs. Organizations of pP2C2S (B) and pGR106/7 (C). The positions of
the duplicated, subgenomic promoter sequences are indicated by arrows. The restriction enzyme
sites in the multiple cloning sites are listed above. Features shown are: viral RNA-dependent
RNA polymerase gene (RdRp); viral movement protein genes (M1, M2, M3); viral coat protein
gene (CP); T7 bacteriophage (T7) and cauliower mosaic virus 35S (35S) promoters; nopaline
synthase transcriptional terminator (NOS); T-DNA left and right borders (LB, RB); -lactamase
gene (ApR ) conferring ampicillin resistance; and neomycin phosphotransferase II gene (KnR )
conferring kanamycin resistance.
positioned between a cauliflower mosaic virus 35S promoter and nopaline synthase
terminator sequence in binary vectors to allow Agrobacterium tumefasciensmediated
initiation of infections. This system provides a more robust and cheaper method for
accomplishing infections than in vitro transcription. Beginning with the insertion
of a foreign gene sequence into the binary pGR106, Basic Protocol 2 describes the
transformation of Agrobacterium, propagation of transformed bacteria, and infiltration
of such bacteria into plant leaves to initiate infection. A similar protocol is found in
UNIT 16B.2. This method is of particular utility when pervasive infections of inoculated
leaves and plants are required.
Use of PVX-Based
Vectors for Gene
Expression and
VIGS
Tubers produced by potatoes are the worlds fourth largest crop. In Basic Protocol 3,
a modification of the Agrobacterium-mediated infection process, stab inoculation, is
used to initiate infections to achieve VIGS in potato microtubers, a model for the crop
tissue. Stab inoculation has previously been used in high-throughput studies, involving
multiple stab inoculations of a single leaf, to screen clones for a phenotype that is
easily assessable at the site of inoculation (Takken et al., 2000) and for facile initiation of
systemic infections of large numbers of plants with individual clones for high-throughput
VIGS studies (Lu et al., 2003).
PVX-based vectors do not in themselves present a risk to human health, but, as genetically
engineered plant pathogens, they present a risk to the environment. So far, PVX-based
16I.1.2
Supplement 8
vectors have been found to be unfit in comparison to wild-type virus and to revert to
wild-type virus through homologous recombination between the duplicated promoter
sequences. PVX is found in potato-growing regions worldwide. Experiments involving
PVX-based vectors are likely to require BL2-P containment; however researchers should
carry out a risk assessment and comply with local and national regulations (see UNIT 1A.1).
BASIC
PROTOCOL 1
This protocol begins with a plasmid vector (pP2C2S, Figure 16I.1.1) containing the
gene of interest inserted in the multiple cloning site between the duplicated coat protein
subgenomic promoters. This recombinant plasmid is then linearized and transcribed in
vitro with T7 RNA polymerase. After confirmation of the integrity of the in vitro run-off
transcripts, they are manually inoculated onto host plant leaves. After incubation to allow
the recombinant virus to move locally and/or systemically, the plants are observed for
symptom development and analyzed for expression of the gene of interest.
Materials
pP2C2S-derived recombinant plasmid (The Sainsbury Laboratory;
http://www.ayeaye.tsl.ac.uk; follow links for David Baulcombe, then for PVX
vectors)
SpeI restriction endonuclease and 10 buffer
25:24:1 (v/v/v) phen4ol:chloroform:isoamyl alcohol saturated with 10 mM TE
buffer, pH 8.0 (APPENDIX 2A)
3 M sodium acetate pH 5.2 (APPENDIX 2A)
100% and 70% ethanol
10 mM TE buffer, pH 8.0 (APPENDIX 2A)
0.8% and 0.7% nondenaturing agarose gels (Voytas, 2000) prepared in 1 TBE
buffer (APPENDIX 2A)
5 T7 RNA polymerase buffer
0.1 M dithiothreitol (DTT)
10 A/C/U/gTP mix (see recipe)
5 mM cap analog (m7G[5 ]ppp[5 ]G)
50 U/l T7 RNA polymerase
20 mM GTP
Plants to be infected (Table 16I.1.1)
Aluminum oxide F400WP (Washington Mills Electro Minerals Company;
http://www.washingtonmills.com/)
0.5-ml microcentrifuge tubes
Erlenmeyer flask
Gauze
Containment glasshouse or growth chamber
Additional reagents and equipment for agarose gel electrophoresis and ethidium
bromide staining of gels (Voytas, 2000)
Linearize plasmid DNA
1. In a 0.5-ml microcentrifuge tube, digest 25 g of plasmid DNA with 25 U SpeI
restriction endonuclease using an appropriate buffer in a final volume of 50 l.
Incubate overnight at 37 C.
The SpeI site is preferred for linearization of pP2C2S as it lies immediately downstream
of the 3 poly(A) tail of the infectious clones. The SphI site is located just downstream of
the SpeI site and can be used instead if an SpeI site is present in the inserted cDNA, but
addition of extra nucleotides after the poly(A) tail is detrimental to transcript infectivity.
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Current Protocols in Microbiology
Supplement 8
5. After incubating at 37 C for 20 min, chase the reaction with GTP by pipetting 5 l
of 20 mM GTP onto the surface of the reaction. Do not mix, and continue incubating
for 40 min at 37 C.
The initial GTP concentration is reduced to promote incorporation of the cap analog, as
uncapped transcripts are not infectious.
16I.1.4
Supplement 8
Optionally, if the transcripts are not to be used immediately for plant inoculation, add 0.1
volume of 3 M sodium acetate and 2.5 volumes of ethanol, mix, and store at 20 C. Prior to
use, microcentrifuge for 15 min at 13,000 g, 4 C, remove supernatant, wash pellet with
cold 70% ethanol, centrifuge briefly, remove all supernatant, evaporate residual ethanol,
and dissolve the RNA pellet in 45 l of RNase-free or DEPC-treated sterile distilled water.
Current Protocols in Microbiology
Inoculate plants
8. Mark leaves to be inoculated by punching a hole in each of them with a pipet tip.
Very lightly dust the leaves with aluminum oxide using an Erlenmeyer flask topped
with a double layer of gauze.
CAUTION: Due to the fine particulate nature of the aluminum oxide suitable respiratory
protection should be worn.
The choice of leaves for inoculation is important. Young leaves are generally more
susceptible to virus infection, but also more easily damaged by the manual inoculation
process. For inoculation of Nicotiana benthamiana, select plants that are about 12 cm
across and at the nine-leaf stage (4 to 5 weeks post sowing), and inoculate the seventh leaf.
9. Pipet 5 l of transcript to an inter-veinal region on the upper side of the leaf near
the base. Wearing gloves, support the leaf from below with one hand. Spread the
inoculum over the leaf surface by drawing it to the leaf apex with a finger.
Ten gentle strokes should be adequate for this; more may result in excessive damage to
the leaf.
10. Place plants under controlled growth conditions (in a containment glasshouse or
growth chamber) and leave until systemic infection results in the development of a
mild, chlorotic mosaic on uninoculated leaves.
IMPORTANT NOTE: Containment level and standard operating procedures required for
propagation of PVX-infected plants must be in accordance with the health and safety
regime defined by local and national regulations covering work with genetically modified
organisms in contained facilities (as an example for the U.K., http://www.hse.gov.uk/
biosafety/gmo/acgm/acgmcomp/).
11. When systemic symptoms are well developed, assess accumulation of foreign protein
in inoculated and systemically infected leaves by suitable analytic methods, e.g.,
through immunoblotting of total protein extracts.
The stability of the recombinant PVX-based vector can be assessed through reverse
transcription of extracted poly(A) RNA and PCR amplification with primers flanking the
duplicated promoter sequences, i.e., a sense primer 5 of position 5580 in the PVX genome
and an antisense primer 3 of position 5680.
BASIC
PROTOCOL 2
This protocol begins with a plasmid vector (pGR106 or pGR107, Figure 16I.1.1) containing the sequence of interest inserted in the multiple cloning site between the duplicated
subgenomic promoters. This recombinant plasmid is then introduced into electrocompetent agrobacteria cells previously transformed with the helper plasmid pSoup. The
transformed agrobacteria are propagated and induced, and their density adjusted prior
to pressure infiltration of leaves. After incubation to allow the recombinant virus to
move locally and/or systemically, the plants are observed for symptom development and
analyzed for expression or down-regulation of the gene of interest.
Plant RNA
Viruses
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Current Protocols in Microbiology
Supplement 8
Materials
Electrocompetent A. tumefasciens strain GV3101 (Hellens et al., 2000) harboring
pSoup plasmid (http://www.pgreen.ac.uk/JIT/JIT fr.htm; follow links for binary
vectors, then for pGreen)
pGR106 or pGR107-derived recombinant plasmid
LB plates (APPENDIX 4A) with 50 g/ml kanamycin
PVX- or insert-specific primers (see information on supply and sequence data at
http://www.ayeaye.tsl.ac.uk/; follow links for David Baulcombe, then for PVX
vector; pGR106 and pGR107 GenBank accession numbers are, respectively,
AY297843 and AY297842)
LB liquid medium (APPENDIX 4A) with 50 g/ml kanamycin
Agromix (see recipe)
N. benthamiana plants (or other Solanaceae; see Table 16.I.1.1) to be inoculated
28 C incubator
Bench-top centrifuge
Spectrophotometer
2-ml syringes
Containment glasshouse or growth chamber
UV lamp: Blak-Ray model B100AP (UVP) for green fluorescent protein
visualization
Additional reagents and equipment for transformation of Agrobacterium
(UNIT 16B.2), the polymerase chain reaction (PCR; Kramer and Coen, 2001), and
agarose gel electrophoresis (Voytas, 2000)
Transform of Agrobacterium tumefasciens
Also see Hellens et al. (2000a) and http://www.pgreen.ac.uk/JIT/JIT fr.htm (follow links
for binary vectors, then for pGreen).
1. Transform electrocompetent A. tumefasciens strain GV3101 harboring helper plasmid pSoup with 1 g of recombinant pGR106/107 plasmid DNA (UNIT 16B.2).
The plasmids pGR106 and pGR107 are derived from the binary vector pGreen0000, which
carries a kanamycin selectable marker and replicates to high copy number in Escherichia
coli. For replication in Agrobacterium, these plasmids require a helper plasmid pSoup (or
pJIC Sa Rep), which provides a replicative function (RepA) in trans and carries a tetracycline selectable marker (Hellens et al., 2000b; http://www.pgreen.ac.uk/JIT/pSoup.htm).
Agrobacteria can be transformed with pSoup and pGR106/107 simultaneously; however, for simplicity, it is recommended that Agrobacteria already harboring pSoup be
transformed with the binary-PVX constructs. Instead of GV3101, Agrobacterium strains
LBA4404 or AGL1 can be used. A. tumefasciens strain GV3101, LBA4404, or AGL1
can be obtained from the e-mail addresses and Web sites mentioned in Hellens et al.
(2000a).
16I.1.6
Supplement 8
Infiltrate leaves
4. Inoculate 20-ml aliquots of LB liquid medium supplemented with 50 g/ml
kanamycin with the positive clones using a sterile pipet tip. Incubate cultures
overnight at 28 C with agitation.
Typically 20 ml of overnight culture is sufficient to inoculate up to 20 plants.
BASIC
PROTOCOL 3
Plant RNA
Viruses
16I.1.7
Supplement 8
new medium to induce microtuber formation; when these have formed, the effects of
silencing are analyzed (Faivre-Rampant et al., 2004).
Materials
pGR106 or pGR107-derived recombinant plasmid in A. tumefasciens GV3101
harboring plasmid pSoup (Basic Protocol 2)
In vitrogrown potato explants (Xu et al., 1998; in U.K. available from Scottish
Agricultural Science Agency; http://www.sasa.gov.uk; info@sasa.gsi.gov.uk)
propagated in potato explants propagation medium (see recipe) by internode
cuttings (Xu et al., 1998; Hendriks et al., 1991)
Microtuberization medium (see recipe)
Plant growth cabinet at 18 to 22 C with 16-hr photoperiod and light intensity
110 E m2 sec1
Plant growth cabinet at 16 C with 8-hr photoperiod and light intensity 80 E
m2 sec1
Nescofilm (Karlan; http://www.karlan.com) or other laboratory film, e.g., Parafilm
Perform stab inoculation in vitro
1. Using sterile toothpicks or pipet tips, pick individual, clonal, transformed agrobacteria colonies.
2. Inoculate all leaves of in vitrogrown potato explants at the two- to four-leaf stage
as follows (Takken et al., 2000):
a. Open a vessel containing a potato explant under sterile conditions in a laminar
flow hood.
b. Wipe gloved hands with 70% ethanol.
c. Supporting a leaf with a gloved finger, stab leaf in several locations with the pipet
tip or toothpick used to pick the agrobacteria colony.
3. Incubate the plants for 4 weeks at 18 to 22 C with a 16-hr photoperiod and light
intensity of 110 E m2 sec1 .
VIGS will occur in the upper, uninoculated leaves within 4 weeks of stab inoculation.
Induce microtuberization
4. Collect plant tissue from above the inoculated leaves under sterile conditions. Cut
off leaves and cut stem into pieces containing single nodes. Place nodal cuttings on
the surface of a Petri dish containing microtuberization medium and seal plate with
Nescofilm. Incubate at 16 C with an 8 hr photoperiod of 80 E m2 sec1 .
5. After 7 days wrap petri dishes in aluminum foil to exclude light and continue
incubating at 16 C for a further 28 days.
6. Harvest the microtubers by pulling them from the agar medium and characterize
the silencing response by appropriate analytical methods, e.g., semiquantitative or
quantitative RT-PCR, northern blotting, and immunoblotting.
A/C/U/gTP mix, 10
Use of PVX-Based
Vectors for Gene
Expression and
VIGS
16I.1.8
Supplement 8
20 mM ATP
20 mM CTP
20 mM UTP
2 mM GTP
Store in aliquots up to 6 months at 20 C
Current Protocols in Microbiology
Agromix
10 mM MgCl2 (add from 1 M filter-sterilized stock; store up to 1 year at 4 C)
150 M acetosyringone (add from 10 mM stock in ethanol; store up to 1 year at
20 C)
10 mM 2-(N-morpholino)ethanesulfonic acid (MES; add from 0.5 M filter-sterilized
stock, pH adjusted to 5.6 with KOH; store up to 1 year at 4 C)
Prepare fresh
Microtuberization medium
Murashige & Skoog (MS) basal medium (Sigma)
6% (w/v) sucrose
0.8% (w/v) Bacto Agar (BD Difco)
Adjust pH to 5.8
Autoclave
Aseptically add filter-sterilized 1 M chlorocholine chloride (Sigma) to final concentration of 7 mM
Aseptically add filter-sterilized 1 mg/ml benzylaminopurine (Sigma) to final concentration of 8 M
Store up to 1 month at 18 to 20 C
Potato explants propagation medium
Murashige & Skoog (MS) basal medium, with macro- and micronutrients and
vitamins (Sigma)
2% (w/v) sucrose
0.8% (w/v) Bacto Agar (BD Difco)
Adjust pH to 5.8
Autoclave
Store up to 6 months at 4 C
COMMENTARY
Background Information
PVX-based vectors were developed to facilitate rapid, transient expression of foreign
genes in plants (Chapman et al., 1992) and
to down-regulate genes through antisense approaches. Plant virusbased vectors were produced to circumvent the lengthy procedures
involved in generating transgenic plants and
to achieve higher levels of foreign protein expression. The rapidity of such vectors is still
one of their primary advantages, and the levels of expression achievable with some tobacco mosaic virus (TMV)based vectors are
still greater than those achievable by any other
means (Marillonnet et al., 2004). The development of Agrobacterium infiltration techniques
has made transient expression and gene silencing possible without virus-based vectors
(Johansen and Carrington, 2001; Voinnet et
al., 2003). However, when virus-based vectors
are used in conjunction with Agrobacterium,
they are still advantageous in terms of levels
of expression, the strength of the silencing responses they induce in host plants, and their
adaptability to high-throughput studies. How-
Plant RNA
Viruses
16I.1.9
Current Protocols in Microbiology
Supplement 8
Use of PVX-Based
Vectors for Gene
Expression and
VIGS
studies, as they give less symptomatic infections and induce more pervasive and sustained
VIGS responses (Ratcliff et al., 2001).
Although PVX is distributed in potatogrowing regions worldwide, the disease that it
causes is not of major economic importance.
In potatoes, PVX causes mild mosaic symptoms or is latent, but can cause much more severe symptoms in mixed infections with other
viruses, such as potato virus Y. PVX is readily,
mechanically transmissible; this is probably
its major route of spread, as it does not have
known vectors. Potato cultivars are protected
against infection by strain-specific hypersensitivity genes, including Nx, and extreme resistance (Rx) genes that protect against all but
two strains of PVX.
16I.1.10
Supplement 8
In vitro transcription
As with most procedures involving RNA,
it is essential to avoid RNase contamination,
and a number of precautions are advised to
avoid this. First, gloves should be worn during
all procedures to prevent transfer of RNases
from hands. Second, nuclease-free solutions
and ultrapure RNase-free water and chemicals should be used. These can be purchased
from suppliers, or where appropriate, solutions can be treated with diethylpyrocarbonate (DEPC). Third, equipment, such as microcentrifuge tubes, pipet tips, and glassware
should be RNase free. The latter can be baked
to inactivate RNases. Fourth, standard laboratory equipment that cannot be baked (e.g., that
used in gel electrophoresis), should be cleaned
thoroughly. Optionally, an RNase decontamination solution, such as RNaseZap (Ambion),
can be used to remove RNase from glassware and plastic surfaces, including microcentrifuge tubes.
In Basic Protocol 1, a method for the production of in vitro transcripts is presented; alternatively, commercially available kits, such
as T7 mMESSAGE mMACHINE (Ambion),
Table 16I.1.1 Host Plants Used for PVX-Based Systemic Expression and Virus-Induced Gene
Silencing
Genus
Species
Vectors
Expression
VIGS
Nicotiana
benthamiana
P2C2S
pGR106
clevelandii
Not tested
paniculata
Not tested
glutinosa
Not tested
rustica
Not tested
tabacum
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Lycopersicon
esculentum cultivars
MoneyMaker, Marmande,
Rossol, Rio Grande
P2C2S
pGR106
pimpinellifolium
Solanum
P2C2S
pGR106
bulbocastanum
Capsicum
annuum
P2C2S
Plant RNA
Viruses
16I.1.11
Current Protocols in Microbiology
Supplement 8
Anticipated Results
The levels of overexpression or VIGS
achieved with PVX-based vectors are not predictable and will vary from gene to gene.
Time Considerations
Once plasmid DNAs or transformed
Agrobacterium have been obtained, the inoculation procedures described here take a few
hours if only a few different constructs are
being tested. Obviously, the time taken will
increase if library screening is undertaken.
A major component of the time taken is required for in planta virus propagation. This
can range from less than 1 week for overexpression studies on inoculated leaves to up to
2 months to achieve VIGS in microtubers. The
time required for analysis will depend on the
gene of interest and the method of analysis. Immunoblotting of extracted proteins in protein
overexpression studies and real-time RT-PCR
in VIGS studies can be expected to take 2 to 3
days.
Literature Cited
Baulcombe, D.C., Chapman, S., and Santa Cruz,
S. 1995. Jellyfish green fluorescent protein as
a reporter for virus infections. Plant J. 7:10451053.
Chapman, S., Kavanagh, T., and Baulcombe, D.C.
1992. Potato virus X as a vector for gene expression in plants. Plant J. 2:549-557.
Faivre-Rampant, O., Gilroy, E.M., Hrubikova, K.,
Hein, I., Millam, S., Loake, G.J., Birch, P.,
Taylor, M., and Lacomme, C. 2004. Potato virus
X-induced gene silencing in leaves and tubers of
potato. Plant Phys. 134:1308-1316.
Hellens, R., Mullineaux, P., and Klee, H. 2000a.
Technical focus: A guide to Agrobacterium
binary Ti vectors. Trends Plant Sci. 5:446451.
16I.1.12
Supplement 8
Key Reference
Chapman et al., 1992. See above.
Seminal paper describing construction and use of
PVX-based vectors for expression.
Internet Resources
http://www.ayeaye.tsl.ac.uk
Provides a source to obtain PVX-based vectors.
Also gives sequence information for vectors and
methods for their use.
http://www.pgreen.ac.uk/JIT/pSoup.htm
Provide a source to obtain pSoup helper plasmid.
Also gives sequence information for pSoup- and
pGREEN-derived vectors.
Plant RNA
Viruses
16I.1.13
Current Protocols in Microbiology
Supplement 8
UNIT 16I.4
ABSTRACT
This unit describes principles and protocols for expressing a gene of interest in plant cells
using gene vectors that are derived from an infectious full-length cDNA plasmid of the
tomato bushy stunt virus (TBSV) genomic RNA, and from defective interfering RNAs
(DIs). The TBSV gene vector system permits convenient cloning, allows modification
and abundant expression of the gene of interest, and facilitates biosecure containment
of the gene vectors. These vectors can be employed for functional genomics studies and
for analyzing the biochemical properties and subcellular distribution of expressed RNAs
and/or their cognate proteins. As with other plant virus gene vectors, recombination
and deletion of the gene of interest during virus multiplication limits the application
of the TBSV gene vectors to the inoculated cells or leaves. Curr. Protoc. Microbiol.
C 2007 by John Wiley & Sons, Inc.
7:16I.4.1-16I.4.16.
Keywords: tomato bushy stunt virus r defective interfering RNAs r gene vectors r
gene of interest
INTRODUCTION
At the present time, expression of foreign genes in plants is routinely achieved in many
laboratories through Agrobacterium-mediated transformation or biolistic delivery followed by the regeneration of transgenic plants. This approach results in a stable, genetically inherited trait. As an alternative methodology, transient expression techniques are
also being used. For this purpose, Agrobacterium-mediated agroinfiltration or biolistic
delivery through (hand-held) gene guns (UNIT 16B.3) can be applied to achieve high levels
of temporary gene expression at local sites. These methods are expedient and do not need
to await the time-consuming process of selecting transgenic lines.
As reviewed in detail in the literature (Pogue et al., 2002; Scholthof et al., 2002), in
order to conveniently obtain expression of foreign genes in a relatively short time span,
plant viruses have also been explored as gene vectors. Advantages of virus-mediated gene
delivery include high levels of replication and concurrent expression of foreign genes, and
the repeatable application of the same gene vector for inoculation on multiple susceptible
plants without the necessity for genetic transformation of each host species. A number of
plant viruses have been investigated for their utility as gene vectors (as described in the
other units of Section 16I), and each has specific advantages and disadvantages. Some of
these can be predicted, but the success often depends on the virus-host combination, and,
more importantly, on the type and size of the insert, because these factors substantially
influence the stability and expression of foreign genes in the recombinant virus. In this
unit, we will describe methods of utilizing gene vectors derived from tomato bushy stunt
virus (TBSV) and its defective interfering RNAs (DIs).
Plant RNA
Viruses
Current Protocols in Microbiology 16I.4.1-16I.4.16, November 2007
Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc16i04s7
C 2007 John Wiley & Sons, Inc.
Copyright
16I.4.1
Supplement 7
TBSV is the type member of the Tombusvirus genus in the Tombusviridae, with a very
broad host range. The TBSV genome consists of a single-stranded, plus-sense RNA
(4.8 kb) that essentially functions as an mRNA for translation of the two replicationassociated proteins, P33 and P92 (Fig. 16I.4.1). The capsid protein (CP) is translated from
p41 on subgenomic messenger RNA1 (sgRNA1), and not only is required for encapsidation, but also plays an active role in virus transport (Desvoyes and Scholthof, 2002; Qu and
Morris, 2002). TBSV encodes two additional proteins, P22 and P19, that are expressed via
a second subgenomic mRNA (sgRNA2) which encodes two nested open reading frames
(ORFs) for translation of the p22 and p19 genes (Figs. 16I.4.1 and 16I.4.2). P22 is required
for cell-to-cell movement, whereas P19 is involved in host-specific systemic invasion and
acts as a suppressor of post-transcriptional gene silencing (Desvoyes et al., 2002; Qiu
et al., 2002; Turina et al., 2003). An intriguing and exploitable property of TBSV is that,
during infection of laboratory hosts, it can generate high levels of DIs that are coreplicating deletion mutants of 400 nucleotides (White and Morris, 1999). DIs are composed
of four contiguous regions (I, II, III, and IV) derived from the helper TBSV genome.
The abundant proliferation of DIs interferes with the accumulation of the genomic and
subgenomic RNAs, which generally results in attenuated symptoms on infected plants.
Using Vectors
Derived from
TBSV and TBSV
DIs
Figure 16I.4.1 TBSV-based gene vectors. Schematic diagram of TBSV RNA- and DNA-based
vectors to illustrate the viral genome and the composition of defective interfering RNAs (DIs). The
function of each protein (numbers represent molecular mass in kDa) is listed on top of the diagram. Rectangles denote coding regions of the TBSV genome, and lines represent untranslated
sequences. The four regions (I, II, III, and IV) of DIs are presented on top of the diagram for the
plasmid pHS21. Unique restriction sites for cloning purposes are listed below each diagram. Abbreviations: MSC, multiple cloning sites; MP, movement protein; sGFP, synthetic green uorescent
protein; HDVagrz, hepatitis delta virus antigenomic ribozyme; CaMV 35S, the cauliower mosaic
virus 35S promoter; T7, the DNA-dependent RNA polymerase promoter of the T7 bacteriophage.
16I.4.2
Supplement 7
Figure 16I.4.2 Levels of TBSV RNA accumulation. (A) An agarose gel image is shown to illustrate that TBSV genomic RNA accumulates abundantly and can be readily visualized upon
ethidium bromide staining. (B) The identity of the single-stranded genomic (gRNA) and subgenomic RNAs (sgRNA1 and sgRNA2) can be conrmed by hybridization with TBSV-specic probes.
These particular results were obtained with wild-type TBSV viral RNAs that accumulate in infected
Nicotiana benthamiana protoplasts. Abbreviations: M, mock-inoculated; T, TBSV-infected. The
double-stranded genomic RNA is visible above the gRNA in the lanes labeled T on the ethidium
bromide-stained gel.
The concept of using TBSV as a gene vector for expressing foreign genes was first
demonstrated by replacement of a large portion of the TBSV CP gene with reporter
genes (Scholthof et al., 1993). This application was based on a discovery that the TBSV
CP is not required to establish an infection of certain Nicotiana species (Scholthof et al.,
1993; Russo et al., 1994); this has since also been found to be the case for other species
(Table 16I.4.1). In the prototype version of the TBSV gene vector, the foreign gene was
inserted as an in-frame fusion with the 5 end of the CP gene. This resulted in high levels
of expression due to the efficient initiation of translation from the native CP start codon.
However, the absence of multiple cloning (restriction enzyme) sites (MCS) precluded
convenient cloning of foreign genes of choice. For this purpose, a new version of the
TBSV gene vector was engineered in which an MCS was incorporated to substitute the
major part of the CP gene, and the native start codon of the CP gene was inactivated (Fig.
16I.4.1). This strategy permits translational initiation from the endogenous start codon
of any inserted foreign gene (Scholthof et al., 1996).
The plasmid of the new TBSV gene vector (referred to as pHST2) contains a 4103-bp
TBSV-derived cDNA (Fig. 16I.4.1). Infection of plants with this gene vector is initiated
by inoculating T7 polymerasegenerated RNA that is transcribed from linearized pHST2
DNA templates. Thus, pHST2 represents an RNA-based TBSV gene vector. To provide
an alternative T7 polymeraseindependent method of infecting plants with TBSV gene
vectors, a DNA-based gene vector was developed. In this vector, the TBSV cDNA
backbone of pHST2 is positioned downstream of the universally applied cauliflower
mosaic virus (CaMV) 35S promoter and upstream of the hepatitis deltavirus antigenomic
ribozyme and the nopaline synthase poly(A) signal (Scholthof, 1999). The DNA-based
TBSV gene vector is referred to as pHST12 (Fig. 16I.4.1). Direct rub-inoculation of the
recombinant pHST12 plasmid DNA results in an infection that also sustains expression
of the foreign gene in inoculated leaves (Scholthof, 1999). For expression of small
Plant RNA
Viruses
16I.4.3
Current Protocols in Microbiology
Supplement 7
Table 16I.4.1 Examples of Using TBSV Gene Vectors to Express Foreign Genesa
Foreign genes
Cloning strategy
References
CaMV gene II
Replacement of CP
CAT
In-frame fusion to CP
N-terminal and
remainder of CP
replaced
N. benthamiana
N. clevelandii
GFP
Replacement of CP
N. benthamiana
GUSb
In-frame fusion to CP
N-terminal and
remainder of CP
replaced.
HIV-1 gp120
V3 loop
N. benthamiana
HIV-1 p24
In-frame fusion to
N-terminal of CP
N. benthamiana
PMV CPb
Replacement of CP
C. quinoa
Scholthof (1999)
RCNMV RNA-2
Replacement of CP
N. benthamiana
sGFP
Replacement of CP
N. benthamiana
SPMV CP
Replacement of CP
N. benthamiana
TEV P1
Replacement of CP
N. benthamiana
a Abbreviations: CaMV, cauliflower mosaic virus; CAT, chloramphenicol acetyltransferase; GFP, green fluorescent
protein; GUS, -glucuronidase; RCNMV, red clover necrotic mosaic virus; PMV, Panicum mosaic virus; sGFP, synthetic
GFP; SPMV, satellite panicum mosaic virus; TEV, tobacco etch virus; HIV, human immunodeficiency virus.
b These foreign genes were cloned in both RNA- and DNA-based TBSV gene vectors.
proteins, TBSV has also been used as an epitope-presentation vector whereby peptides
of interest decorate the surface of the particles (Joelson et al., 1997; Zhang et al., 2000).
The examples in Table 16I.4.1 illustrate various applications of the TBSV-derived gene
vectors.
Using Vectors
Derived from
TBSV and TBSV
DIs
This unit focuses on strategies employing RNA- and DNA-based TBSV gene vectors to
express a gene of interest in plants. The protocols below represent the general procedures
for expressing a gene of interest from TBSV gene vector pHST2 or pHST12 in Nicotiana
benthamiana, a model plant species for testing the infectivity of plant viruses and for
expression of foreign genes. Figure 16I.4.1 illustrates the main important features of
these gene vectors. The Basic Protocol details the application of the RNA-based gene
vector system, starting with the purification of pHST2 plasmid DNA and going on
to describe strategies for cloning the gene of interest into the pHST2 or its derived
plasmids, generating RNA transcripts in vitro, and inoculation of these transcripts onto
N. benthamiana plants. The Strategic Planning section introduces two assays for detecting
the presence of the gene of interestencoded RNA and/or protein in the infected plants.
Alternate Protocol 1 briefly describes the additional unique features associated with the
DNA-based gene vector system, and Alternate Protocol 2 introduces a method of cloning
a gene-of-interest fragment into a DI gene vector (pHS21) for RNA interference (RNAi)
assays.
16I.4.4
Supplement 7
STRATEGIC PLANNING
Protoplasts as Alternative Expression Systems
In addition to whole plants, RNA transcripts can also be inoculated directly into protoplasts for expression of a foreign gene (Fig. 16I.4.3). For general-application protocols
to prepare protoplasts from host plants and for inoculating protoplasts with RNA transcripts, we refer to Section 16D (particularly UNIT 16D.4). However, it is worthwhile to
include a few salient notes for transfecting N. benthamiana protoplasts with TBSV RNA
transcripts: (1) use 4 105 protoplasts for transfection; (2) treat RNA transcripts
with RNase-free DNase after the completion of in vitro transcription reaction; (3) use a
polyethylene glycol (PEG, mol. wt. 1540)mediated transfection procedure (40% PEG,
pH 5.6, 3 mM CaCl2 ); (4) harvest protoplasts at 24 hr after inoculation and extract total
RNAs using protoplast RNA extraction buffer (see recipe in Reagents and Solutions),
followed by standard phenol/chloroform extraction and ethanol precipitation. Users can
also refer to UNIT 16E.2 for extracting viral RNA from protoplasts.
When preparing the PEG solution, no chemicals should remain insoluble (giving a slight
cloudy appearance). It is best to autoclave PEG and a stock of CaCl2 separately prior to
preparation of the final solution.
Alternative Hosts
This unit mostly focuses on the use of N. benthamiana plants, but an apparent advantage
of the system relates to the broad host range of TBSV. The virus will establish infections upon rub-inoculation of transcripts onto a variety of plants (see Table 16I.4.1 for
examples). Best results are obtained when using young plants with three or four fully
Figure 16I.4.3 TBSV-mediated expression of green uorescent protein (GFP) from a TBSV vector reminiscent of pHST2
(construct kindly provided by Teresa Rubio and Andy Jackson). (A) Protoplasts prepared from leaf tissues of Nicotiana
benthamiana transfected with a TBSV vector expressing GFP. The protoplasts shown in the image were illuminated with
blue light. The red-colored cells (appearing as dark in the black and white reproduction) represent uninfected protoplasts
containing autouorescent chloroplasts. The green and yellow color (green superimposed on red; gray in the black and
white version) that is due to GFP expression can be observed in some of the protoplasts. The image serves as an
example to illustrate that protoplasts can be infected with RNA transcripts of TBSV gene vectors for transient expression
of a foreign gene and for inspecting infectivity of a recombinant TBSV virus. (B) N. benthamiana leaves 5 days postinoculation with TBSV expressing GFP (right), compared to mock-inoculated leaves (left). The bright-eld images (top)
show the slight chlorotic symptoms typical upon inoculation of this host with TBSV vectors; the UV-illuminated panels
(bottom) show the GFP uorescence. Images courtesy of Yi-Cheng Hsieh. For the color version of this gure go to
http://www.currentprotocols.com.
Plant RNA
Viruses
16I.4.5
Current Protocols in Microbiology
Supplement 7
Using Vectors
Derived from
TBSV and TBSV
DIs
To enable the application of standard purification protocols, it is advisable to fuse a Histag to the gene of interest protein. Protein affinity chromatography purification protocols
can then be applied to isolate the protein (see Kimple and Sondek, 2004). For example,
the authors have used this methodology to purify TBSV-expressed His-tagged GFP from
infected plants (W. Qiu and H.B. Scholthof, unpub. observ.). Extraction buffers and other
reagents are the same as those prescribed by the suppliers of chromatography materials
(e.g., GE Healthcare), with the only variation being that the plant material is pulverized
in a mortar and pestle or in a blender, which is followed by a low speed-centrifugation
to remove insoluble debris.
16I.4.6
Supplement 7
BASIC
PROTOCOL
Materials
TBSV gene vector (pHST2) DNA (can be obtained from the authors at
http://scholthoflab.tamu.edu)
E. coli DH5 competent cells
LB medium containing 50 to 100 g/ml ampicillin (APPENDIX 4A)
Column-based plasmid purification kit (e.g., QIAprep Spin Miniprep kit; Qiagen)
Gene-of-interest DNA fragment
Restriction endonucleases XhoI, SacI, and SnaBI (and other restriction enzymes as
needed) and corresponding buffers
Forward and reverse PCR primers for gene of interest (see Kramer and Coen, 2001)
PfuUltraII Fusion HS DNA polymerase (Stratagene, cat. no. 600670; optional)
Klenow fragment of DNA polymerase I
Plasmid harboring Kanr gene (e.g., pKAN-2 or Gateway pENTR)
1% agarose gel prepared in TBE buffer (Voytas, 2000)
TE buffer (APPENDIX 2A)
50 U/l T7 RNA polymerase (Invitrogen, cat. no. 18033-19) and 5 T7 buffer
100 mM dithiothreitol (DTT)
10 mM NTP mix (Invitrogen, cat. no. 18109-017)
RNAsin ribonuclease inhibitor (Promega)
RNA inoculation buffer (see recipe)
Nicotiana benthamiana plants
Climate-controlled growth chambers (Conviron; http://www.conviron.com)
Additional reagents and equipment for transformation of E. coli (Seidman et al.,
1997), plasmid DNA isolation (Engebrecht et al., 1991), polymerase chain
reaction (PCR; Kramer and Coen, 2001), insertion of DNA fragments into
plasmids (Struhl, 1991), use of Klenow fragment of DNA polymerase I to
generate blunt ends (Tabor et al., 1997), phenol/chloroform extraction and
ethanol precipitation of DNA (Moore and Dowhan, 2002), agarose gel
electrophoresis (Voytas, 2000), spectrophotometric quantitation of nucleic acids
(Gallagher and Desjardins, 2006), and inoculation of RNA transcripts into whole
plants (UNIT 16B.1 in this manual)
Amplify and purify the plasmid
1. Amplify pHST2 DNA as a regular plasmid in E. coli DH5 competent cells cultured
in LB medium containing 50 to 100 g/ml ampicillin overnight.
Heat shock (Seidman et al., 1997) is the preferred method of transformation.
Plant RNA
Viruses
16I.4.7
Supplement 7
a modified version (sGFP) of the green fluorescent protein (GFP) gene in place of the
CP gene. The pTBSV-sGFP/p19 construct is similar, but with the added capability of
expressing P19. Both plasmids have additional BglII, MluI, and PstI sites to increase
cloning flexibility (Fig. 16I.4.1). The sGFP gene can simply be substituted with the gene
of interest, and the resultant recombinant plasmid is essentially the same as the one made
by inserting the gene of interest fragment into the pHST2 plasmid.
3. Design a strategy for cloning the gene of interest into the pHST2 plasmid based on
the restriction sites flanking the gene of interest (either derived from another plasmid
or as a PCR product) and the available MCS of pHST2 (Fig. 16I.4.1; also see Ausubel
et al., 2007).
To permit detection of protein expression (Palmer and Wingfield, 2004) it might be practical to fuse a tag sequence to the open reading frame of the gene of interest prior to
insertion into the TBSV vectors.
For restriction enzyme digestion of plasmids, gel purification of DNA fragments containing
the gene of interest, ligation of the gene of interest insert into pHST2, transformation of E.
coli, screening of recombinant plasmids, and other routine cloning methods, see relevant
units in Current Protocols in Molecular Biology (Ausubel et al., 2007).
IMPORTANT NOTE: A condition for using the pHST2 gene vector is that the gene-ofinterest DNA fragment not contain either an SmaI or a SphI site. Those sites are located
at the 3 end of the TBSV cDNA (Fig. 16I.4.1), and one of those will be used to linearize
the plasmid to prepare a DNA template for the in vitro transcription reaction (step 13).
Using Vectors
Derived from
TBSV and TBSV
DIs
16I.4.8
Supplement 7
i. PCR amplification (Kramer and Coen, 2001) with a polymerase (e.g., the highfidelity PfuUltra II Fusion HS DNA polymerase) that does not generate single
nucleotide overhangs.
ii. Treatment of cohesive ends or overhangs at the termini of the gene-of-interest
DNA fragments with DNA polymerase Klenow fragment (utilizing either the 3 -5
exonuclease or 5 -3 polymerase activity; Tabor et al., 1997).
iii. By designing blunt-end restriction enzyme sites on both ends of the gene-of-interest
DNA fragment.
5b. Digest pHST2 with SnaBI to obtain a linearized plasmid with blunt ends.
6b. Insert the blunt-end gene-of-interest DNA fragment into the plasmid (Struhl, 1991).
Current Protocols in Microbiology
7b. Check the orientation of the gene of interest fragment after cloning by using an
internal unique restriction enzyme site and the unique SmaI or SphI site located 3
distal to the location of the insertion (Fig. 16I.4.1).
Alternative cloning method to avoid purification of DNA fragments from agarose gels
4c. First, clone the gene of interest into an intermediate plasmid harboring the Kanr gene
(Struhl, 1991)e.g., pUC-derived Kanr vectors such as pKAN-2 that was used in a
previous study (Qiu and Scholthof, 2004) or the Gateway pENTR vectors that allow
directional cloning of PCR products into a Kan background.
5c. Subsequently, digest the intermediate pKAN-gene of interest and pHST2 using the
same restriction enzymes (such as XhoI and SacI).
6c. Perform phenol/chloroform extraction and ethanol precipitation of the products
(Moore and Dowhan, 2002).
7c. Mix the DNA fragments and perform a ligation reaction (Struhl, 1991).
8. Transform E. coli, then select transformed bacterial colonies on ampicillin-containing
medium (Seidman et al., 1997).
11. Perform two phenol/chloroform extractions, then ethanol precipitate the linearized
plasmids. Collect precipitated DNA by centrifugation, vacuum dry the preparation,
and dissolve the DNA template in 20 l of TE buffer.
The above procedures are described in Moore and Dowhan (2002).
Plant RNA
Viruses
16I.4.9
Supplement 7
Inoculate RNA transcripts onto whole plants (also see UNIT 16B.1)
15. Mix RNA transcripts 1:5 with RNA inoculation buffer, and gently rub-inoculate
the mixture onto N. benthamiana plants as described in UNIT 16B.1. Allow plants to
recover 16 hr in the dark at room temperature with 100% humidity.
The rub-inoculation with the mixture containing Celite will introduce microscopic wounds
that serve as the entry points for the RNA transcripts present in the inoculum.
16. After the 16-hr recovery period, maintain the inoculated plants in a climate-controlled
growth chamber with 14 hr daytime at 24 C, and 20 C night temperature.
The 100% humidity during recovery improves infectivity of RNA transcripts, and this can
simply be achieved by covering the RNA-inoculated plants with moist paper sheets. It is
advisable to inoculate plants with transcripts from empty pHST2 plasmids to provide
a negative control sample for subsequent analysis of the gene of interest RNA and/or
protein expression. At the same time, inoculation of plants with these empty transcripts
will serve as a positive control to verify that the transcription reaction yields infectious
RNA transcripts. If RNA transcripts are not infectious on plants, it is recommended to
test their ability to infect protoplasts (see Strategic Planning) to determine if the gene of
interest insert has detrimental effects on replication and/or viral transcription.
ALTERNATE
PROTOCOL 1
Using Vectors
Derived from
TBSV and TBSV
DIs
16I.4.10
Supplement 7
interest is cloned into pHST12 or pHST34 as in the Basic Protocol. The pHST12 contains
the same TBSV cDNA as pHST2, while pHST34 is a modified version of pHST12 with
the added feature of expressing P19. Figure 16I.4.1 illustrates the main features of
pHST12 and pHST34. After confirming that the gene of interest is correctly inserted
into the vector, purify plasmid DNAs and directly rub-inoculate these onto leaves of host
plants as in the Basic Protocol for transcript inoculation. Normally, 10 g of plasmid
DNA is sufficient for rub-inoculation of a Celite-dusted leaf. The subsequent phases of
recovery, monitoring infectivity, and evaluating expression of the gene of interest, are
the same as in Basic Protocol 1.
How the rub-inoculated DNA is transferred to the nucleus for in vivo transcription is
not known, but it most likely involves a nonspecific translocation event in the damaged
cell. Upon transfer into the nucleus, transcription occurs and the viral RNA is properly
processed and transported out of the nucleus for translation in the cytoplasm. This is then
followed by viral transcription and replication to establish an infection. The DNA-based
gene vector can also be used in combination with agroinfiltration (see UNIT 16B.2), whereby
Agrobacterium is used to transfer and integrate the viral DNA into the plant nucleus.
Subsequent transcription is then followed by nuclear exit of the RNA into the cytoplasm
to initiate an infection.
ALTERNATE
PROTOCOL 2
16I.4.11
Current Protocols in Microbiology
Supplement 7
COMMENTARY
Background Information
Using Vectors
Derived from
TBSV and TBSV
DIs
16I.4.12
Supplement 7
Critical Parameters
Cloning strategy
There are several options for cloning a gene
of interest into the MCS of TBSV gene-vector
constructs (Fig. 16I.4.1). Transferring the gene
of interest from the recombinant plasmid directly into the TBSV gene vector is preferred
because this method ensures the true-to-origin
sequences of the gene of interest. If restriction
sites are incompatible or unavailable, cloning
gene-of-interest PCR products amplified by a
high-fidelity DNA polymerase into the TBSV
gene vector is an option. To further improve
general utility of the TBSV gene vector system, TBSV gene vector can be converted into
a Gateway-compatible destination vector with
the Gateway Vector Conversion System (Invitrogen, cat. no, 11828-029). In this novel
cloning system, the gene of interest can first
be cloned into pENTR/TOPO plasmid, and
then transferred to the TBSV gene vector by
the convenient recombination reaction. Refer
to http://www.invitrogen.com for details about
the Gateway technology
Another factor to be considered is the size
of the gene of interest. Smaller-sized inserts of
<1 kb are generally preferred, but secondarystructure features of the inserted RNA are
also influential, yet thus far mostly unpredictable. These structural properties are important within the context of gene-vector stability
in plants.
16I.4.13
Current Protocols in Microbiology
Supplement 7
Troubleshooting
Using Vectors
Derived from
TBSV and TBSV
DIs
Anticipated Results
Nucleic acid biochemistry
If the TBSV gene-vector infection successfully establishes in inoculated plants, then it
is expected that relatively high and readily detectable levels of viral RNAs will accumulate.
When the gene-of-interest insert is maintained,
then the size of the genomic RNA and sgRNA1
that hybridize with gene of interest-specific
probes should be of the same size as those
that hybridize with TBSV-specific probes in
parallel tests.
Protein biochemistry
If the objective involves expression of protein, then the high levels of TBSV accumulation will ensure detection of the foreign protein by the gene of interestspecific antiserum
in immunoblot assays, and the proteins (if soluble) can be purified.
RNAi
Even when the gene-of-interest inserts are
deleted during infection, studies with N. benthamiana showed that the initial rounds of replication can be sufficient to induce RNAi (Qiu
et al., 2002; Hou and Qiu, 2003).
Time Considerations
In anticipation of cloning success, the first
action should involve planting seed. For hosts
such as N. benthamiana, it takes 6 to 8 weeks
before plants have grown to a suitable size with
three or four fully expanded leaves for inoculation. As little as 2 days can be sufficient to
clone the gene of interest into the TBSV gene
vector plasmid and confirm the insertion of the
16I.4.14
Supplement 7
Literature Cited
Ausubel, F.M., Brent, R., Kingston, R.E., Moore,
D.D., Seidman, J.G., Smith, J.A. and Struhl, K.
(eds.). 2007. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
Blanc, S., Schmidt, I., Vantard, M., Scholthof,
H.B., Kuhl, G., Esperandieu, P., Cerutti, M., and
Louis, C. 1996. The aphid transmission factor
of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant
cells. Proc. Natl. Acad. Sci. U.S.A. 93:1515815163.
Qiu, W.P. and Scholthof, K.-B.G. 2004. Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes
with a suppressor of virus-induced gene silencing. Mol. Plant-Microbe Interact. 17:263-271.
Qu, F. and Morris, T.J. 2002. Efficient infection of
Nicotiana benthamiana by Tomato bushy stunt
virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing. Mol. Plant-Microbe Interact. 15:193202.
Rubio, T., Borja, M., Scholthof, H.B., Feldstein,
P.A., Morris, T.J., and Jackson, A.O.
1999. Broad-spectrum protection against
Tombusviruses elicited by defective interfering RNAs in transgenic plants. J. Virol.
73:5070-5078.
Russo, M., Burgyan, J., and Martelli, G.P. 1994.
Molecular biology of Tombusviridae. Adv. Virus
Res. 44:381-428.
Scholthof, H.B. 1999. Rapid delivery of foreign
genes into plants by direct rub-inoculation with
Plant RNA
Viruses
16I.4.15
Current Protocols in Microbiology
Supplement 7
Key References
Scholthof, 1999. See above.
Describes features and methodology of DNA-based
TBSV gene vectors pHST12 and pHST34.
Scholthof, et al. 1996. See above.
A detailed review on background, principles, and
application of virus gene vectors.
Qiu, et al. 2002. See above.
Presents an example of using TBSV gene vectors to
induce silencing, allowing for functional studies of
coexpressed suppressor protein.
Hou and Qiu, 2003. See above.
A case study of applying both TBSV and defective interfering RNA (DI) as gene vectors in genesilencing research.
Using Vectors
Derived from
TBSV and TBSV
DIs
16I.4.16
Supplement 7
UNIT 16I.5
The unit describes the application of satellite RNA virus vectors for induction of transient
gene silencing in the tobacco species Nicotiana tabacum. It demonstrates the power of
using RNA virus vectors for highly efficient gene function discovery or validation studies
in plants.
Satellite tobacco mosaic virus (STMV) is a plus single-stranded RNA virus of 1059
nucleotides. It encodes its own coat protein but depends on helper virus proteins for
replication and movement. The combination of native autonomous helper viruses, such
as TMV-U2 (which is used in this protocol), with a tailor-made infectious vector of a
naturally occurring satellite virus, such as STMV, permits the uncoupling of the components needed for virus replication from those components that are essential for durable
silencing induction. Such a two-component silencing system enhances stable and highly
efficient performance. The authors refer to this TMV and STMV silencing system as a
satellite virusinduced silencing system (SVISS).
The selected virus combination used in these protocols works efficiently in Nicotiana
tabacum SR1 plants, producing gene silencing phenotypes in 10 to 12 days after inoculation (see Basic Protocol 3). In this tobacco species, the helper virus (TMV-U2;
see Support Protocol 2) causes no or only minor symptoms (see Critical Parameters
and Troubleshooting). It is crucial for the successful use of the system that the tobacco
plants are cultivated under good standard growth conditions (see Support Protocol 1)
before and after virus infection. Physiologically weak or stressed plants are less prone to
allowing the accumulation of high levels of viruses and therefore will not induce gene
silencing to a desired extent. It is also crucial that the target gene insert size does not
exceed 250 bp. Insertion of fragments >250 bp results in significantly lower levels of
satellite virus RNA and thus less-efficient silencing induction. In vitro transcription (see
Basic Protocol 1) and RNA isolation and analysis (see Basic Protocol 4) follow standard
protocols. Because satellite virus RNA is neither 5 capped nor 3 polyadenylated in vivo,
satellite virus RNA that is synthesized in vitro does not require end protection. This is
highly advantageous with respect to time and resource constraints.
In this unit, the authors describe only the TMV-STMV-based system (see Basic Protocol
2). They expect that other naturally occurring helper virussatellite virus combinations
can, however, be converted into transient silencing systems and can be implemented for
plant species other than tobacco. They also include a short description of combining
helper virus inoculation and satellite virus agroinfiltration (see Alternate Protocol 1).
This combination permits the delivery of the satellite virus RNA, including the target
gene insert, in a form that is protected by bacterial structures and becomes transiently
expressed in the inoculated leaves via the strong CaMV 35S promoter. The authors have
observed that this approach results in less variability of the obtained silencing phenotype
among individual plants. Therefore, the combination of helper virus inoculation with
satellite virus agroinfiltration can be a preferred method when highly uniform silencing
phenotypes of a larger number of plants are required. A method for infecting plants
with sap or total RNA from leaves that show a silencing phenotype is also included (see
Alternate Protocol 2).
CAUTION: Although TMV and STMV are not pathogenic to humans and animals, both
viruses can easily be spread by direct contact from humans to plants, or from infected
Plant RNA
Viruses
Contributed by Veronique Gossele and Michael Metzlaff
Current Protocols in Microbiology (2005) 16I.5.1-16I.5.17
C 2005 by John Wiley & Sons, Inc.
Copyright
16I.5.1
BASIC
PROTOCOL 1
Using Satellite
TMV Vectors for
Gene Silencing
Figure 16I.5.1 Physical map of satellite tobacco mosaic virus (STMV) vector pVE349. The
authors have obtained the best results with pVE349 and recommend using this vector rst when
setting up SVISS. Abbreviations: bla, -lactamase; PSP6, SP6 promoter sequence; PT7, T7
promoter sequence; stmv 5 and 3 , full-length STMV cDNA sequence, with a polylinker inserted.
16I.5.2
Current Protocols in Microbiology
Figure 16I.5.2 Physical map of STMV vector pVE350. Abbreviations: bla, -lactamase; PSP6,
SP6 promoter sequence; PT7, T7 promoter sequence; stmv 5 and 3 , STMV cDNA sequence,
without 275 bp of the STMV coat protein, with a polylinker inserted.
Fragments of plant genes targeted for gene silencing can be inserted into the STMV
vectors using the SbfI and NotI sites. The most straightforward way is to amplify the
target gene fragment using primers that contain the SbfI and NotI recognition sites at the
5 and 3 ends, respectively, and to insert the fragment into the SbfI-NotI double-digested
vector.
Materials
SbfI and NotI and BsaI or HindIII restriction enzymes and appropriate buffers
Satellite tobacco mosaic virus (STMV) vectors pVE349 or pVE350 (Figs. 16I.5.1
and 16I.5.2)
Buffered phenol (APPENDIX 2A)
25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (APPENDIX 2A)
24:1 (v/v) chloroform/isoamyl alcohol
5 M sodium perchlorate (NaClO4 )
Isopropanol
RNase-free H2 O
MEGAscript high-yield T7 transcription kit (Ambion)
Additional reagents and equipment for PCR, subcloning DNA fragments, DNA
sequencing (optional), agarose gel electrophoresis, measuring DNA and RNA
concentration by UV spectrophotometry, denaturing formaldehyde agarose gel
electrophoresis (UNIT 16E.3)
1. Choose a target gene fragment of 50 to 250 bp for which an optimal primer combination can be designed. Add an SbfI recognition sequence to the 5 primer and a
NotI recognition sequence to the 3 primer.
The recognition sequence of SbfI is 5 -CCTGCAGG-3 , and that of NotI is 5 GCGGCCGC-3 .
Plant RNA
Viruses
16I.5.3
Current Protocols in Microbiology
3. Digest PCR fragment with SbfI and NotI and digest vector pVE349 or pVE350 with
the same enzymes. Gel purify the PCR fragment.
The digested vector can be used without gel purification.
4. Ligate vector and insert fragments following standard protocols. Check the correct insertion of the fragment in the resulting clones by restriction enzyme and/or
sequence analysis.
5. Digest 10 g STMV vector plasmid DNA with BsaI or HindIII. Check on a gel
whether digest is complete.
For efficient synthesis of chimeric STMV in vitro transcripts, a fully linear plasmid DNA
template should be used. The STMV vectors pVE349 and pVE350 contain a BsaI and
HindIII recognition site downstream of the STMV coding sequence and upstream of the
SP6 promoter sequence (Figs. 16I.5.1 and 16I.5.2). Depending on the sequence of the
inserted target, either BsaI or HindIII is chosen for linearization of the template. The
chosen enzyme recognition site should not be present in the target sequence.
The HindIII digest creates a single band of a fully linear plasmid. The BsaI digest generates
two bands (1300 and 2300 bp) because of the presence of a second BsaI site further
downstream in the vector (Figs. 16I.5.1 and 16I.5.2). The smaller band is needed, but the
presence of the larger band does not affect the following steps. No fragment purification
is needed.
7. Carry out an in vitro transcription reaction using a MEGAscript high-yield T7 transcription kit. Recover the in vitro transcript RNA using lithium chloride precipitation
solution as described by the manufacturer. Resuspend RNA in RNAse-free water and
measure the concentration.
The authors use the Ambion kit because it produces the highest yields in their hands.
They have also obtained good yields with in vitro transcription kits from other suppliers.
Alternatively, transcripts can be prepared using T7 polymerase without a kit (e.g., see
UNIT 16D.5).
Using Satellite
TMV Vectors for
Gene Silencing
16I.5.4
Current Protocols in Microbiology
8. Check the quality of the RNA on a denaturing formaldehyde agarose gel. Load 0.5
to 1 g transcript.
A single 1300-nucleotide band should be visible for the full-length in vitro RNA. If partial
degradation has occurred during in vitro synthesis, the band would be accompanied by
a smear. Partially degraded in vitro RNA results in poor performance of satellite virus
induced silencing system (SVISS), and the transcripts should be remade.
9. Store intact in vitro transcripts indefinitely at 20 C until they are needed for
inoculation.
BASIC
PROTOCOL 2
The authors normally co-inoculate five to ten plants per construct with in vitro satellite
tobacco mosaic virus (STMV) RNA and TMV-U2 helper virus leaf sap. As controls,
one or two plants are infected with helper virus only and one or two plants are mock
inoculated with phosphate buffer only. To avoid degradation of in vitro transcripts, the
time taken to manipulate the in vitro transcript should be kept as short as possible.
NOTE: Before thawing STMV and TMV-U2 stock solutions (see Basic Protocol 1 and
Support Protocol 2), check and choose the plants to be inoculated carefully. Use only
well-developed, nondamaged plants for infection. Label the pots and decide in advance
for each plant which leaf should be inoculated. Choose the youngest fully developed leaf
for inoculation. Labeling the pots at the side of the inoculated leaf will help to identify
the inoculated leaf later during phenotype screening.
NOTE: Plants should be cultivated before and after virus infection under the most optimal
growth conditions to obtain physiologically fit and unstressed plants. Do not use plants
that show signs of physiological weakness, such as chlorosis, atonic leaves, or extended
internodes. This is the major prerequisite for stable and optimal performance of the
satellite virusinduced silencing system (SVISS).
Materials
STMV in vitro transcript of interest (see Basic Protocol 1)
0.2 M sodium phosphate buffer, pH 7.0 (APPENDIX 2A), autoclaved
Carborundum powder (e.g., silicon carbide, 400 grinding compound; Alfa Aesar
cat. no. 39800)
Nicotiana tabacum SR1 plants (see Support Protocol 1), each with leaf chosen for
inoculation
TMV-U2-containing sap (see Support Protocol 2), store on ice
Plastic squirt bottle containing water
0.5-cm (diameter) brush, does not need to be sterilized
1. Dilute STMV in vitro transcript of interest in 0.2 M sodium phosphate buffer to a
concentration of 50 g/ml and a final volume of 50 l per each plant to be inoculated.
Keep diluted in vitro transcript on ice for 1 hr while preparing plants for inoculation.
2. Using a 0.5-cm brush, apply carborundum powder on the upper side of a Nicotiana
tabacum SR1 leaf or leaves (for multiple plants) that will be inoculated in one series.
Carefully brush over the complete leaf surface.
Always inoculate five plants per construct in one go. If more than five plants need to be
infected at a time, they should be inoculated in series of five.
Plant RNA
Viruses
16I.5.5
Current Protocols in Microbiology
3. Immediately pipet 50 l in vitro transcript on each brushed leaf. Spread RNA over
the complete surface of the leaves with your gloved finger. Remove glove and replace
with a clean glove.
A clean glove needs to be used when applying the helper virus in the next step.
The virus-contaminated gloves, together with all used pipet tips and rinsing water, should
be collected for safe disposal.
4. Pipet 50 l TMV-U2-containing sap on the same leaves. Again, spread the sap
over the complete surface. Remove glove and replace with a clean glove.
Because the sap can not be easily pipetted with small (yellow) pipet tips, the larger (blue)
pipet tips should be used.
5. Wait for a few minutes and then rinse inoculated leaves thoroughly with water using
a plastic squirt bottle. Rinse leaves before they are completely dry.
The water in the squirt bottle does not need to be sterile.
6. Apply carborundum powder as described (step 2) to leaves from one or two plants
each to inoculate with TMV-U2 alone and to mock inoculate.
7. Pipet 50 l TMV-U2-containing sap on a carborundum-brushed leaf or leaves for
the TMV-U2 control. Spread the virus with your gloved finger, wait a few minutes,
and rinse thoroughly. Remove glove and replace with a clean glove.
8. Mock inoculate one or two plants by pipetting 50 l of 0.2 M sodium phosphate
buffer on a carborundum-brushed leaf. Spread the buffer with your gloved finger,
wait a few minutes, and rinse.
SUPPORT
PROTOCOL 1
Materials
Nicotiana tabacum SR1 seeds (available from many seed suppliers)
5% (v/v) bleach solution
Tween 20
H2 O, sterile
14-cm sterile polystyrene plates (e.g., Falcon 3025) containing 100 ml N. tabacum
germination medium (see recipe)
12-cm flowerpots containing soil
Plant growth room (UNIT 16A.3), 24 C, equipped with timer-controlled grow lights
and controlled relative humidity
Using Satellite
TMV Vectors for
Gene Silencing
16I.5.6
Current Protocols in Microbiology
5. Grow plants in a growth room at 24 to 25 C, with the same light/dark cycle and relative air humidity of 60% to 80% (UNIT 16A.4). Water regularly but avoid continuously
wet soil, which leads to water stress and root damage.
Depending on the growth conditions, the plants reach the 5- to 6-leaf stage in 2 to 3
weeks. This is the best stage for virus inoculation.
Young plants should be used for inoculation. Using older plants will result in lower efficiency of inoculation and gene silencing induction. Because of this, silencing phenotypes
that are less pronounced may result.
Suboptimal conditions can result in slower plant growth. As long as plants remain healthy,
this should not affect the infection and silencing efficiency.
SUPPORT
PROTOCOL 2
The leaf sap of TMV-U2-infected plants contains high concentrations of virus particles
and can therefore be used for infection of new tobacco plants (as described in Section
16B). The inoculated leaf and all upper leaves can be harvested 1 to 2 weeks after
inoculation of plants with leaf sap from infected plants. The harvested leaves must be
frozen immediately in liquid nitrogen. They can be stored for up to 10 years or longer at
70 C. For longer storage and shipment of material, the leaf material should be freezedried. The authors can supply interested users of SVISS with freeze-dried tobacco leaves
infected with TMV-U2 to start with.
Materials
Leaf material from TMV-U2-infected plants, frozen at 70 C or freeze-dried
0.2 M sodium phosphate buffer, pH 7.0 (APPENDIX 2A), autoclaved
Mortar and pestle, prechilled
Additional reagents and equipment for preparing total RNA from leaves (UNIT
16E.1), measuring RNA concentration by UV spectrophotometry, and denaturing
agarose gel electrophoresis
1. Transfer an 4-cm2 portion of frozen or freeze-dried leaf material from TMV-U2infected plants into a prechilled mortar.
An additional amount of leaf material will be needed to measure the amount of TMV RNA
that is present (step 3).
2. Add 0.5 ml of 0.2 M sodium phosphate buffer and grind leaf material with a
prechilled pestle. Add 2 ml more sodium phosphate buffer so fluid can easily be
pipetted with an automatic pipet. Transfer sap into 1.5- or 2.2-ml microcentrifuge
tubes and keep on ice.
This sap can be used for inoculation of new tobacco plants (see Basic Protocol 2) or can
be stored indefinitely as a stock at 70 C. For storage, the sap should be divided into
small aliquots; repeated freeze-thawing should be avoided.
Plant RNA
Viruses
16I.5.7
Current Protocols in Microbiology
Figure 16I.5.3 Physical map of the agroinltration vector pTVE334. Abbreviations: ColE1, origin of replication of the naturally occurring E. coli plasmid ColE1; LB, left border of T-DNA;
P35S3, cauliower mosaic virus 35S promoter; Rb, right border of T-DNA; Sm/Sp, streptomycin/spectomycin resistance gene; stmv, full-length satellite tobacco mosaic virus cDNA sequence; 3 nos, 3 untranslated region of the nopaline synthase gene.
3. To check whether the sap contains sufficient levels of TMV, prepare total RNA from
an additional sample of the leaf material (UNIT 16E.1).
4. Run 5 to 10 g RNA, as determined by UV spectrophotometry, on a denaturing
(formaldehyde) agarose gel.
The TMV-U2 RNA should be visible after ethidium bromide staining as a separate band
that runs slower than all ribosomal bands (the length of TMV RNA is 6355 nucleotides).
ALTERNATE
PROTOCOL 1
Using Satellite
TMV Vectors for
Gene Silencing
16I.5.8
Current Protocols in Microbiology
4. After the 3-hr incubation, brush the tobacco leaves with carborundum and inoculate
with TMV-U2-containing sap as described (see Basic Protocol 2, step 2 and step 4).
TMV-only and mock-inoculated controls should be set up as described (see Basic Protocol
2, steps 6 to 8).
6. After a few minutes, rinse off carborundum and excess TMV-U2-containing sap
with water using a plastic squirt bottle.
ALTERNATE
PROTOCOL 2
Plants can also be efficiently infected with sap or with total RNA from leaves that
show a silencing phenotype. This is very easy to perform and results in highly uniform
phenotypes of all inoculated plants. Prepare sap of the silenced leaf tissue (see Support
Protocol 2) and inoculate the plants as described for TMV-U2 control inoculations (see
Basic Protocol 2). Because the sap contains TMV particles, there is no separate TMV
inoculation needed. Alternatively, isolate total RNA from infected leaves (e.g., UNIT 16E.1)
and directly apply the total RNA preparations to tobacco plants as described earlier for
in vitro transcripts (see Basic Protocol 2). Because the total RNA preparations contain
both STMV and TMV-U2 RNAs, no separate TMV infection is needed. The authors
have obtained good results with a volume of 50 to 100 l and a concentration of 100 g
total RNA/ml 0.2 M sodium phosphate buffer, pH 7.0.
Plant RNA
Viruses
16I.5.9
Current Protocols in Microbiology
BASIC
PROTOCOL 3
BASIC
PROTOCOL 4
Using Satellite
TMV Vectors for
Gene Silencing
CAUTION: When working with 32 P, investigators should use appropriate shielding and
frequently check themselves and the work area for radioactivity using a hand-held monitor. All proper guidelines for radioisotopes should be followed. Radioactive waste should
be placed in appropriately designated areas for disposal. Follow the guidelines provided
by your local radiation safety adviser. See APPENDIX 1C for more information.
16I.5.10
Current Protocols in Microbiology
Materials
Frozen leaf samples, harvested at appropriate time points (see Basic Protocol 3),
including noninoculated leaf samples for transcript quantification only
STMV in vitro transcript (see Basic Protocol 1), for transcript quantification only
Hybond N+ filter (e.g., Amersham Biosciences)
STMV or target gene probe, either subcloned in an appropriate vector or as PCR
fragment
Additional reagents and equipment for preparing RNA from leaf samples
(UNIT 16E.1) and northern analysis of plant RNA (UNIT 16E.3)
1. Prepare total RNA from frozen leaf samples (including TMV-only and mockinoculated samples) using a standard protocol (UNIT 16E.1). Prepare RNA from noninoculated leaves if quantification of transcripts is desired (step 3).
Usually, the third upper leaf is the first leaf that shows a clear silencing phenotype and
still has high STMV RNA levels. The next three emerging upper leaves show a stronger
phenotype but lower STMV RNA levels. This indicates that gene silencing is at its height
in these upper leaves, degrading most of the target gene mRNA and chimeric STMV RNA.
2a. For detection of STMV RNA: Load 1 g total RNA on a denaturing agarose gel.
2b. For detection of target gene mRNA: Load 5 to 10 g total RNA on a denaturing
agarose gel.
3. Optional: Load a dilution series of transcripts in the same gel for quantification. Use
STMV in vitro transcript diluted 0.5 pg to 5 ng and each mixed with 1 g total RNA
isolated from noninoculated tobacco leaves. For mRNA detection, adjust dilution
series to lower quantities, depending on the abundance of the mRNA in leaves.
It is especially important to carry out target gene mRNA detection when no silencing
phenotype is observed. This will determine whether the absence of a phenotype is due to
loss of STMV replication.
4. Run the gel and blot onto a Hybond N+ filter using standard procedures.
TMV RNA can be seen after electrophoresis in ethidium bromidestained gels as an extra
slow-moving band that is located above all rRNA bands. STMV RNA runs within the
region of rRNA and can be seen only after hybridization with an STMV-specific probe.
5. Hybridize the filter with an STMV probe or a probe for detecting the target gene
mRNA.
For STMV hybridization, the authors use an STMV 5 leader sequence riboprobe. This
probe contains the first 200 nucleotides of the STMV cDNA. The authors cloned this
fragment into a pGEM vector for general use as a probe, but PCR fragments can also be
used directly.
For detecting the target gene mRNA, an appropriate target gene probe should be designed
and prepared. A 3 end fragment of the STMV sequence is not recommended for use as a
probe because it is partially homologous with a specific region of the TMV-U2 sequence.
An STMV 3 end probe will therefore also hybridize with TMV-U2 RNA.
6. Expose the hybridized filter after washing to an X-ray film. The levels of STMV RNA
are usually very high and therefore short exposures (for a few hours) are sufficient.
For mRNA detection, longer exposures will be needed.
Plant RNA
Viruses
16I.5.11
Current Protocols in Microbiology
Agroinfiltration medium
4.3 g/liter Murashige and Skoog salts (Duchefa Biochemie)
20 g/liter sucrose
500 M 2-(N-morpholino)ethanesulfonic acid (MES)
Adjust pH to 5.6 with 3 M KOH
Autoclave for 15 min
Add 10 mM acetosyringone (filter sterilized with 0.45-m filter) before use
Store up to 6 months at room temperature
This medium can be used for most plant species.
COMMENTARY
Background Information
Using Satellite
TMV Vectors for
Gene Silencing
16I.5.12
Current Protocols in Microbiology
Plant RNA
Viruses
16I.5.13
Current Protocols in Microbiology
RNA secondary structures that are formed differentially depending on the orientation of the
insert. The authors therefore suggest producing constructs in the sense and antisense orientation for each target gene and inoculating
separate plants with each construct. For subsequent experiments, the construct with the
highest frequency of gene silencing induction
can be used.
The authors have also produced chimeric
vectors for simultaneous silencing of two target genes by inserting small fragments for both
target genes in tandem orientation. Again, the
total insert size should not exceed 250 bp. This
approach resulted in simultaneous downregulation of the expression of both genes, although
the frequency and extent of gene silencing
were lower than for vectors with a single target
gene insert.
In vitro transcription of infectious STMV
clones
For in vitro transcription, the authors use
standard kits and have obtained the best results with the Ambion MEGAscript T7 kit.
The template plasmid DNA should be fully
linear. Contamination with circular plasmid
significantly reduces the final concentration of
in vitro transcripts. Therefore, all restriction
enzyme digests of template DNA should be
checked on agarose gels.
STMV RNA forms stable stem-loop structures at both ends and is not 5 capped or
3 polyadenylated. Therefore, STMV in vitro
transcripts do not need in vitro capping and
polyadenylation. It is important, however, that
only sterilized equipment and RNase-free solutions are used for synthesis and subsequent
handling of STMV in vitro RNA. Always
check the quality of the STMV in vitro transcripts on denaturing formaldehyde agarose
gels. If STMV in vitro transcripts are partially
degraded, a smear occurs. Use only fully intact in vitro transcripts for inoculation to obtain best results for SVISS. In vitro transcripts
can be stored at 20 C for up to 12 weeks.
For long-term storage (up to several years),
the transcripts should be stored at 70 C.
Using Satellite
TMV Vectors for
Gene Silencing
16I.5.14
Current Protocols in Microbiology
Anticipated Results
SVISS is an easy-to-perform, transient
gene-silencing tool. As discussed earlier, the
performance of the system is very much dependent on the efficiency of infection with the
helper and satellite viruses, which in turn depends on the physiological conditions of the
plants used for infection. Once these conditions are established and all steps of the protocol are standardized, the output of gene silencing data can be very high, considering that
most of the experimental steps described in
the protocols can be performed in parallel for
numerous target genes. It must be remembered
that SVISS is a transient, virus-based system.
Thus, the silencing phenotype will in most
cases disappear over time because of recovery of the plant from virus infection, and the
silencing phenotype will not occur in all tissues (e.g., in the case of leaf-expressed genes,
most leaves will show a variegation phenotype). When used to determine gene function,
SVISS should be regarded as a screening tool
for identifying candidate genes, which can
then be channeled into detailed gene function
validation studies.
Time Considerations
Depending on the containment space
that is available for growing virus-infected
plants, hundreds of transient gene silencing
experiments can be performed in a few
months. PCR amplification of target gene
Plant RNA
Viruses
16I.5.15
Current Protocols in Microbiology
Figure 16I.5.4 Satellite virusinduced silencing system (SVISS) tobacco knockout plants, 4 weeks after inoculation with
chimeric satellite tobacco mosaic virus (STMV) in vitro transcripts and TMV helper virus leaf extract. The wild-type TMVand STMV-inoculated plant shows almost no viral symptoms, whereas plants co-inoculated with wild-type TMV leaf extract
and target genespecic chimeric STMV in vitro transcripts show numerous changes in pigmentation, leaf structure, and
development. The targeted genes are indicated for each photograph. (This black and white facsimile of the gure is
intended only as a placeholder; for full-color version of gure go to http://www.interscience.wiley.com/c p/colorgures.htm.)
Table 16I.5.1 Timeline for Satellite VirusInduced Silencing System
Using Satellite
TMV Vectors for
Gene Silencing
Procedure
Time required
1 day
2 days
4-6 weeks
/2 day
/2 day
10-12 days
postinoculation
Analyzing virus RNA and target gene mRNA (see Basic Protocol 4)
4 days
inoculation in parallel with many other constructs. For a single transient silencing experiment, the time estimation provided in Table 16I.5.1 can be used as a general guideline. In summary, within 14 days after
16I.5.16
Current Protocols in Microbiology
Key References
Literature Cited
Internet Resources
http://www.ambion.com/catalog
Provides much information on RNA, RNAi, and enzymes for in vitro RNA procedures.
http://www.esi-topics.com/genesil
Summarizes publication and citation data from ISI
Essential Science Indicators for the analysis of research trends and performance in gene silencing.
http://tgi.ncsu.edu
Provides information on the progress of the Tobacco
Genome Initiative, launched in 2003, with the aim to
sequence the whole genome of Nicotiana tabacum.
Plant RNA
Viruses
16I.5.17
Current Protocols in Microbiology
UNIT 16I.6
Over the past decade, researchers have developed various methods for exploiting viruses
to study gene function. This unit will explain how viral vectors are used for virus-induced
gene silencing (VIGS) of an endogenous gene. Many different VIGS vectors have been
developed over the past decade, including vectors based on tobacco mosaic virus (TMV),
potato virus X (PVX), tomato golden mosaic virus (TGMV), and tobacco rattle virus
(TRV). TRV-based silencing provides the most robust, persistent, and consistent silencing results without causing severe viral symptoms; therefore, protocols optimized for
TRV-based silencing in plant species belonging to the family Solanaceae, of which tobacco and the tomato are representatives, are provided in this unit. TRV is a bipartite
virus and requires two vectors containing either RNA1 or RNA2. RNA1 encodes two
replicases, a movement protein, and a small (16-kDa) protein of unknown function (Fig.
16I.6.1). RNA2 encodes the coat protein and two nonessential proteins. The nonessential
proteins in RNA2 were removed and replaced with a multiple cloning site that can be
used to insert the gene of interest (Fig. 16I.6.2). The protocols below rely on transforming Agrobacterium tumefaciens with a modified TRV vector containing a segment of a
gene targeted for VIGS. Agrobacterium is used as delivery system for the TRV-based
binary vectors to avoid laborious in vitro transcription of viral RNA and inoculation
Figure 16I.6.1 Physical map of the tobacco rattle virus RNA1 vector pYL192. The RNA1 vector in
Agrobacterium must be coinltrated with an RNA2 vector. Abbreviations: RB, right border; LB, left
border; 2X35S promoter, two copies of the cauliower mosaic virus (CaMV) 35S promoter; 134K,
134-kDa replicase; 194K, 194-kDa replicase translated as a read-through of the 134K stop codon;
MP, movement protein; 16K, 16-kDa protein; Rz, ribozyme; NOS terminator, nopaline synthase
terminator.
Plant RNA
Viruses
Contributed by Jeffrey Caplan and S.P. Dinesh-Kumar
Current Protocols in Microbiology (2006) 16I.6.1-16I.6.13
C 2006 by John Wiley & Sons, Inc.
Copyright
16I.6.1
Supplement 1
Figure 16I.6.2 Physical map of the tobacco rattle virus RNA2 vector pYL156. A fragment of the
gene of interest will be inserted into the multiple cloning site. Abbreviations: RB, right border; LB,
left border; 2X35S promoter, two copies of the cauliower mosaic virus (CaMV) 35S promoter;
CP, coat protein; MCS, multiple cloning site; Rz, ribozyme; NOS terminator, nopaline synthase
terminator.
with unstable RNA. The four protocols described below differ in the method used for
inoculation of Agrobacterium into the plant. Basic Protocol 1 introduces the Agrobacterium via syringe infiltration (agroinfiltration), and is the most robust method for VIGS
in Nicotiana benthamiana. Basic Protocol 2 uses an Agrobacterium drench (agrodrench)
method that has been tested in seven different Solanaceae species. This unit also describes two alternative methods which result in higher success rates when silencing in
Lycopersicon esculentum (tomato). Alternate Protocol 1 inoculates Agrobacterium by
a spray-inoculation method, and Alternate Protocol 2 inoculates Agrobacterium via a
vacuum-infiltration method. These protocols are complemented by support protocols for
culturing Agrobacterium for VIGS (Support Protocol 1), construction of a TRV vector
containing a segment of target gene for silencing (Support Protocol 2), and growing the
two plants most commonly used for VIGS, N. benthamiana (Support Protocol 3) and
L. esculentum (Support Protocol 4).
CAUTION: Agrobacterium is a Biosafety Level 1 (BSL-1) organism. Such organisms
are not known to consistently cause disease in healthy adult humans, and are of minimal
potential hazard to laboratory personnel and the environment. Standard microbiological
practices should be followed when working with these organisms. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for more information.
Using Viral
Vectors to
Silence
Endogenous
Genes
16I.6.2
Supplement 1
BASIC
PROTOCOL 1
Materials
Agrobacterium GV2260 containing pTRV1 vector (Support Protocol 1)
Agrobacterium GV2260 containing pTRV2 with target gene (pTRV2-TARGET;
Support Protocol 1)
VIGS agroinfiltration medium (see recipe)
N. benthamiana or tomato plants (Support Protocol 2 or 3)
1-ml syringe without needle
1. Separately resuspend pelleted Agrobacterium GV2260 (see last step of Support
Protocol 1) containing the TRV1 or TRV2-TARGET vector in 5 ml VIGS agroinfiltration medium.
For discussion of controls, see Critical Parameters.
2. Using VIGS agroinfiltration medium, adjust the OD600 to 1.0 for N. benthamiana or
1.5 to 2.0 for tomato.
3. Incubate cultures 4 to 6 hr at room temperature.
4. Mix the cultures of Agrobacterium containing pTRV1 and pTRV2 vectors in a 1:1
ratio.
5. Load a 1-ml syringe (without needle) with the mixture of Agrobacterium.
6. Using the corner of a new razor blade, make a 0.1-mm hole in the two lower leaves
of N. benthamiana or tomato plants.
7. Place the opening of the 1-ml syringe (without needle) containing the Agrobacterium
mixture over the hole.
8. Using a finger from the other hand, apply light pressure on the opposite face of the
leaf to form a seal between the tip of the syringe and the leaf. Squeeze the syringe to
infiltrate the Agrobacterium mixture into the leaf.
The penetration of the Agrobacterium into the plant cells can be clearly seen on the
underside of the leaf.
9. Repeat steps 5 to 8 until the entire two lower leaves are infiltrated with the Agrobacterium mixture.
10. Maintain the growth temperature at 25 C for N. benthamiana and between 18 to
21 C for tomato.
11. Quantify or visualize VIGS of the target gene (see Critical Parameters) at 6 to
10 days post infiltration of Agrobacterium.
BASIC
PROTOCOL 2
Plant RNA
Viruses
16I.6.3
Current Protocols in Microbiology
Supplement 1
systematically tested in many different members of the Solanaceae family. The silencing
success rate is near 100% in N. benthamiana and between 60% and 70% in tomato, N.
tabacum (tobacco), Petunia hydrida, Solanum tuberosum (potato), Capsicum annuum
(pepper), and eggplant (Ryu et al., 2004). The agrodrench method does not require fully
expanded leaves; consequently, VIGS can be conducted on plants as young as 1 week old.
Furthermore, because the agrodrench method is faster and requires fewer materials than
the other protocols described in this unit, it may be the best method for high-throughput
VIGS-based forward genetic screens.
Materials
Agrobacterium GV2260 containing pTRV1 vector (Support Protocol 1)
Agrobacterium GV2260 containing pTRV2 with target gene (pTRV2-TARGET;
Support Protocol 1)
VIGS agroinfiltration medium (see recipe)
Plant of Solanaceae family: N. benthamiana (Support Protocol 3), tomato (Support
Protocol 4), pepper, potato, petunia, or eggplant
10-ml pipet
1. Separately resuspend pelleted Agrobacterium GV2260 (see last step of Support
Protocol 1) containing the TRV1 or TRV2-TARGET vector in 5 ml VIGS agroinfiltration medium.
For discussion of controls, see Critical Parameters.
2. Adjust the OD600 of each suspension to 1.0 with VIGS agroinfiltration medium.
3. Incubate cultures 4 to 6 hr at room temperature.
4. Mix the cultures of Agrobacterium containing pTRV1 and pTRV2 vectors in a 1:1
ratio.
5. Fill a 10-ml pipet with the Agrobacterium mixture.
6. Apply 3 to 5 ml of the Agrobacterium mixture to the crown of the plant.
7. Maintain temperature between 18 C to 21 C for tomato and between 24 to 26 C
for all other Solanaceae species.
8. Quantify or visualize VIGS of the target gene (see Critical Parameters) 10 to 14 days
post drenching of Agrobacterium.
ALTERNATE
PROTOCOL 1
Materials
Using Viral
Vectors to
Silence
Endogenous
Genes
16I.6.4
Supplement 1
2. Adjust the OD600 of each suspension to 2.0 with VIGS agroinfiltration medium.
3. Incubate the cultures for 4 to 6 hr at room temperature.
4. Mix the cultures of Agrobacterium containing pTRV1 and pTRV2 vectors in a 1:1
ratio.
5. Add a pinch ( 75 to 100 mg) of carborundum to the mixed culture.
6. Attach an artists airbrush to a pressure compressor set to 80 psi. Load the Agrobacterium mixture into the reservoir of the airbrush.
7. Spray the underside of the two lower leaves of each tomato plant for 1 to 5 sec
from a distance of 8 in. (20 cm). Shake the suspension in the airbrush reservoir
periodically to prevent settling of the carborundum.
8. Maintain growth temperature between 18 to 21 C.
9. Visualize or quantify VIGS of the target gene (see Critical Parameters) at 8 to 10
days post infiltration of Agrobacterium.
ALTERNATE
PROTOCOL 2
Materials
Agrobacterium GV3101 containing pTRV1 vector (Support Protocol 1)
Agrobacterium GV3101 containing pTRV2-TARGET vector (Support Protocol 1)
VIGS agroinfiltration medium (see recipe)
Tomato plants (see Support Protocol 4)
Silwet L-77
500-ml plastic beaker
Desiccator (240 mm diameter; Bel-Art Co.)
Vacuum pump (Model FB DVP0352; Fisher)
1. Separately resuspend pelleted Agrobacterium GV3101 (see last step of Support
Protocol 1) containing the TRV1 or TRV2-TARGET vector in 5 ml VIGS agroinfiltration medium.
For discussion of controls, see Critical Parameters.
2. Adjust the OD600 of the pTRV1 culture to 0.4 and that of the pTRV2 culture to 0.2
with VIGS agroinfiltration medium.
3. Incubate the cultures 4 to 6 hr at room temperature.
Current Protocols in Microbiology
Plant RNA
Viruses
16I.6.5
Supplement 1
4. Mix the cultures containing pTRV1 and pTRV2 vectors in a 1:1 ratio. Transfer the
culture mixture to a 500-ml plastic beaker. Add Silwet L-77 to a final concentration
of 0.04% (v/v).
It is not necessary to completely fill the beaker. Mixed Agrobacterium from two 50-ml
cultures is sufficient.
5. Completely submerge the tomato plant by placing it upside down in the plastic beaker
and immediately continue to the next step.
6. Place the beaker containing the submerged plant into a vacuum desiccator. Attach
the desiccator to the vacuum pump and pull with a maximum vacuum of 29.5 in. Hg
(749.3 mmHg) for 90 sec.
7. Maintain growth temperature between 18 to 21 C.
8. Visualize or quantify VIGS of the target gene (see Critical Parameters) at 6 to
10 days post infiltration of Agrobacterium.
SUPPORT
PROTOCOL 1
Materials
TRV1 or TRV2 vector (available by request from Dr. S. Dinesh-Kumar;
savithramma.dinesh-kumar@yale.edu)
Kit for BP Gateway or LR Gateway reactions (Invitrogen)
Agrobacterium strain GV2260 or GV3101
LB plates (APPENDIX 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin,
50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or
50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
LB medium (APPENDIX 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin,
50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or
50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
200 mM acetosyringone (see recipe)
1 M MES (see recipe)
Additional reagents and equipment for transformation of Agrobacterium (UNIT 16B.2)
1. Transform the TRV1 or TRV2 vector into either Agrobacterium strain GV2260 or
GV3101.
2a. For TRV vectors in Agrobacterium GV2260: Streak Agrobacterium containing either
the TRV1 or TRV2 vector on LB plates containing 50 mg/liter kanamycin, 25 mg/liter
rifampicin, 50 mg/liter streptomycin, and 50 mg/liter carbenicillin.
2b. For TRV vectors in Agrobacterium GV3101: Streak Agrobacterium containing either
the TRV1 or TRV2 vector on LB plates containing 50 mg/liter kanamycin and
15 mg/liter gentamycin.
3. Grow plates for 1 to 2 days at 28 C.
Using Viral
Vectors to
Silence
Endogenous
Genes
4a. For TRV vectors in Agrobacterium GV2260: Inoculate a colony from the plate into
5 ml LB medium containing kanamycin (50 mg/liter), rifampicin (25 mg/liter),
streptomycin (50 mg/liter), and carbenicillin (50 mg/liter).
4b. For TRV vectors in Agrobacterium GV3101: Inoculate a colony from the plate into
5 ml LB medium containing kanamycin (50 mg/liter) and gentamycin (15 mg/liter).
16I.6.6
Supplement 1
SUPPORT
PROTOCOL 2
A gene of interest may be silenced by inserting a piece of the target gene sequence into
a TRV2 vector such as pYL156 or pYL279 (Liu et al., 2002a,b). The following protocol
provides brief instructions for creating a TRV2-TARGET vector and transforming it into
Agrobacterium.
The pYL156 vector is an improved TRV2 cloning vector. It contains a double 35S
promoter and a ribozyme at the 3 end, which increase silencing efficiency and infectivity,
respectively. pYL279 is designed to work with the Gateway cloning system (Invitrogen)
and is optimal for high-throughput cloning of genes for VIGS.
Materials
Target gene of interest
TRV2 vector pYL156 or pYL279 (available by request from Dr. S. Dinesh-Kumar;
savithramma.dinesh-kumar@yale.edu)
pDONR221 (Invitrogen)
Competent Agrobacterium strain GV2260 or GV3101 (available by request from
Dr. S. Dinesh-Kumar; savithramma.dinesh-kumar@yale.edu)
LB plates (APPENDIX 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin,
50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or
50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
Additional reagents and equipment for PCR (Kramer and Coen, 2001), restriction
digestion (Bloch and Grossman, 1995) and ligation (Tabor, 1987), and
transformation of Agrobacterium (UNIT 16B.2)
1. Design primers to amplify a 500 to 700 bp segment of the target gene to be silenced.
Amplify the gene (Kramer and Coen, 2001).
Cloning into pYL156 or pTV00 will require the addition of restriction sites to the 5
ends of the primers. Use unique restriction sites located in the multiple cloning site (Fig.
16I.5.2).
Cloning into pYL279 will require the addition of att-B1 (GGGG-ACA-AGT-TTG-TACAAA-AAA-GCA-GGC-TNN) to the forward primer and att-B2 (GGG-AC-CAC-TTT-GTACAA-GAA-AGC-TGG-GTN) to the reverse primer.
2. Clone the fragment into the TRV2 vector pYL156 or pYL279. If cloning into TRV2
vector pYL156, cut the fragment and the vector with appropriate restriction enzymes
(Bloch and Grossman, 1995) and ligate (Tabor, 1987). If cloning into pYL279,
transfer the fragment into pDONR221 entry clone using a BP Gateway reaction.
Next, transfer the fragment from pDONR221 to pYL279 using an LR Gateway
reaction.
Refer to http://www.invitrogen.com/content/sfs/manuals/pdonr man.pdf for more information about using the pDONR221 plasmid in Gateway reactions.
Plant RNA
Viruses
16I.6.7
Current Protocols in Microbiology
Supplement 1
Materials
N. benthamiana seeds
Super Fine Germinating Mix (Conrad Fafard, Inc.; http://www.fafard.com)
Professional Growth Medium No. 2 (Conrad Fafard, Inc.; http://www.fafard.com)
1-pint (500-ml) pots, 4 in. (10 cm) square
Pot trays
Clear plastic domes
Light source (40-W Gro-Lux fluorescent light bulb, Sylvania)
1. Place germination growth medium into a 1-pint pot. Spread approximately 40 to 50
N. benthamiana seeds onto the surface of the germination growth medium. Place pot
on a tray and cover with a clear plastic dome.
2. Remove plastic dome immediately after seeds germinate.
3. Grow seedlings under continuous light at 24 to 26 C for 10 to 12 days. Water plants
every 2 to 3 days by adding 0.5 to 1 in. (1.25 to 2.5 cm) of water to the pot trays.
4. Transplant seedlings into individual 1-pint pots containing professional growth
medium and cover with clear plastic dome for 2 to 3 days.
5. Remove plastic dome and grow plants until they reach the four-leaf stage (3 weeks).
SUPPORT
PROTOCOL 4
Materials
Using Viral
Vectors to
Silence
Endogenous
Genes
16I.6.8
Supplement 1
1. Plant 1 to 2 VF36 or MicroTom tomato seeds per 1-pint pot containing professional
growth media. Cover with 0.5 in. (1.27 cm) of vermiculite. Place pots on a tray and
cover with a clear plastic dome.
2. Remove plastic dome immediately after seeds germinate.
3. Germinate seeds under a 16-hr light/8-hr dark cycle at 24 to 26 C for 12 to 14 days.
4. Keep one seedling per 1-pint pot.
5. Grow plants to the two-leaf stage (4 weeks).
Acetosyringone, 200 mM
Dissolve 0.1962 g of 3 ,5 -dimethoxy-4 -hydroxyacetophenone (acetosyringone) in
5 ml dimethylformamide (DMF).
It is advisable to make this stock fresh; however, it may be stored for up to one month at
20 C.
MES, 1 M
Add 19.62 g of 2-[N-morpholino]ethanesulfonic acid (MES) to 100 ml water. Store
up to several months at room temperature.
VIGS agroinfiltration medium
10 ml 1 M MgCl2 (10 mM final)
10 ml 1 M MES (see recipe; 10 mM final)
0.75 ml 200 mM acetosyringone (see recipe; 150 M final)
Prepare fresh (do not store)
A 1 M solution of MgCl2 can be prepared by adding 20.33 g MgCl2 6H2 O to 100 ml of
water. Store this stock up to several months at room temperature.
COMMENTARY
Background Information
Viruses are some of the most insidious and
devastating plant pathogens. They harbor extraordinary diversity, ranging from DNA to
RNA genomes and from small monopartite
to large tripartite capsulation (Hull, 2002).
Millions of years of plant evolution have resulted in a sophisticated arsenal of antiviral
defense mechanisms. One of the most effective antiviral defenses is post-transcriptional
gene silencing (PTGS), which is similar
to quelling and RNA interference (RNAi;
Waterhouse and Helliwell, 2003). The PTGS
defense system is activated by dsRNA originating from transgenes or viruses. The dsRNA
is cleaved into short, 23-nucleotide segments
called short interfering RNA (siRNA) that
associate with an RNAi silencing complex
(RISC). The RISC complex is guided by the
siRNA to degrade RNA containing 100% homology to the siRNA (Meister et al., 2004).
Plant RNA
Viruses
16I.6.9
Current Protocols in Microbiology
Supplement 1
Using Viral
Vectors to
Silence
Endogenous
Genes
16I.6.10
Supplement 1
Growth conditions
Growth conditions can affect silencing efficiency and reproducibility. Light and temperature are the two most important variables and
should be kept constant between experimental
sets. Conduct seed germination, plant growth,
and gene silencing in growth chambers that
have controlled light and temperature. VIGS in
Nicotiana sp. is favored by continuous light,
while VIGS in other Solanaceae species requires a 16 hr light/8 hr dark cycle.
The optimal temperature for Agrobacterium T-DNA insertion and VIGS may differ. Lower temperatures between 16 to 21 C
may result in greater efficiency of T-DNA insertion, and, therefore, a higher success rate
of VIGS. VIGS in tomato is more sensitive
to high temperature because the silencing success rate drops dramatically at temperatures
above 21 C. Conversely, higher temperatures,
above 24 C, have been shown to increase the
abundance of siRNAs that are associated with
VIGS (Szittya et al., 2003). Thus, the protocols
provided here may be optimized by inoculating the Agrobacterium at 16 to 21 C and then
shifting the plants to a higher temperature 24
to 26 C after 1 to 2 days. The authors of this
unit have not tested this protocol, but it should
be considered if optimizing VIGS for a new
plant species.
Age or developmental stage
The age of the plant inoculated with
Agrobacterium can affect silencing efficiency.
The four-leaf and two-leaf stages are the optimal ages for N. benthamiana and tomato
respectively. Syringe infiltration of younger
leaves in N. benthamiana may result in severe
necrosis of the inoculated leaf. If the VIGS
phenotype needs to be observed at a younger
stage in development, then use the agrodrench
method (Basic Protocol 2).
Choosing a gene segment for silencing
In order for VIGS to efficiently silence an
endogenous gene, a fragment of the gene must
be chosen. In theory, 23 nucleotides of complete identity should be sufficient for silencing. In practice, a fragment between 300 and
1500 bp should be cloned into the TRV2 vector. Below 300 bp in length, the silencing efficiency is greatly reduced. Above 1500 bp in
length, the insert may be lost from the virus
and viral movement or replication may be impaired. The optimal size of the insert is 500 to
700 bp. At least 30 bp of the mRNA target sequence should be excluded from the construct,
so RT-PCR may be used to check the silencing efficiency. Design two primers to check
Current Protocols in Microbiology
Anticipated Results
It is nearly impossible to obtain complete
knockout of a gene using VIGS. A genes transcript level is often reduced by 75% to 95%
with VIGS. This level of down-regulation is
often sufficient to disrupt a genes function.
The phenotype may be a loss of function or a
reduction of function. The results may vary depending on the plant species, age, or the gene
targeted by VIGS.
Plant RNA
Viruses
16I.6.11
Current Protocols in Microbiology
Supplement 1
VIGS has the highest success rate and silencing efficiency in N. benthamiana. If the
syringe-infiltrate method for VIGS in N. benthamiana is used, then a success rate near
100% and a silencing efficiency between 85%
and 95% can be expected. VIGS of NbCTR1
(constitutive triple response 1) and NbEDS1
(enhanced disease susceptibility 1) resulted in
a >95% reduction of their endogenous mRNA
(Liu et al., 2002a,b). However, the silencing efficiency varies with different genes and
constructs. For example, a reduction of only
78% was obtained when silencing with TRV2NPR1/NIM1 (Liu et al., 2002a).
If the spray-infiltration method is used for
VIGS in tomato, a success rate >90% and a
silencing efficiency between 75% and 85%
can be obtained. Endogenous CTR1 was reduced by 85% and the highly transcribed
ribulose bisphosphate carboxylase (RbcS) was
reduced by 76% using TRV-based VIGS
Using Viral
Vectors to
Silence
Endogenous
Genes
(Liu et al., 2002b). Similar results were obtained in Solanum species and other members
of the Solanaceae family (Brigneti et al., 2004;
Ryu et al., 2004).
Figure 16I.5.3 shows the effects of VIGS of
endogenous genes in N. benthamiana plants.
Time Considerations
The amount of time it takes to complete a
VIGS experiment is dependent on the plants
generation time and the developmental stage
at which the silencing phenotype can be observed. Silencing in N. benthamiana or tomato
can be completed in 4 to 5 weeks.
It is important to consider the amount of
time it takes to transform the TRV2 vector
into Agrobacterium. Transformations should
be done 1 week before the plants are ready
for silencing. A time commitment of 1 day
will be required for the transformation, 2 days
for the transformed Agrobacterium to grow,
Figure 16I.6.3 VIGS of endogenous genes in N. benthamiana. Pictures were taken 9 days post
syringe inltration of TRV1- and TRV2-vector-containing Agrobacterium. All of the TRV2 constructs
were coinltrated with TRV1. Empty TRV2 vector was used as a control. TRV2 containing phytoene
desaturase (PDS) resulted in bleaching of the leaves. TRV2 containing elongation factor 1a (EF1a)
or an uncharacterized gene caused severe stunting and deformation of the leaf structure. For the
color version of this gure go to http://www.currentprotocols.com.
16I.6.12
Supplement 1
Literature Cited
Bloch, K.D. and Grossman, B. 1995. Digestion of
DNA with restriction endonucleases. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G.
Seidman, J.A. Smith, and K. Struhl, eds.) pp.
3.1.1-3.1.21. John Wiley & Sons, Hoboken, N.J.
Waterhouse, P.M. and Helliwell, C.A. 2003. Exploring plant genomes by RNA-mediated gene
silencing. Nat. Rev. Genet. 4:29-38.
Plant RNA
Viruses
16I.6.13
Current Protocols in Microbiology
Supplement 1
UNIT 16K.1
Many plant viruses have positive-strand RNA as their genetic material. These viral RNAs
serve as messenger RNAs upon infection and are the templates for synthesis of proteins
necessary for replication of the virus. Analysis of the translation of plant viral RNAs
using in vitro protein synthesis systems are very useful for biochemical analysis of the
efficiency of the RNAs and the effects of 5 UTRs, 3 UTRs, m7 GpppG cap groups, and
other elements. The protocols described here are for the large-scale preparation of a wheat
germ extract (see Basic Protocol 1) and in vitro transcription of plant viral RNAs from
phage promoters (see Basic Protocol 2). The wheat germ extract is a preparation of all
the soluble components of the translational apparatus and includes ribosomes, initiation
factors, elongation factors, and aminoacyl tRNA synthetases. Wheat germ, the embryo
of the wheat kernel, is a dormant system and as such has a very low level of endogenous
messenger RNA. The use of in vitro transcription to prepare RNAs is an excellent way to
prepare large amounts of RNA to translate in the wheat germ system. Variations of RNA,
capped or uncapped, changes in UTRs, or mutations may be easily prepared by in vitro
transcription and compared in the wheat germ extract. The translation assay described
(see Basic Protocol 3) may be used to optimize the amount of extract, amount of RNA,
and concentrations of Mg2+ and K+ for efficient translation in vitro.
NOTE: All solutions that have been stored should be mixed well by inversion before use.
Use a graduated cylinder of appropriate size for measurement and preparation. Unless
otherwise indicated, store all solutions at 20 C.
BASIC
PROTOCOL 1
Materials
Wheat germ column elution buffer (see recipe)
Wheat germ extraction buffer (see recipe)
14.3 M 2-mercaptoethanol (2-ME; Sigma)
2- liter Sephadex G-25 column (see Support Protocol)
Wheat germ (e.g., Randolph & James Flax Mills), chilled: store up to 6 months at
20 C
Soybean trypsin inhibitor (STI; Sigma)
50 mM PMSF (see recipe)
Powdered dry ice or dry ice/isopropanol bath or liquid nitrogen
Blender, chilled
Slender kitchen spatula (optional)
50-ml centrifuge tubes, chilled
High-speed centrifuge (capacity up to 250 ml)
Cheese cloth
1. One day before beginning the preparation, prepare wheat germ column elution and
wheat germ extraction buffers. Chill overnight at 4 C.
Plant RNA
Viruses
Contributed by Karen S. Browning and Laura Mayberry
Current Protocols in Microbiology (2006) 16K.1.1-16K.1.13
C 2006 by John Wiley & Sons, Inc.
Copyright
16K.1.1
Supplement 1
7. Spoon the mixture into chilled 50-ml centrifuge tubes (14 tubes) and centrifuge
20 min at 25,700 g, 4 C.
The extract should be kept at 4 C or on ice at all times.
8. Gently push aside the fatty layer on the top of the supernatant with a slender spatula
and pour the liquid through a layer of cheese cloth to catch any residual fat.
Approximately 200 ml should be recovered in a chilled beaker or graduated cylinder.
9. Layer the supernatant on top of the G25 column (step 2), taking care not to disturb
the top of the resin bed. Once all of the supernatant has entered the G25 matrix,
gently layer the column elution buffer on top. Reconnect buffer reservoir containing
column elution buffer.
The column separates a brown fraction containing the macromolecular components for
protein synthesis from a bright yellow fraction of small molecules.
10. Collect 20-ml column fractions when the brown fraction begins to elute from the
bottom of the column and eluate appears cloudy (0.5 hr).
After the column run is completed, do not forget to wash the column as described (see
Support Protocol). A fraction collector may be used if desired.
11. Measure the absorbance at 260 nm on a 1:100 dilution of every other fraction. Pool
fractions containing >90 A260 .
An alternative method is to take six to eight 20-ml fractions until the A260 is >90, collect
200 ml in batch form in a beaker or graduated cylinder, and then start collecting 20-ml
fractions again. Read the A260 of the last fractions and only keep those >90.
12. Centrifuge the pooled eluate for 20 min at 30,000 g, 4 C to generate the S30
(supernatant of 30,000 g) fraction.
13. Prepare S30 aliquots of appropriate size for storage. Fast freeze by placing container
into powdered dry ice if using small aliquots (<10-ml) or into a dry ice/isopropanol
bath or liquid nitrogen if using large aliquots (>10-ml). Store at 70 C.
Powdered dry ice should be deep enough to cover the sample. The dry ice/isopropanol
bath should be prepared and allowed to cool for 15 min. Care should be taken to
prevent the isopropanol from seeping into the tubes by keeping the tube top well above
the isopropanol level. Liquid nitrogen should also not be allowed to seep into the tubes.
In Vitro
Translation of
Plant Viral RNA
For long-term storage (i.e., several months to years before use), 50-ml aliquots are used.
These are then thawed and subaliquoted to 10 ml, 1 ml, or smaller aliquots as desired.
16K.1.2
Supplement 1
SUPPORT
PROTOCOL
If after the column has been poured, it runs dry within reach of a sturdy glass rod, stir the
bed and allow to resettle. If the crack is 8 to 12 inches from the top, add water or buffer
to the column, seal the ends of the adaptors, remove the column from the clamps and
gently tilt it back and forth until the crack in the column disappears. Allow the column
to resettle. If the column runs completely dry or >50% down the column, as soon as
possible, add buffer or water to the top, remove the bottom adaptor, and allow the liquid
to displace the resin into a 4-liter beaker placed under the column; repour the column.
CAUTION: Dispose of sodium azide as described in UNIT 1A.3.
NOTE: The column should be prepared at least one day prior to use.
Materials
Sephadex G-25 slurry (Sigma, cat. no. G-25-150)
Wheat germ column wash buffer (see recipe)
2.35-liter (50 1200mm) ACE glass column (e.g., Ace Glass, cat. no. 5820-58;
http://www.aceglass.com)
Top and bottom Luer adaptors (Ace Glass, cat. no. 5837-21) with appropriate
tubing and stopcock
Buffer reservoir (e.g., 6-liter flat-style carboy) with appropriate tubing
1. Weigh out 425 g of Sephadex G-25 and allow to swell in 2.5 liters deionized water
for at least 3 hr at room temperature or overnight at 4 C. If at any time during the
swelling process there is not an excess of water, add more.
2. Set up a clean 2.35-liter ACE glass column with top and bottom Leur adaptors in a
cold room.
The fully loaded column is heavy so adequate clamping to a column rack is necessary.
3. Close the bottom adaptor and pour 200 ml water into the column. Check for leaks.
The column must not leak or the column will run dry and have to be repoured.
4. Gently swirl the G25 resin in a large beaker to resuspend, minimizing the amount of
liquid, but having enough to allow the resin to move freely. Immediately pour about
half of the G-25 slurry into the column. Allow 10 to 15 min for the resin to begin
settling. Open the bottom adaptor to allow a slow flow rate (1.0 ml/min).
5. Continue to add the slurry of G-25 until all is poured into the column.
Make every effort to prevent the bed of G-25 from settling completely until all the slurry
is added. If the bed does settle, use a very sturdy glass rod to stir the top of the bed before
adding more G-25.
6. After all the G-25 is hand-poured, allow the bed to completely settle. Turn off the
column until needed (up to 24 hr) or proceed immediately to step 7.
Do not leave column for more than 24 hr without adding 0.5% sodium azide to prevent
microbial growth.
Current Protocols in Microbiology
Plant RNA
Viruses
16K.1.3
Supplement 1
7. Attach a reservoir of buffer 12 to 18 in. above the column using appropriate tubing
to attach it to the top Luer adaptor.
8. Wash the column with 6 to 8 liters of wheat germ column wash buffer (24 hr).
9. Close the bottom adaptor and store (no more than 24 hr) until use at 4 C.
BASIC
PROTOCOL 2
Materials
Plasmid DNA
Restriction enzyme and buffer appropriate for linearizing plasmid DNA
Sterile water
Ambion MegaScript (uncapped RNA) or MessageMachine (capped RNA) T7 or
SP6 transcription kit or equivalent
Sephadex G100 slurry (see recipe)
RNA column elution buffer (see recipe)
ACE-saturated phenol (see recipe)
Chloroform
80% and 100% ethanol, 20 C
20-ml plastic columns and frits (BioRad)
Stopcocks (Baxter), sterile
1.5-ml microcentrifuge tubes, sterile
Tabletop centrifuge
13-ml sterile centrifuge tubes
Additional reagents and equipment for purifying DNA (Moore and Dowhan, 2002)
and separating RNA on a urea gel (Ellington and Pollard, 1998)
NOTE: All reagents must be prepared with ultrapure, sterile water to prevent nuclease
contamination. Autoclave all buffers, resin, columns, tubes, and other reagents and equipment to prevent nuclease contamination. Gloves should be worn and all efforts made to
prevent nuclease contamination from personnel or air sources.
1. Linearize 100 to 200 g plasmid DNA with an appropriate restriction enzyme
(5 overhang or blunt end) for run-off transcription.
2. Phenol-chloroform extract the reaction, ethanol precipitate the DNA in the presence
of ammonium acetate or other salt, and collect by centrifugation (Moore and Dowhan,
2002). Resuspend in 100 to 200 l sterile water.
The author has not found it necessary to use diethylpyrocarbonate (DEPC)-treated water,
as autoclaved, ultrapure water appears to be sufficient. However, if nuclease is a problem,
DEPC-treated water may help (see APPENDIX 2A). Plasmids can be prepared by several
different methods for use as template; however, methods that avoid RNase in the procedure
are preferred. PCR-derived template also works well.
In Vitro
Translation of
Plant Viral RNA
3. Following the manufacturers instructions for the transcription kit, assemble components for a 300- to 1000-l transcription reaction (1/2 of a 25-reaction kit or up to a
full 40-reaction kit). Add enzyme mixture to start the transcription reaction. Incubate
2 hr at 37 C.
16K.1.4
Supplement 1
4. While the transcription reaction is incubating, pour 20 ml Sephadex G100 resin into
a sterile 20-ml plastic column fitted with a sterile stopcock.
5. After the bed has settled, push a sterile frit down on top of the resin. Wash the column
with 30 to 40 ml of sterile RNA column elution buffer (0.5 hr) and turn off the
column.
This procedure is carried out at room temperature. Make every effort to ensure that
nothing falls into the column or buffer during the procedure. The frit on top of the column
protects the top from being disturbed when washing, loading, and eluting.
6. Following completion of the transcription reaction (step 3), degrade the template by
adding DNase (included in the kit) for 15 min at 37 C.
7. Adjust volume to 0.5 to 1.0 ml with sterile water if necessary.
8. Load the entire reaction mixture onto the G100 column (step 5) and elute with 10
to 15 ml of RNA column elution buffer at a flow rate of 1 ml/min.
The G100 column removes unincorporated ribonucleotides, deoxynucleotides from degradation of the template, and salts. The column elution buffer contains potassium chloride
as the salt to precipitate the RNA. Potassium chloride is compatible with protein synthesis
assays.
10. Prepare a 1:300 dilution (2 l to 598 l water) of each fraction and measure the A260 .
Discard the dilutions and pool the fractions (generally three) with the highest A260
in a 13-ml centrifuge tube.
The RNA is usually in fractions six to eight and the yield should be 1 to 2 mg for an
uncapped mRNA and 0.5 to 1 mg for a capped mRNA. The columns are only used once
for RNA work; however, the resin and column may be reused for non-RNA purposes.
11. Extract pooled fractions with an equal volume of ACE-saturated phenol. Centrifuge
the extraction mixture in a tabletop centrifuge 10 min at 3500 g, room temperature.
Remove the supernatant, avoiding the interface.
12. Extract the supernatant two times with an equal volume of chloroform. Precipitate
the RNA in the supernatant by adding 2.5 vol of 100% ethanol at 20 C. Store 1 to
2 hr at 20 C.
The phenol extraction removes T7 or SP6 polymerase, deoxyribonuclease, and any
ribonuclease contamination that may have been introduced during the handling procedures. No additional salt is necessary for precipitation of the RNA. The 150 mM KCl in the
column elution buffer is sufficient. Potassium is the preferred salt, as it is compatible with
in vitro translation conditions. Sodium or ammonium ion may be inhibitory under certain
conditions. The RNA should be visible as a snowy, fluffy, white precipitate in the ethanol.
Overnight precipitation is also acceptable, but additional salt or other contaminants may
also precipitate.
13. Collect the RNA by centrifuging 20 min at 8000 g, 4 C. Rinse the precipitate
gently with 80% ethanol at 20 C. Remove as much liquid as possible and dry the
pellet for 10 to 15 min at 37 C.
Plant RNA
Viruses
16K.1.5
Current Protocols in Microbiology
Supplement 1
15. Make a 1:300 dilution (2 l to 598 l water) and read the A260 to confirm the
concentration. Check the integrity of 1 to 2 g RNA by electrophoresis on an
appropriate percentage (e.g., 4% for large mRNAs, >1000 nucleotides) urea gel
(Ellington and Pollard, 1998).
The ultimate assay is to use the mRNA in in vitro protein synthesis assays (Basic
Protocol 3).
The activity, or how much protein is synthesized from the mRNA of the mRNA preparation, will vary depending on several factors, including the level of contaminants from
phenol extraction (phenol, chloroform, ethanol, excess salt) and, more importantly, the
integrity of the RNA. At least three preparations should be compared to get an idea of
the range of an mRNAs activity in in vitro protein synthesis. A low yield of RNA may
lead to poor protein synthesis as the contaminants present become inhibitory at higher
concentrations of RNA.
BASIC
PROTOCOL 3
Materials
In Vitro
Translation of
Plant Viral RNA
27 C water bath
90 C heating block (for filter binding analysis)
Glass fiber filters (Schleicher and Schuell; for filter binding analysis): wet with 5%
TCA prior to use
Vacuum filtration manifold (for filter binding analysis)
100 C drying oven (for filter binding analysis)
Scintillation vials (for filter binding analysis)
Additional reagents and equipment for SDS-PAGE (Gallagher, 1999)
16K.1.6
Supplement 1
Contribution to concentration
a
Component
l/tube
Water
Supermix (Leu)
K+
Mg2+
19
b
7.5
c
1 M potassium acetate
4.7
0.25
14
[ C]leucine
mRNA (0.510 pmol)
0.5 mM
3
0.5
Contribution from 15 l
S30 column bufferd
Total
94 mMc
35
36 mM
1.5 mM
130 mM
2 mM
a Always prepare one more tube than is actually needed so as not to run out of reaction mixture. For
example, the assay in Table 16K.1.2 would require a 7 reaction mixture. Note that the mixture
specified in this recipe is for a single reaction.
b The supermix may also be made with 19 amino acids but without methionine if [35 S]methionine
labeling is desired.
c Be sure to check that the amount of K+ and Mg2+ being added is compensated correctly for a
final concentration of 130 mM and 2 mM, respectively, in 50 l. Avoid adding more than 30 mM
chloride from any source, as chloride ions become inhibitory above 30 mM.
d The amount of S30 to be added depends upon the quality of the extract: a range of 7.5, 10, 12,
and 15 l or higher should be assayed to determine the optimum amount.
2a. Add the reaction components shown in Table 16K.1.2 in order from left to right.
Gently flick the tube to mix components well.
See Table 16K.1.2 for an example of an assay for activity of S30.
2b. Add the reaction components shown in Table 16K.1.4 in order from left to right.
Gently flick tube to mix components well. Proceed to step 3.
See Table 16K.1.4 for an example of an assay for RNA activity.
Current Protocols in Microbiology
Plant RNA
Viruses
16K.1.7
Supplement 1
Reaction
mixtureb (l)
S30 (l)
15c
35
7.5
35
7.5
35
10
2.5
35
12.5
35
15
35
Tube no.
15
Contribution to concentration
a
l/tube
Water
Supermix (Leu)
Mg2+
12.1
b
7.5
c
1 M potassium acetate
0.1 M magnesium
acetate
0.4
[14 C]leucine
3.0
K+
101.2 mMc
0.8 mM
S30
12
28.8 mM
1.2 mM
Total
40.0
130 mM
2 mM
a Always prepare one additional tube than is actually needed so as not to run out of reaction
mixture. For example, the assay in Table 16K.1.4 would require a 6 reaction mixture. Note
that the mixture specified in this recipe is for a single reaction.
b The supermix may also be made with 19 amino acids but without methionine if
[35 S]methionine labeling is desired.
c Be sure to check the amount of K+ and Mg2+ being added is compensated correctly if the
amount of S30 changes.
d The amount of S30 (in column elution buffer) to be added depends upon the quality of the
extract: a range should be assayed to determine the optimum amount (see Table 16K.1.2).
3. Remove the reaction tubes from ice and incubate 30 min in a 27 C water bath or the
desired time(s) for a time-course experiment.
16K.1.8
Supplement 1
Water (l)
mRNA (l)b
10
40
7.5
40
2.5
40
40
10
10
40
5a. Pass the mixture through a glass fiber filter on a vacuum filtration manifold. Rinse
the tubes two times with 3 ml of 1% TCA, each time pouring the TCA through the
filter.
The 5% and 1% TCA are placed in acid-resistant squeeze bottles for convenience and
the 3 ml may be estimated. The vacuum manifold should be set up while the tubes are
heating and the filters wetted with 5% TCA prior to the pouring of the terminated reaction
mixtures.
6a. Rinse the filters again with 3 ml of 1% TCA and allow to slightly dry in the
manifold (2 to 3 min).
7a. Remove the filters with forceps, being sure to only handle the edges of filters. Dry
10 min in a small 100 C drying oven. Place in 10 ml scintillation fluid and count in
a scintillation counter.
The filters must be completely dry. Wet spots may cause anomalies in the scintillation
counting.
ACE buffer
1.67 ml 3 M sodium acetate, pH 5.1 (final 10 mM)
1.46 g NaCl (final 50 mM)
3 ml 0.5 M EDTA (final 3 mM)
Adjust the volume to 500 ml with H2 O
Autoclave and store up to 1 year at room temperature
Plant RNA
Viruses
16K.1.9
Current Protocols in Microbiology
Supplement 1
ACE-saturated phenol
Combine 6 ml ACE buffer (see recipe) and 20 ml molecular-biology-grade phenol
(Fisher). Store up to 1 year at 20 C in the dark.
Amino acid mix minus leucine (AALeu), 5 mM each
Combine 200 l of a 0.1 M solution of each of the amino acids (store each up to
1 year at 20 C) except leucine and tryptophan. Add 400 l of a 0.05 M stock of
tryptophan. Divide into 0.25-ml aliquots and store up to 1 year at 20 C. Use once
and discard.
Amino acid mix minus methionine (AAMet), 5 mM each
Combine 200 l of a 0.1 M solution of each of the amino acids (store each up to
1 year at 20 C) except methionine and tryptophan. Add 400 l of a 0.05 M stock
of tryptophan. Divide into 0.25-ml aliquots and store up to 1 year at 20 C. Use
once and discard.
HEPES (1 M)/KOH, pH 7.6
Place 1600 ml water in a 2-liter beaker. Add a magnetic stir bar and place on a
magnetic stirrer. Slowly add 476.6 g of HEPES (USB) and stir until dissolved.
Slowly add 65 g KOH pellets and stir until dissolved. Make a 1:10 dilution and
measure the pH, which should be 7.6. If necessary, add more KOH and check the
pH of a 1:10 dilution (pH of a 1 M solution will be 8.1 when the pH of a 1:10
dilution is 7.6). Adjust volume to 2 liters with water and let sit overnight at 4 C.
Recheck pH of 1:10 dilution at room temperature; adjust pH if necessary. Store 1
liter for short term (1 to 3 weeks) at 4 C and 1 liter at 20 C. If used for RNA work,
autoclave the buffer for 20 min. Unopened autoclaved solutions may be stored 1 to
2 months at room temperature in the dark.
Magnesium acetate, 1 or 0.1 M
Prepare a 1 M solution by dissolving 107.2 g magnesium acetate (Sigma) in 500 ml
water. Prepare a 0.1 M solution by dissolving 21.5 mg magnesium acetate per
milliliter water. Store up to 1 year at 20 C. Mix thoroughly after thawing.
The solid is very hydroscopic and is stored in a metal can with desiccant.
16K.1.10
Supplement 1
Plant RNA
Viruses
16K.1.11
Current Protocols in Microbiology
Supplement 1
In Vitro
Translation of
Plant Viral RNA
16K.1.12
Supplement 1
Anticipated Results
The amount of protein synthesis will be
dependent on the efficiency of the mRNA
template. Some mRNAs, especially viral
RNAs, are very efficient in vitro and several
rounds of initiation will occur; however, other
mRNAs are not as efficient and may only
be translated one time. The amount of protein product (pmol) may be estimated from
the radioactivity incorporated based on the
number of [14 C]leucine or [35 S]methionine
residues in the protein product. The efficiency of the RNA is calculated as picomoles protein synthesized per picomoles
RNA. For example, if a viral RNA template
(10 pmol) encodes a protein containing 18
leucine residues, and the amount of radioactive [14 C]leucine obtained by filter binding is
45,000 cpm, then the calculation would be as
follows:
45,000 cpm/200 cpm/pmol [14 C]leucine
= 225 pmol [14 C]leucine
225 pmol [14 C]leucine/18 leucine per pmol
protein = 12.5 pmol protein synthesized
12.5 pmol protein synthesized/10 pmol
mRNA = 1.25 pmol protein synthesized
per pmol of mRNA
Time Considerations
The preparation of the wheat germ extract
requires 2 days (solution preparation on day
1 and extraction/purification on day 2). The
preparation of the RNA requires 4 to 5 hr
if the solutions are prepared/autoclaved ahead
of time. The assays generally take 1.5 to
2 hr (depending on incubation time) if all the
reagents are prepared.
Literature Cited
Plant RNA
Viruses
16K.1.13
Current Protocols in Microbiology
Supplement 1
UNIT 16K.2
Viral messenger RNAs must compete with cellular RNAs to be translated on ribosomes
and to support viral gene expression. Translational expression of an RNA is typically
controlled by its 5 - and 3 - untranslated regions (UTRs). Replacing all or most of the
viral coding region with a reporter gene is the most convenient way to study the role(s)
of UTRs in directing translation. Among available reporter systems, firefly luciferase
(LUC) has strong advantages, most notably an easy assay procedure and superior sensitivity (also see Brasier and Fortin, 1996). Two protocols for introduction of in vitro
transcribed RNAs into protoplasts are described in this section. The Basic Protocol illustrates electroporation of cowpea mesophyll protoplasts, and the Alternate Protocol
describes polyethylene glycol (PEG)mediated transfection. In addition, LUC activity
measurement and normalization by protein concentration, as well as experimental design
considerations and data interpretation, are described.
BASIC
PROTOCOL
In this protocol, luciferase (LUC) reporter RNA transcripts are introduced into cowpea
protoplasts by electroporation. The RNA constructs are mixed with protoplasts in cold
electroporation buffer and are then subjected to an electrical pulse with high capacitance.
After electroporation, the protoplasts are transferred to growth medium and incubated
to allow LUC expression. LUC activity and total protein are assayed in extracts made
from harvested protoplasts. The procedure describes assays based on 96-well plates
for accommodating large numbers of determinations. Detailed discussion of protoplast
preparation and electroporation are found in Section D of Chapter 16 (e.g., UNIT 16D.4).
Materials
8- to 10-day-old cowpea (Vigna unguiculata) seedlings, e.g., California
Blackeye 46
30% ethanol
Protoplast digestive enzyme solution (see recipe)
MMC buffer (see recipe)
Protoplast electroporation buffer (see recipe)
5 mg/ml fluorescein diacetate in acetone (store indefinitely at 20 C)
In vitrotranscribed RNA (2 pmol/10 l; see Critical Parameters and
Troubleshooting) containing luciferase reporter gene, suspended in H2 O: chill
on ice
Protoplast growth medium (see recipe)
Passive Lysis Buffer (Promega cat. no. E1941)
Luciferase assay reagent (see recipe)
Bradford dye-binding protein assay reagent
BSA protein standards: dilute BSA in 1 Passive Lysis Buffer to produce stock
concentrations of 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml
Whatman 3MM filter paper
Fine-point tweezers
Petri dish, 15 cm diameter
Platform rotator
30 C incubator
Miracloth (Calbiochem)
50-ml polypropylene conical tubes
Contributed by Daiki Matsuda and Theo W. Dreher
Current Protocols in Microbiology (2005) 16K.2.1-16K.2.11
C 2005 by John Wiley & Sons, Inc.
Copyright
Plant RNA
Viruses
16K.2.1
Clinical centrifuge
Hemacytometer
UV/visible fluorescence microscope (UNIT 2A.1)
Electroporation cuvettes (2-mm gap), chilled on ice
Electroporation system with capacitance extender (CE) module (Bio-Rad
GenePulser Xcell)
Pasteur pipets, autoclaved (one per electroporation)
Refrigerated centrifuge
Standard 96-well plate (optional)
Clear-bottom black 96-well plates (Costar #3631)
Microplate luminometer: e.g., Wallac Microbeta 1450 or Turner BioSystems
Veritas (http//www.turnerdesigns.com)
Microplate spectrophotometer (e.g., Molecular Devices SpectraMax 250)
Additional reagents and equipment for electroporation of protoplasts (UNIT 16D.4)
and protein assay (APPENDIX 3A)
NOTE: Avoid exposure to ribonucleases in all procedures. Use gloved hands at all times,
including when handling chemical ingredients of solutions, and make all solutions with
MilliQ-grade (Millipore) water. Alternatively, treat solutions with DEPC as described in
Appendix 2A.
2. On Whatman 3MM filter paper, cut each leaf into halves and remove the midrib.
Hold a pair of sharp fine-point tweezers near-horizontal to the leaf. Lift a minor vein
and peel parallel to it to remove the epidermis.
The lower epidermis is peeled from the leaves to permit access of the protoplast digestive
enzyme solution to mesophyll cells. This is a more gentle procedure than dicing, which
is necessary for the leaves of some types of plants. Cowpea leaves are easy to peel
and the protoplasts are particularly reliable to prepare; however, the authors have used
essentially the same procedures described here to study LUC expression in protoplasts
made from other plants and from suspension cell culture.
3. Put eight to ten half-leaves in a large petri dish containing 25 ml protoplast digestive
enzyme solution.
About 106 protoplasts can be obtained from two half-leaves.
In Vivo
Translation
Studies of Plant
Viral RNAs
Using Reporter
Genes
5. Swirl the petri dish gently three to four times to release the digested protoplasts into
the medium.
16K.2.2
Current Protocols in Microbiology
6. Strain the protoplast suspension through one layer of Miracloth (prewetted with
MMC buffer) into a 50-ml polypropylene conical tube.
Tilt the conical tube so that the protoplast suspension moves down the wall of the tube.
Gentle treatment of protoplasts will prevent rupture.
11. Adjust the protoplast concentration to 1 106 cells/ml with protoplast electroporation buffer. Aliquot 300 l (i.e., 0.3 106 cells) into a 1.7-ml microcentrifuge tube
for each electroporation to be performed.
This amount is sufficient for up to eight harvest time points (see step 16).
12. At 35- or 45-sec intervals, place each microcentrifuge tube on ice. Incubate 15 min
before electroporation.
This step establishes equal timing of samples on ice (i.e., 15 min 45 sec if working alone),
which is especially important when handling large numbers of samples. Steps 13 and 14
take 45 sec per sample when working alone or 35 sec if another person is available to
help. If early harvest time points (e.g., 15 to 20 min) are taken, they may overlap with
the electroporation points of later samples. This should be taken into consideration when
planning the work schedule. Also see Time Considerations.
Plant RNA
Viruses
16K.2.3
Current Protocols in Microbiology
15. Use a fresh, sterile, disposable glass Pasteur pipet to transfer protoplasts from the
cuvette to a microcentrifuge tube containing 1.1 ml protoplast growth medium.
To recycle cuvettes, submerge the cuvettes in water overnight, brush the inside with a test
tube brush, rinse six times with distilled, deionized water, and leave them in 70% alcohol
until use. Three hours before use, remove the ethanol and dry completely.
16. Incubate under fluorescent light at room temperature. Aliquot the protoplasts at this
time into separate microcentrifuge tubes according to the desired number of harvest
time points (e.g., eight aliquots of 170 l for eight harvest times).
Fluorescent lights should be used because photosynthesis is thought to be necessary for
the reaction.
19. Add at least 20 l of 1 Passive Lysis Buffer per 4 104 protoplasts. Vortex briefly
and shake vigorously for 5 min at room temperature.
The minimal volume of protoplast lysis buffer to lyse all the 4 104 cowpea protoplasts
was found to be 20 l. A greater volume of protoplast lysis buffer is sometimes used in order
to perform enough replicates of luciferase readings, protein concentration determinations, etc. However, using a larger volume results in dilution of expressed luciferase,
which correspondingly gives lower signals. This becomes particularly crucial if the
sample is from an early time point or if the RNA has low ability to support translation.
In Vivo
Translation
Studies of Plant
Viral RNAs
Using Reporter
Genes
The luminometer that the authors use (Wallac Microbeta 1450) reads a new sample every
3 sec when set to read for 1 sec; other instruments may advance to the next sample with
a different timing that should be reflected in the above interval. At most, 24 wells can be
read without losing LUC signal in an instrument that lacks a reagent injector. The signal
declines by <10% over 2 min.
16K.2.4
Current Protocols in Microbiology
26. Using a multichannel pipettor, place 195 l Bradford protein assay reagent in
96-well plates. Fill enough wells to perform at least duplicate determinations for
each standard (i.e., ten wells; see step 28) and sample, as well as eight wells to allow
determination of the appropriate volume of sample to use (see step 29)
See APPENDIX 3A for more information regarding the Bradford assay.
27. Let stand 5 min to allow bubbles to settle down. Read the background at 595 nm
using a microplate spectrophotometer.
28. Add 5 l BSA standard to each well set aside for this purpose to produce a standard
curve from 2.5 to 0.156 g BSA.
29. Add 2, 3, 4, and 5 l of two of the experimental samples to separate wells.
These determinations will be used to find the appropriate volume of sample to use, i.e., the
volume which will produce measurements falling within the linear range of the standard
curve.
30. Read the plate and plot the standard curve. Determine the linear range by linear
regression, obtaining the slope, y-axis intercept, and regression coefficient (R2 ).
31. Referring to the standard curve, decide the appropriate sample volume to assay based
on the resulting measurements from the wells prepared in step 29.
32. Using a multichannel pipettor, in duplicate add that amount of sample into the
appropriate wells containing Bradford reagent (see step 26). Determine the protein
concentration by reading in a microplate spectrophotometer.
33. Using the equation obtained from the standard curve, determine the protein concentration for each sample.
34. Divide LUC readings in relative light units (RLU) obtained for each sample by the
total protein (mg) present in the same volume of sample (typically 10 l). LUC
activity is thus reported as RLU/mg protein.
ALTERNATE
PROTOCOL
The use of polyethylene glycol (PEG) is an alternative way to introduce RNA into
protoplasts when an electroporator is not available. Although this method is economical,
it requires more time and is thus not recommended if the early time course of reporter
gene expression is to be studied.
Plant RNA
Viruses
16K.2.5
Current Protocols in Microbiology
4. Aliquot 0.52 106 protoplasts for each transfection into separate 15-ml roundbottom glass or polypropylene tubes. Centrifuge 5 min at 50 g, room temperature.
Remove sufficient supernatant to leave 170 l total volume in the tube. Place on ice
until use.
5. Using a wide-mouth pipet tip, take 500 l PEG solution and leave the pipet on the
bench.
The aliquot of PEG solution must be taken in advance because of the viscosity of the
solution, which makes pipetting a slow process. The PEG should be added immediately
after the RNA is mixed with protoplasts in step 6.
7. Add 5 ml MMC buffer at once (allow to run down the wall of the tube), and mix the
contents gently by inverting the tube five times. Let the tubes sit at room temperature
for 15 min.
8. Pellet protoplasts by centrifuging 5 min at 50 g, room temperature. Remove the
supernatant and resuspend the pellet in 3 ml protoplast growth medium.
9. Pellet the protoplasts by centrifuging 5 min at 50 g, room temperature. Remove
2 ml supernatant (leaving about 1 ml).
10. Incubate under fluorescent light at room temperature (see step 16 of the Basic
Protocol).
11. Harvest protoplasts (see Basic Protocol, steps 17 to 21) and perform LUC assay (see
Basic Protocol, steps 22 to 25) and protein concentration assay (see Basic Protocol,
steps 26 to 32).
In Vivo
Translation
Studies of Plant
Viral RNAs
Using Reporter
Genes
MMC buffer
0.6 M D-mannitol
0.1% (w/v) MES
5 mM CaCl2
Adjust to pH 5.8 with KOH
Autoclave for 15 min and store at room temperature up to 6 months
16K.2.6
Current Protocols in Microbiology
PEG solution
40% (w/v) PEG 8000 (RNase-free)
100 mM Ca(NO3 )2
0.5 M D-mannitol
Heat to 50 C to dissolve, but do not overheat
Filter-sterilize; do not autoclave
Store at room temperature in the dark up to 6 months
Protoplast digestive enzyme solution
1% (w/v) cellulase (Onozuka)
0.05% (w/v) macerase-pectinase (Calbiochem)
Dissolve in MMC buffer (see recipe) by stirring in the dark 30 min
Filter-sterilize and store at 20 C for up to 1 month in aliquots for single use
Protoplast electroporation buffer
0.6 M D-mannitol
0.1% (w/v) MES
5 mM CaCl2
40 mM KCl
Adjust to pH 5.8 with KOH and autoclave for 20 min
Store up to 6 months at room temperature
This buffer (400 l) in a 2-mm electroporation cuvette should have a resistance of 37
at room temperature.
and Kobayashi, 1994; Guo et al., 2000; Gallie, 2002; Matsuda et al., 2004), and the reporter protein coding region is introduced in
place of the viral coding region, between the
5 - and/or 3 -UTRs. The reporter mRNA construct can be expressed in vitro (see UNIT 16K.1)
and in vivo (this unit) to investigate the translational properties of the UTRs. Although the
coding regions usually do not influence translational expression, this possibility will need
to be borne in mind when drawing conclusions from studies with reporter constructs (see
below).
Several reporter genes have been used
to study translational regulation, including
-glucuronidase, -galactosidase, chloramphenicol acetyltransferase, and firefly (Photinus) and sea pansy (Renilla) luciferases.
Plant RNA
Viruses
16K.2.7
Current Protocols in Microbiology
In Vivo
Translation
Studies of Plant
Viral RNAs
Using Reporter
Genes
troporator and the continuous expense of disposable cuvettes, although they may be reused
to some extent. PEG-mediated transfection is
a viable alternative when an electroporator is
not available.
16K.2.8
Current Protocols in Microbiology
Figure 16K.2.1 A typical LUC expression prole in protoplasts and determination of mRNA functional halflife. Early in the time course, LUC expression enters a near-linear stage of accumulation (usually by 40 min
post electroporation) and reaches plateau at around 4 hr. The LUC expression half-life can be determined as
(A) the time for the maximal rate to fall to half-maximal (interval x) or (B) the time to reach the half-maximum
accumulation of LUC less the lag time, represented by l (interval y). When studying reporter RNAs making
stable forms of LUC, the LUC expression half-life represents the mRNA functional half-life. However, when
comparisons involve the expression of modied forms of LUC with shorter half-lives, the mRNA functional
half-life is under-estimated.
Protoplasts
The quality of protoplasts can be critical
for optimal results. The viability of the protoplasts is typically 70% to 80% after overnight
digestion of the leaves at room temperature
and >85% after 4 to 5 hr digestion at 30 C. Although overnight digestion is convenient, the
LUC yield per cell is usually lower than from
protoplasts prepared with the short digestion
period.
LUC fusion proteins
In some instances, part of the coding region
in mRNAs may be involved in translational
control (Kozak, 1990; Frolov and Schlesinger,
1996; Fang et al., 2004). When investigating
such cases, a portion of the coding region can
be fused in-frame to the LUC coding region to
make N-terminal fusion proteins. This translational fusion can potentially result in altered
specific activity of the LUC enzyme, which
can be determined upon in vitro translation
by comparing its light emission activity to incorporation of radiolabeled amino acids into
the LUC band on an SDS-PAGE gel (Matsuda
et al., 2004). Very little variation in specific
activity for a range of N-terminal LUC fusion proteins has been seen, but new variants
are routinely tested. LUC fusion proteins may
also possess an altered stability or turnover
rate, which may result in underestimated expression unless a time course of LUC expres-
Plant RNA
Viruses
16K.2.9
Current Protocols in Microbiology
In Vivo
Translation
Studies of Plant
Viral RNAs
Using Reporter
Genes
Anticipated Results
LUC activity can be detected as early as
15 min post electroporation, in the authors
experience. More RNA and protoplasts can
be used in order to observe the signal earlier if desirable. In most cases, the LUC
expression profile enters a steady stage of nearlinear increase at 40 min post transfection
and reaches plateau or maximum at 4 to 6
hr (Fig. 16K.2.1). The time taken to reach the
maximal rate of increase in LUC activity (lag
time) can differ from one RNA construct to
another; a short lag time for a well translated
RNA suggests that the RNA may have a superior ability to actively recruit translation initiation factors and ribosomes (Gallie, 2002).
The functional half-life of input RNAs
varies from 0.7 (uncapped RNAs) to 2.5 hr in
the authors system. Translation can be said
to be enhanced if the difference in LUC
expression from two mRNAs is significantly
greater than the difference in their functional
half-lives.
Time Considerations
The preparation and analysis of input
RNA may take 5 to 6 hr total. The leafpeeling process takes 0.5 to 1.5 hr, depending on the number of leaves and the experimenters experience. Digestion to produce protoplasts takes 4.5 hr or overnight
(see Critical Parameters and Troubleshooting for discussion of the two choices). Another 1.5 hr are expected after digestion in
preparing the cells for electroporation. With
a 35-sec interval, it takes 20 min to electroporate 24 samples into protoplasts and dispense samples for multiple harvest time points.
Harvesting and lysis of protoplasts takes
20 min.
16K.2.10
Current Protocols in Microbiology
Literature Cited
Bradshaw, R.A., Brickey, W.W., and Walker, K.W.
1998. N-terminal processing: The methionine
aminopeptidase and N alpha-acetyl transferase
families. Trends Biochem. Sci. 23:263-267.
Brasier, A.R. and Fortin, J.J. 1995. Nonisotopic
assays for reporter gene activity. In Current
Protocols in Molecular Biology (F.M. Ausubel,
R. Brent, R.E. Kingston, D.D. Moore, J.G.
Seidman, J.A. Smith, and K. Struhl, eds.) pp.
9.7.12-9.7.21. John Wiley & Sons, Hoboken,
N.J.
Dreher, T.W. 1999. Functions of the 3 -untranslated
regions of positive strand RNA viral genomes.
Annu. Rev. Phytopathol. 37:151-174.
Fang, P., Spevak, C.C., Wu, C., and Sachs,
M.S. 2004. A nascent polypeptide domain that
can regulate translation elongation. Proc. Natl.
Acad. Sci. U.S.A. 101:4059-4064.
Frolov, I. and Schlesinger, S. 1996. Translation of
Sindbis virus mRNA: Analysis of sequences
downstream of the initiating AUG codon that
enhance translation. J. Virol. 70:1182-1190.
Gallie, D.R. 1991. The cap and poly(A) tail function
synergistically to regulate mRNA translational
efficiency. Genes Dev. 5:2108-2116.
Gallie, D.R. 2002. The 5 -leader of tobacco mosaic
virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res. 30:34013411.
Gallie, D.R. and Kobayashi, M. 1994. The
role of the 3 -untranslated region of nonpolyadenylated plant viral mRNAs in regulating translational efficiency. Gene 142:159165.
Internet Resources
http://www.promega.com
The Promega Inc. Web site is a searchable resource
with information on various aspects of the use of
LUC as a reporter.
Plant RNA
Viruses
16K.2.11
Current Protocols in Microbiology
APPENDIX 1
APPENDIX 1A
Bq Becquerel
BrdU 5-bromodeoxyuridine
BSA bovine serum albumin
BSC biological safety cabinet
BSL Biosafety Level
C cytosine or cytidine; one-letter code for
cysteine
CA3 chromomycin A3
cAMP adenosine
3 ,5 -cyclic-monophosphate
CAPS cyclohexylaminopropane sulfonic
acid
CAT chloramphenicol acetyltransferase
CDC Centers for Disease Control and
Prevention
cDNA complementary deoxyribonucleic
acid
CDP cytidine 5 -diphosphate
CFA complete Freunds adjuvant
cfu colony forming units
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate
CHES 2-(Ncyclohexylamino)ethanesulfonic acid
CHO Chinese hamster ovary
Ci curie
CLSM confocal laser scanning microscopy
cM centimorgans
CMFDA chloromethylfluoroscein diacetate
CMP cytidine 5 -monophosphate
CMV cytomegalovirus
CNBr cyanogen bromide
CPC cetylpyridinium chloride
CPE cytopathic effect
cpm counts per minute
CSF colony-stimulating factor
CTAB cetyltrimethylammonium bromide
CTD C-terminal domain
CTP cytidine 5 -triphosphate
CFP cyan fluorescent protein
D dextrorotatory
Da Dalton
dA deoxyadenosine
DAB diaminobenzidine
DABCO 1,4-diazabicyclo[2,2,2]-octane
dAMP deoxyadenosine monophosphate
DAPI 4 ,6-diamidino-2-phenylindole
dATP deoxyadenosine triphosphate
dC deoxycytosine
Useful
Information
and Data
A.1A.1
Supplement 2
Abbreviations
Used in this
Manual
A.1A.2
Supplement 2
Useful
Information
and Data
A.1A.3
Current Protocols in Microbiology
Supplement 2
Pu purine
Py pyrimidine
Q-RT-PCR quantitative reverse
transcription PCR
RB reticulate body
RBS ribosome-binding site
RCF relative centrifugal force
RFP red fluorescent protein
RI refractive index
RNA ribonucleic acid
RNAi RNA interference
RNase ribonuclease
RP reversed phase (HPLC)
rRNA ribosomal ribonucleic acid
RT reverse transcriptase
RTF reduced transport fluid
RT-PCR reverse transcription PCR
SD standard deviation
SDS sodium dodecyl sulfate or sodium
lauryl sulfate
SDS-PAGE sodium dodecyl sulfate
polyacrylamide gel electrophoresis
SED standard enzyme diluent
siRNA short interfering RNA
SLS sodium lauryl sulfate
SRBC sheep red blood cells
ss single stranded
SSC sodium chloride/sodium citrate (buffer)
STI soybean trypsin inhibitor
T thymine or thymidine; one-letter code for
threonine
TAE Tris/acetate (buffer)
Taq Thermus aquaticus DNA (polymerase)
TB terrific broth
TBE Tris/borate (buffer)
TBS Tris-buffered saline
TBST Tris-buffered saline containing
Tween-20
TCA trichloroacetic acid
TCP Transmission Control Protocol
TE Tris/EDTA (buffer)
TEA triethanolamine acetate
TEAE triethylaminoethyl
TEM transmission electron microscopy
TEMED N,N,N ,N -tetramethylethylenediamine
TEN sodium chloride in TE buffer
TFA trifluoroacetic acid
TIFF Tagged-Image File Format
TK thymidine kinase
TLC thin-layer chromatography
TLCK N-p-tosyl-L-lysine chloromethyl
ketone
Tm melting (or midpoint) temperature;
thermal denaturation
TMAC tetramethylammonium chloride
TMP trimethyl phosphate
TPCK N-p-tosyl-L-phenylalanine
chloromethyl ketone
Tris tris(hydroxymethyl)aminomethane
TrisCl Tris hydrochloride
tRNA transfer ribonucleic acid
TSB trypticase soy broth
TTP thymidine 5 -triphosphate
U unit (enzyme); uracil or uridine
UDP uridine 5 -diphosphate
UMP uridine 5 -monophosphate
USDA United States Department of
Agriculture
UTP uridine 5 -triphosphate
UTR untranslated leader region
UV ultraviolet
V0 void volume
Vent Thermococcus litoralis DNA
(polymerase)
vol/vol, v/v volume/volume
WHO World Health
Organization
wt/vol; w/v weight/volume
WT wild-type
WWW World Wide Web
Xgal 5-bromo-4-chloro-3-indolyl--Dgalactoside
YFP yellow fluorescent protein
Abbreviations
Used in this
Manual
A.1A.4
Supplement 2
APPENDIX 1B
Determining the appropriate safety guidelines and regulations relevant to a given experiment is the first step in any research study. To facilitate this process when working
with microorganisms, the reader is refered to http://www.absa.org/resguides.html, the
Biosafety Guidelines website maintained by the American Biological Safety Association
(ABSA). This comprehensive site not only provides direct links to several high-impact
references, but also to the compendium maintained by the International Biosafety Working Group (IBWG), which researchers can use to search through an extensive list of
international biosafety resources, including other databases appropriate to the investigation. Note that the IBWG compendium is currently hosted by the European Biosafety
Association (EBSA; http://www.ebsa.be), which is itself a valuable resource.
The reader is also strongly advised to explore the ABSA home page (http://
www.absa.org/index.shtml) and Resources and Tools page (http://www.absa.org/
restool.html), both of which provide a wealth of biosafety information.
Table A.1B.1 provides a list of the resources available on the ABSA website. For general
safety information, the reader is referred to the first section of Chapter 1, including
UNIT 1A.1, which provides a discussion of general biosafety.
IMPORTANT NOTE: While an effort has been made to provide a starting point for the
reader to begin their investigation into the appropriate safety precautions for their experiment, the resources presented here and at the websites mentioned should not be considered comprehensive. Ultimate responsibility for safety lies with the individual researcher.
Please consult all appropriate safety resources prior to beginning any experiment.
Table A.1B.1 Resources Available from the American Biological Safety Association Biosafety Guidelines Websitea
Resource
Responsible Agency
a Available at http://www.absa.org/resguides.html.
b This site is currently hosted by the European Biosafety Association (EBSA; http://www.ebsa.be) and is accessed by selecting International Biosafety
Useful
Information
and Data
Current Protocols in Microbiology (2005) A.1B.1
C 2005 by John Wiley & Sons, Inc.
Copyright
A.1B.1
APPENDIX 2
APPENDIX 2A
Table A.2A.1 Molarities and Specific Gravities of Concentrated Acids and Basesa
Acid/base
Mol. wt.
% by weight
Molarity
(approx.)
Specific
gravity
1 M solution
(ml/liter)
Acetic acid
(glacial)b
60.05
99.6
17.4
1.05
57.5
Ammonium
hydroxide
35.0
28
14.8
0.90
67.6
Formic acidb
46.03
90
23.6
1.205
42.4
98
25.9
1.22
38.5
Hydrochloric acid
36.46
36
11.6
1.18
85.9
Nitric acid
63.01
70
15.7
1.42
63.7
Perchloric acid
100.46
60
9.2
1.54
108.8
72
12.2
1.70
82.1
Phosphoric acidb
98.00
85
14.7
1.70
67.8
Sulfuric acid
98.07
98
18.3
1.835
54.5
a CAUTION: Handle strong acids and bases carefully (see UNIT 1A.3).
b Also see Table A.2A.3.
Commonly
Used Reagents
and Equipment
Current Protocols in Microbiology (2005) A.2A.1-A.2A.15
C 2005 by John Wiley & Sons, Inc.
Copyright
A.2A.1
Alsevers solution
20.5 g dextrose (114 mM)
7.9 g sodium citrate2H2 O (27 mM)
4.2 g NaCl (71 mM)
H2 O to 1 liter
Adjust to pH 6.1 with 1 M citric acid (Table A.2A.3) and filter sterilize
Store indefinitely at 4 C
Ammonium hydroxide, concentrated stock solution
See Table A.2A.1
Ammonium acetate, 10 M
Dissolve 385.4 g ammonium acetate in 150 ml H2 O
Add H2 O to 500 ml
Ammonium sulfate, saturated
76 g ammonium sulfate
100 ml H2 O
Heat with stirring to just below boiling point
Let stand overnight at room temperature
ATP, 100 mM
1 g ATP (adenosine triphosphate)
12 ml H2 O
Adjust pH to 7.0 with 4 M NaOH
Adjust volume to 16.7 ml with H2 O
Store in aliquots indefinitely at 20 C
Base, concentrated stock solutions
See Table A.2A.1.
BBS (BES-buffered solution), 2
50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; Calbiochem)
280 mM NaCl
1.5 mM sodium phosphate buffer, pH 6.95 (see recipe)
800 ml H2 O
Adjust pH to 6.95 with room temperature 1 N NaOH
H2 O to 1 liter
Filter sterilize through a 0.45-m nitrocellulose filter (Nalgene)
Store in aliquots at 20 C (can be frozen and thawed repeatedly)
The pH of this solution is critical (pH 6.95 to 6.98). When a new batch of 2 BES buffer is
prepared, its pH should be checked against a reference stock prepared (and tested) earlier.
BCIP, 5% (w/v)
Dissolve 0.5 g 5-bromo-4-chloro-3-indolyl phosphate disodium salt (stored at
20 C) in 10 ml of 100% dimethylformamide (DMF). Store wrapped in aluminum
foil up to 6 months at 4 C.
The BCIP may not dissolve completely. Vortex the solution immediately before use and
pipet with a wide-mouth pipet tip.
Discard solution if it turns pinkish.
Commonly Used
Reagents
A.2A.2
Current Protocols in Microbiology
CaCl2 , 1 M
147 g CaCl2 2H2 O
H2 O to 1 liter
Carbonate buffer
1.6 g Na2 CO3 (15 mM final)
2.9 g NaHCO3 (35 mM final)
0.2 g NaN3 (3.1 mM final)
H2 O to 1 liter
Adjust to pH 9.5
CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling, storage, and disposal.
Commonly
Used Reagents
and Equipment
A.2A.3
Current Protocols in Microbiology
Base
Adenine
15,200
Cytosine
7050
Guanosine
12,010
Thymine
8400
pH 7.0 with 1 M NaOH (to prevent acid-catalyzed hydrolysis). Determine the actual
concentration of each dNTP by UV spectrophotometry at 260 nm, referring to the
molar extinction coefficients given in Table A.2A.2.
Working solutions: Prepare working solutions of desired concentration (commonly
2 mM) for each dNTP by diluting concentrated stocks appropriately. Remember
that the molarity of the 3dNTP and 4dNTP mixes refers to the concentration of
each precursor present in the solution.
4dNTP mixes: Prepare mixed dNTP solutions containing equimolar amounts of all
four DNA precursors; e.g.:
DTT (dithiothreitol), 1 M
Dissolve 1.55 g DTT in 10 ml water
Filter sterilize
Store in aliquots at 20 C
Commonly Used
Reagents
A.2A.4
Current Protocols in Microbiology
Commonly
Used Reagents
and Equipment
A.2A.5
Current Protocols in Microbiology
HCl, 1 M
Mix the following in order:
913.8 ml H2 O
86.2 ml concentrated HCl (Table A.2A.1)
HeBS (HEPES-buffered saline) solution, 2
16.4 g NaCl
11.9 g HEPES acid (Table A.2A.3)
0.21 g Na2 HPO4
800 ml H2 O
Titrate to pH 7.05 with 5 M NaOH
Add H2 O to 1 liter
Filter sterilize through a 0.45-m nitrocellulose filter
Store in 50-ml aliquots at 20 C
If the solution is to be used for transfection, the pH should be between 7.05 and 7.12, and
should be tested for transfection efficiency.
KCl, 1 M
74.6 g KCl
H2 O to 1 liter
2-ME (2-mercaptoethanol), 50 mM
Prepare 1 M stock:
0.5 ml 14.3 M 2-ME
6.6 ml H2 O
Prepare 50 mM stock:
5 ml 1 M 2-ME
95 ml H2 O
Store at 4 C
MgCl2 , 1 M
20.3 g MgCl2 6H2 O
H2 O to 100 ml
Commonly Used
Reagents
MgSO4 , 1 M
24.6 g MgSO4 7H2 O
H2 O to 100 ml
A.2A.6
Current Protocols in Microbiology
MOPS buffer
0.2 M MOPS [3-(N-morpholino)-propanesulfonic acid], pH 7.0 (Table A.2A.3)
0.5 M sodium acetate
0.01 M EDTA
Store in the dark and discard if it turns yellow
NaCl, 5 M
292 g NaCl
H2 O to 1 liter
NaOH, 10 M
Dissolve 400 g NaOH in 450 ml H2 O
Add H2 O to 1 liter
PCR amplification buffer, 10
500 mM KCl
100 mM TrisCl, pH 8.3 (see recipe)
x mM MgCl2
0.1% (w/v) gelatin
Store in aliquots at 20 C
This solution can be sterilized by autoclaving. Alternatively, it can be made from sterile
water and stock solutions, and the sterilization omitted.
15 mM MgCl2 is the concentration (x) used for most PCR reactions. However, the optimal concentration depends on the sequence and primer of interest and may have to be
determined experimentally.
Phenol, buffered
Add 0.5 g of 8-hydroxyquinoline to a 2-liter glass beaker containing a stir bar. Gently
pour in 500 ml liquefied phenol or melted crystals of redistilled phenol (melt in a
water bath at 65 C). The phenol will turn yellow due to the 8-hydroxyquinoline,
which is added as an antioxidant. Add 500 ml of 50 mM Tris base. Cover the
beaker with aluminum foil and stir 10 min at room temperature using a magnetic
stirrer on low speed. Let phases separate at room temperature. Gently decant the top
(aqueous) phase into a suitable waste receptacle (UNIT 1A.3). Remove what cannot
be decanted with a 25-ml glass pipet and a suction bulb. Add 500 ml of 50 mM
TrisCl, pH 8.0 (see recipe). Repeat equilibration with 500 ml of 50 mM TrisCl,
pH 8.0, twice. Check the pH of the phenol phase with indicator paper to determine
if it is 8.0. If it is not, repeat equilibration until this pH is obtained. Add 250 ml of
50 mM TrisCl, pH 8.0, or TE buffer, pH 8.0 (see recipe), and store at 4 C in brown
glass bottles or clear glass bottles wrapped in aluminum foil.
CAUTION: Phenol can cause severe burns to skin and damage clothing. Gloves, safety
glasses, and a laboratory coat should be worn whenever working with phenol, and all
manipulations should be carried out in a fume hood. A glass receptacle should be available
exclusively for disposing of used phenol and chloroform (see UNIT 1A.3).
Phenol prepared with 8-hydroxyquinoline as an antioxidant can be stored 2 months at
4 C.
Phenol must be redistilled before use, because oxidation products of phenol can damage
and introduce breaks into nucleic acid chains. Redistilled phenol is commercially available.
Regardless of the source, phenol must be buffered before use.
Commonly
Used Reagents
and Equipment
A.2A.7
Current Protocols in Microbiology
Table A.2A.3 pKa Values and Molecular Weights for Some Common Biological Buffersa
pKa
Useful pH
range
Mol. wt.
(g/mol)
H3 PO4
2.12 (pKa1 )
98.00
C6 H8 O7 (H3 Cit)
3.06 (pKa1 )
192.1
3.75
46.03
4.19 (pKa1 )
118.1
4.74 (pKa2 )
4.75
C6 H6 O7 (HCit2 )
5.40 (pKa3 )
5.57 (pKa2 )
Name
Phosphoric acidb
Citric acidc
b
Formic acid
HCOOH
Succinic acid
C4 H6 O4
C6 H7 O7 (H2 Cit )
Citric acid
Acetic acid
c
Citric acid
CH3 COOH
60.05
Succinic acid
C4 H5 O4
MES
2-(N-Morpholino]ethanesulfonic acid
6.15
5.5-6.7
195.2
Bis-Tris
bis(2-Hydroxyethyl)iminotris
(hydroxymethyl)methane
6.50
5.8-7.2
209.2
ADA
N-(2-Acetamido)-2-iminodiacetic acid
6.60
6.0-7.2
190.2
PIPES
6.80
6.1-7.5
302.4
ACES
N-(Carbamoylmethyl)-2-aminoethanesulfonic acid
6.80
6.1-7.5
182.2
Imidazole
1,3-Diaza-2,4-cyclopentadiene
7.00
68.08
Diethylmalonic acid
C7 H12 O4
7.20
160.2
MOPS
3-(N-Morpholino)propanesulfonic acid
7.20
6.5-7.9
209.3
Sodium phosphate,
monobasic
NaH2 PO4
7.21 (pKa2 )
120.0
Potassium phosphate,
monobasic
KH2 PO4
7.21 (pKa2 )
136.1
TES
N-tris(Hydroxymethyl)methyl-2aminoethanesulfonic acid
7.40
6.8-8.2
229.3
HEPES
7.55
6.8-8.2
238.3
HEPPSO
7.80
7.1-8.5
268.3
GlycinamideHCl
C2 H6 N2 OHCl
8.10
7.4-8.8
110.6
Tricine
N-tris(Hydroxymethyl)methylglycine
8.15
7.4-8.8
179.2
Glycylglycine
C4 H8 N2 O3
8.20
7.5-8.9
132.1
Tris
Tris(hydroxymethyl)aminomethane
8.30
7.0-9.0
121.1
Bicine
N,N-bis(2-Hydroxyethyl)glycine
8.35
7.6-9.0
163.2
Boric acid
H3 BO3
9.24
61.83
CHES
2-(N-Cyclohexylamino)ethane-sulfonic acid
9.50
8.6-10.0
207.3
CAPS
3-(Cyclohexylamino)-1-propane-sulfonic
acid
10.40
9.7-11.1
221.3
continued
Commonly Used
Reagents
A.2A.8
Current Protocols in Microbiology
Table A.2A.3 pKa Values and Molecular Weights for Some Common Biological Buffersa , continued
pKa
Useful pH
range
Mol. wt.
(g/mol)
Na2 HPO4
12.32 (pKa3 )
142.0
K2 HPO4
12.32 (pKa3 )
174.2
Name
Sodium phosphate,
dibasic
Potassium phosphate,
dibasic
a Some data reproduced from Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems (Mohan, 1997) with permission of
Calbiochem.
b See Table A.2A.1 for more information.
c Available as a variety of salts, e.g., ammonium, lithium, sodium.
Commonly
Used Reagents
and Equipment
A.2A.9
Desired pH
Solution A (ml)
Solution B (ml)
3.6
46.3
3.7
3.8
44.0
6.0
4.0
41.0
9.0
4.2
36.8
13.2
4.4
30.5
19.5
4.6
25.5
24.5
4.8
20.0
30.0
5.0
14.8
35.2
5.2
10.5
39.5
5.4
8.8
41.2
5.6
4.8
45.2
A.2A.10
Current Protocols in Microbiology
Desired pH
Solution A (ml)
Solution B (ml)
5.7
93.5
6.5
5.8
92.0
8.0
5.9
90.0
10.0
6.0
87.7
12.3
6.1
85.0
15.0
6.2
81.5
18.5
6.3
77.5
22.5
6.4
73.5
26.5
6.5
68.5
31.5
6.6
62.5
37.5
6.7
56.5
43.5
6.8
51.0
49.0
6.9
45.0
55.0
7.0
39.0
61.0
7.1
33.0
67.0
7.2
28.0
72.0
7.3
23.0
77.0
7.4
19.0
81.0
7.5
16.0
84.0
7.6
13.0
87.0
7.7
10.5
90.5
7.8
8.5
91.5
7.9
7.0
93.0
8.0
5.3
94.7
Saline, 0.9%
9 g NaCl (154 mM final; 0.9% w/v)
H2 O to 1 liter
Saponin, 10% (w/v)
Dissolve 1 g saponin in 10 ml PBS (see recipe)
Store in 500-l aliquots at 20 C
Once thawed, the 10% solution is stable for several months when stored at 4 C.
Commonly
Used Reagents
and Equipment
A.2A.11
Current Protocols in Microbiology
Final conc. in
1 buffer
2.5 ml
5.0 ml
62.5 mM
SDS
0.4 g
0.8 g
2% (w/v)
Glycerol
2.0 ml
4.0 ml
10% (v/v)
Bromphenol blue
20 mg
40 mg
0.1% (w/v)
2-Mercaptoethanola,b,c
400 l
800 l
300 mM
to 10 ml
to 10 ml
Ingredient
H2 O
a See recipe.
b Alternatively, dithiothreitol (DTT; see recipe), at a final concentration of 100 mM, can be
continued
A.2A.12
Current Protocols in Microbiology
Referring to Table A.2A.5 for desired pH, mix the indicated volumes of solutions
A and B, then dilute with water to 200 ml. Filter sterilize if necessary. Store up to
3 months at room temperature.
This buffer may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium
phosphate in the same final volume. Phosphate buffers show concentration-dependent
changes in pH, so check the pH by diluting an aliquot of the concentrate to the final
concentration.
To prepare buffers with pH intermediate between the points listed in Table A.2A.5, prepare
the closest higher pH, then titrate with solution A.
Silanized glassware
For smaller items: In a well-vented fume hood, place glassware or plasticware (e.g.,
tubes, tips) in a dedicated vacuum desiccator with an evaporating dish containing
1 ml dichlorodimethylsilane. Apply vacuum with an aspirator and allow 50%
of the liquid to evaporate (several minutes). Turn off aspirator and allow items to
remain under vacuum for 30 min. Remove the lid and allow fumes to vent into the
hood for 30 min. If desired, autoclave silanized items.
Do not leave the desiccator attached to the vacuum pump. This will suck away the silane,
minimizing deposition and damaging the pump.
For larger items: Silanize items that do not fit in a desiccator by briefly rinsing with
or soaking in a solution of 5% dichlorodimethylsilane in a volatile organic solvent (e.g., chloroform, heptane). Remove organic solvent by evaporation, allowing
deposition of dichlorodimethylsilane.
This approach is particularly useful for treating glass plates for denaturing polyacrylamide
sequencing gels.
Treatment of glassware, plasticware, or equipment with dichlorodimethylsilane introduces a short polymer of dimethylsiloxane onto its surface. Polydimethylsiloxane
is silicone oil. Autoclaving or rinsing with water removes the reactive chlorosilane
end of the dimethylsiloxane polymer generated by dichlorodimethylsilane.
CAUTION: Dichlorodimethylsilane vapors are toxic and highly flammable. Always perform
in a fume hood.
Commonly
Used Reagents
and Equipment
A.2A.13
Current Protocols in Microbiology
Commonly Used
Reagents
IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1 C. Tris-buffered solutions should be adjusted
to the desired pH at the temperature at which they will be used. Because of the pKa , Tris
should not be used as a buffer below pH 7.2 or above pH 9.0 (see Table A.2A.3).
A.2A.14
Current Protocols in Microbiology
Commonly
Used Reagents
and Equipment
A.2A.15
Current Protocols in Microbiology
APPENDIX 2B
Listed below are pieces of equipment that are standard in the modern microbiology
laboratoryi.e., items used extensively in this manual and thus not usually included in
the individual materials lists. No attempt has been made to list all items required for each
procedure in the Materials list of each protocol; rather, those lists present items that might
not be readily available in the laboratory or that require special preparation. See SUPPLIERS
APPENDIX for contact information for commercial vendors of laboratory equipment.
Aluminum foil
Applicators, cotton-tipped and wooden
Autoclave
Bag sealer
Balances, analytical and preparative
Beakers
Bench protectors, plastic-backed
(including blue pads)
Biohazard disposal containers and bags
Biological safety cabinet (BSC), must be
appropriate for the organism under
study
Bottles, glass, plastic, and squirt
Bunsen burners
Cell culture dishes, 60 and 100 mm
Cell culture flasks, T75
Cell spreader
Centrifuges, low-speed (to 20,000 rpm)
refrigerated, ultracentrifuge (20,000 to
80,000 rpm), large-capacity
low-speed, tabletop, with appropriate
rotors and adapters
Centrifuge tubes and bottles, plastic and
glass, various sizes
Clamps
Conical centrifuge tubes, plastic and
glass
Containers, assortment of glass and
plastic, for gel and membrane washes
Coplin jars, glass, for 25 75mm slides
Cryovials, sterile (e.g., Nunc)
Cuvettes
Desiccator and desiccant
Dry ice
Electrophoresis equipment, agarose and
acrylamide, full-size and mini, with
power supplies
Film developing system and darkroom
Filtration apparatus
Flasks
Forceps
Fraction collector
Freezers, 20 C, 70 C, and liquid
nitrogen
Fume hood
Geiger counter
Gel dryer
Gloves, disposable plastic and heat
resistant
Graduated cylinders
Heating blocks, thermostatically
controlled for test tubes and
microcentrifuge tubes
Hemacytometer and/or electronic cell
counter
Homogenizer
Humidified CO2 incubator
Ice bucket
Ice maker
Immersion oil for microscopy
Incubator, 37 C
Inoculating loop
Kimwipes
Lab coats
Laboratory glassware
Light box
Light microscope with 10x , 40x , and
100x oil immersion objectives
Liquid nitrogen
Lyophilizer
Magnetic stirrer, with and without
heater, and stir bars
Markers, including indelible markers,
china-marking pens, and luminescent
markers
Microcentrifuge, Eppendorf-type with
12,000 to 14,000 rpm maximum speed
Microcentrifuge tubes, 0.2-, 0.5-, 1.5-,
2-ml
Microscope slides, glass, 25 75mm,
and coverslips
Microtiter plate reader
Commonly Used
Reagents and
Equipment
A.2B.1
Microwave oven
Mortar and pestle
Ovens, drying and microwave
Paper cutter, large
Paper towels
Parafilm
Pasteur pipets and bulbs
PCR thermal cycler and tubes
pH meter
pH paper
Pipets, graduated
Pipettors, adjustable delivery, 0.5- to
10-l, 10- to 200-l, and 200- to
1000-l
Polaroid camera or video documentation
system
Power supplies, 300-V for
polyacrylamide gels, 2000- to 3000-V
for other applications
Racks, for test tube and microcentrifuge
tubes
Radiation shield, Lucite or Plexiglas
Radioactive waste containers for liquid
and solid wastes
Refrigerator, 4 C
Ring stand and rings
Rubber policemen or plastic scrapers
Rubber stoppers
Safety glasses
Scalpels and blades
-Scintillation counter
Scissors
Shakers, orbital and platform, room
temperature or 37 C
Sharps disposal container
Spectrophotometer, visible and UV range
Speedvac evaporator
Syringes and needles
Tape, masking, electricians black,
autoclave, and Time tape
Test tubes, glass and plastic, various
sizes, with and without caps
Timer
Toolbox with common tools
Trays, plastic and glass, various sizes
Tubing, rubber and Tygon
UV light sources, cross-linking and longand short-wavelength
UV transilluminator
UV transparent plastic wrap (e.g., Saran
Wrap)
Vacuum desiccator
Vacuum oven
Vacuum supply
Vortex mixers
Waring blendor
Water bath with adjustable temperature
Water purification system
X-ray film cassettes and intensifying
screens
Standard
Laboratory
Equipment
A.2B.2
Current Protocols in Microbiology
APPENDIX 2C
Table A.2C.1 presents a list of media used throughout this manual and the unit or appendix
in which the formulation can be found. It is important to note that while the names of
two media may be identical, the formulations may differ, sometimes greatly.
This appendix will be revised regularly; however, due to the constantly updating nature
of Current Protocols in Microbiology, at any given time there may be media formulations
which exist in the book but are not listed here. Likewise, listings may be provided for
recipes in units which are in preparation and expected to be published soon. Readers are
urged to refer to the Main Index and Supplement Index for the most current information.
CAUTION: Prior to beginning any experiment, the reader is strongly urged to read
through Section 1A of this manual, in particular UNIT 1A.1 which presents information
regarding biosafety and UNIT 1A.3 which details safe handling of commonly encountered
chemicals.
NOTE: For formulations of commonly used reagents, refer to APPENDIX 2A. For common
culture techniques, refer to APPENDIX 4.
Table A.2C.1 Media Formulations Detailed in Current Protocols in Microbiology
Medium
Location
199V medium
UNIT 14E.1
7H9 liquid culture medium (for M. marinum; also see Middlebrook UNITS 10B.1 & 10B.2
media)
7H10 agar plates (for M. marinum; also see Middlebrook media)
UNIT 10A.5
AB medium
UNIT 1C.1
Agroinfiltration medium
UNIT 161.5
UNIT 16I.6
UNIT 6A.5
UNIT 13B.1
UNIT 3A.4
UNIT 1B.5
Artificial saliva
UNIT 1B.5
AT medium
UNIT 1C.2
UNIT 11A.1
UNIT 8B.1
UNIT 13C.2
UNIT 9D.5
UNIT 9B.4
UNIT 12C.1
UNIT 12C.1
Caco-2 medium
UNIT 9B.4
continued
Commonly Used
Reagents and
Equipment
Current Protocols in Microbiology A.2C.1-A.2C.5, February 2008
Published online February 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mca02cs8
C 2008 John Wiley & Sons, Inc.
Copyright
A.2C.1
Supplement 8
Medium
Location
UNIT 16D.1
UNIT 1B.5
UNIT 4A.1
UNIT 4A.1
UNIT 11A.1
UNIT 13A.1
Columbia broth
UNIT 13A.1
UNIT 13A.1
UNIT 14E.2
UNIT 15B.1
UNIT 14E.2
UNIT 16D.2
CVE plates
UNIT 13A.1
UNIT 7B.1
UNIT 14E.1
UNIT 15F.2
DMEM-5
UNIT 14E.1
DMEM-10, complete
UNIT 15J.1
UNIT 15G.1
DMEM/HEPES
UNIT 5A.1
UNIT 14E.2
UNIT 14E.2
UNIT 15B.1
UNIT 15B.1
UNIT 12E.4
EMJH, base
UNIT 12E.4
Where to Find
Recipes for Media
FEA plates
UNIT 13A.1
UNIT 15B.1
GC medium base
UNIT 4A.2
GCB plates
UNIT 4A.2
GCBL medium
UNIT 4A.2
UNIT 4A.1
UNIT 4A.1
Gelatin agar
UNIT 6A.5
UNIT 5A.1
UNIT 5A.1
HM buffer
UNIT 7B.1
continued
A.2C.2
Supplement 8
Medium
Location
HM plates
UNIT 7B.1
HM top agar
UNIT 7B.1
UNIT 13B.1
APPENDIX 4B
UNIT 15G.1
UNIT 15G.1
UNIT 15G.1
UNIT 9B.4
UNIT 6A.5
UNIT 9B.4
APPENDIX 4A
UNIT 16B.2
UNIT 12E.1
UNIT 12E.2
UNIT 12E.1
UNIT 12E.1
UNIT 15G.1
UNIT 10B.1
M9/glucose/bicarbonate medium
UNIT 5A.1
M9 minimal salts, 5
UNIT 9C.4
UNIT 13B.1
UNIT 15B.1
UNIT 6A.5
Microtuberization medium
UNIT 16I.1
UNIT 10A.1
UNIT 10A.1
UNIT 10A.1
UNIT 10A.2
UNIT 1C.3
UNIT 15B.1
UNIT 6A.5
UNIT 12E.1
UNIT 6A.5
UNIT 12B.1
UNIT 16D.3
continued
Commonly Used
Reagents and
Equipment
A.2C.3
Current Protocols in Microbiology
Supplement 8
Where to Find
Recipes for Media
Medium
Location
UNIT 16D.3
UNIT 10A.1
UNIT 161.5
UNIT 15F.2
UNIT 15F.2
UNIT 12E.4
UNIT 12B.1
UNIT 12B.1
UNIT 12B.1
OADC plates
UNIT 10A.1
UNIT 12B.1
UNIT 3B.1
UNIT 7B.1
UNIT 7B.1
UNIT 7B.1
UNIT 16I.1
UNIT 3B.1
UNIT 16D.1
UNIT 16D.4
UNIT 16D.4
UNIT 16K.2
UNIT 16D.1
UNIT 6A.5
PYE medium
UNIT 7B.1
PYE plates
UNIT 7B.1
UNIT 15J.1
UNIT 13B.1
SOB medium
UNIT 16B.2
SOC medium
APPENDIX 4A
UNIT 16B.2
UNIT 14C.1
Stabilizing medium
UNIT 3B.1
SWYE plates
UNIT 7B.1
UNIT 6A.5
UNIT 6A.5
UNIT 12B.1
T80/40/LH medium
UNIT 12E.1
TB (Terrific broth)
APPENDIX 4A
continued
A.2C.4
Supplement 8
Medium
Location
UNIT 9D.5
UNIT 8B.1
UNIT 3B.1
UNIT 3B.1
UNIT 13C.2
UNIT 13B.1
UNIT 12B.1
UNIT 1C.3
YEP plates
UNIT 16B.2
UNIT 10B.2
Commonly Used
Reagents and
Equipment
A.2C.5
Current Protocols in Microbiology
Supplement 8
APPENDIX 2C
Table A.2C.1 presents a list of media used throughout this manual and the unit or appendix
in which the formulation can be found. It is important to note that while the names of
two media may be identical, the formulations may differ, sometimes greatly.
This appendix will be revised regularly, however, due to the constantly updating nature
of Current Protocols in Microbiology, at any given time there may be media formulations
which exist in the book but are not listed here. Likewise, listings may be provided for
recipes in units which are in preparation and expected to be published soon. Readers are
urged to refer to the Main Index and Supplement Index for the most current information.
CAUTION: Prior to beginning any experiment, the reader is strongly urged to read
through Section 1A of this manual, in particular UNIT 1A.1 which presents information
regarding biosafety and UNIT 1A.3 which details safe handling of commonly encountered
chemicals.
NOTE: For formulations of commonly used reagents, refer to APPENDIX 2A. For common
culture techniques, refer to APPENDIX 4.
Table A.2C.1 Media Formulations Detailed in Current Protocols in Microbiology
Medium
Location
199V medium
UNIT 14E.1
7H10 plates
AB medium
UNIT 1C.1
Agroinfiltration medium
UNIT 161.5
UNIT 16I.6
UNIT 6A.5
UNIT 3A.4
AT medium
UNIT 1C.2
UNIT 11A.1
UNIT 8B.1
UNIT 9D.5
UNIT 9B.4
UNIT 12C.1
UNIT 12C.1
Caco-2 medium
UNIT 9B.4
UNIT 16D.1
UNIT 11A.1
UNIT 13A.1
Columbia broth
UNIT 13A.1
UNIT 13A.1
UNIT 15B.1
continued
Commonly Used
Reagents and
Equipment
Current Protocols in Microbiology (2007) A.2C.1-A.2C.4
C 2007 by John Wiley & Sons, Inc.
Copyright
A.2C.1
Supplement 4
Where to Find
Recipes for Media
Medium
Location
UNIT 16D.2
CVE plates
UNIT 13A.1
UNIT 7B.1
UNIT 14E.1
UNIT 15F.2
DMEM-5
UNIT 14E.1
DMEM-10, complete
UNIT 15J.1
UNIT 15G.1
UNIT 15B.1
UNIT 15B.1
FEA plates
UNIT 13A.1
UNIT 15B.1
GC medium base
UNIT 4A.2
GCB plates
UNIT 4A.2
GCBL medium
UNIT 4A.2
Gelatin agar
UNIT 6A.5
HM buffer
UNIT 7B.1
HM plates
UNIT 7B.1
HM top agar
UNIT 7B.1
APPENDIX 4B
UNIT 15G.1
UNIT 15G.1
UNIT 15G.1
UNIT 9B.4
UNIT 6A.5
UNIT 9B.4
APPENDIX 4A
UNIT 16B.2
UNIT 12E.1
UNIT 12E.2
UNIT 12E.1
UNIT 12E.1
UNIT 15G.1
UNIT 10B.1
M9 minimal salts, 5
APPENDIX 4A
UNIT 9C.4
UNIT 15B.1
continued
A.2C.2
Supplement 4
Medium
Location
UNIT 6A.5
UNIT 1C.3
UNIT 15B.1
UNIT 6A.5
UNIT 12E.1
UNIT 6A.5
UNIT 12B.1
UNIT 16D.4
UNIT 161.5
UNIT 15F.2
UNIT 15F.2
UNIT 12B.1
UNIT 12B.1
UNIT 12B.1
UNIT 12B.1
UNIT 3B.1
UNIT 7B.1
UNIT 7B.1
UNIT 7B.1
UNIT 3B.1
UNIT 16D.1
UNIT 16D.4
UNIT 16D.4
UNIT 16K.2
UNIT 16D.1
UNIT 6A.5
PYE medium
UNIT 7B.1
PYE plates
UNIT 7B.1
UNIT 15J.1
SOB medium
UNIT 16B.2
SOC medium
APPENDIX 4A
UNIT 16B.2
UNIT 14C.1
Stabilizing medium
UNIT 3B.1
SWYE plates
UNIT 7B.1
UNIT 6A.5
UNIT 6A.5
UNIT 12B.1
Commonly Used
Reagents and
Equipment
continued
A.2C.3
Supplement 4
Medium
Location
T80/40/LH medium
UNIT 12E.1
TB (Terrific broth)
APPENDIX 4A
UNIT 9D.5
UNIT 8B.1
UNIT 3B.1
UNIT 3B.1
UNIT 12B.1
UNIT 1C.3
YEP plates
UNIT 16B.2
UNIT 10B.2
Where to Find
Recipes for Media
A.2C.4
Supplement 4
APPENDIX 3
APPENDIX 3A
Total protein assays are used to analyze hundreds of industrial, agricultural, and biotechnology products. They are also basic for research purposes, especially for determining
the specific activity (i.e., total activity/total protein) of enzymes, antibodies, and lectins.
Clearly, accuracy and precision of specific activity measurements depend as much on
accurate measurement of total protein as on determination of total activity.
In performing total protein assays, there are five issues of concern: (1) sensitivity and technique of the method, (2) clear definition of units, (3) interfering compounds, (4) removal
of interfering substances before assaying samples, and (5) correlation of information
from various techniques. These concerns are discussed at length in the Commentary
section.
This appendix describes two copper-based assays to quantitate total protein: a variation of
the Lowry method (Hartree-Lowry method; see Basic Protocol 1) and the bicinchoninic
acid (BCA) assay (see Basic Protocol 2). Ultraviolet spectrophotometry is used to measure total protein (see Basic Protocol 3) and evaluate samples for the presence of contaminants. The Coomassie-dye binding, or Bradford, assay (see Basic Protocol 4) is quite
simple and frequently quite sensitive, although it sometimes gives a variable response
depending on how well or how poorly the protein binds the dye in acid pH. TCA precipitation to precipitate and concentrate proteins, and to remove low-molecular-weight
contaminants is also described (see Basic Protocol 5).
Table A.3A.1 presents a brief summary of the assays described in this unit, including
detection range and required sample size.
BASIC
PROTOCOL 1
Assay
Hartree-Lowry
BCA
UV absorption
Coomassie dye binding
Sample size
1 ml
100-600
100 l
200-1000
1 ml
30-300
100 to 200 l
60-300
Commonly Used
Techniques
A.3A.1
overcomes the reagent salt coprecipitation problem encountered with Lowry reagents.
Finally, the reagent formulation offers some advantages in storage stability.
Materials
Calibration standard: 300 g/ml crystalline BSA in water
Buffer or solvent used to prepare the protein-containing sample
Sample containing protein at 100 to 600 g/ml
Hartree-Lowry reagents A, B, and C (see recipes)
50 C water bath
Spectrophotometer and 1-cm cuvettes
1. Prepare a dilution series of calibration standard in the same buffer or solvent used to
prepare the sample to give concentrations of 30 to 150 g/ml. Also include reference
standard (blank) containing no protein.
Concentrations of albumin in the final assay volume (f.a.v.) of 5 ml may range from 30
to 300 g protein/5 ml f.a.v. to produce A650 readings from 0.20 to 0.80.
6. Measure the net absorbance of the sample(s), calibration standards, and reference
standard at 650 nm (A650 ) in 1-cm cuvettes.
7. If the spectrophotometer used does not automatically give net absorbance readings,
subtract the value for the reference solution from those obtained for the sample and
calibration standards.
8. Prepare a calibration plot by graphing the net A650 values for the standards versus
protein concentration (micrograms protein/5 ml f.a.v.).
9. Determine the protein concentration of the sample by interpolation from the plot,
accounting for any dilutions made in sample preparation.
Use only the linear portion of the plot. The absorbance plateaus at high protein concentrations, resulting in erroneous calculations.
BASIC
PROTOCOL 2
A.3A.2
Commonly Used Techniques
The BCA assay for total protein is somewhat variable: it has differing sensitivities in
response to incubation time, incubation temperature, standard protein used for calibration,
and other factors (Smith et al., 1985). Certain classes of compounds, such as reducing
sugars and ammonium ions, interfere with the assay, sometimes severely. However, if
interfering compounds are eliminated (e.g., by dialysis), the BCA assay has a good
combination of sensitivity and simplicity, and it has some advantages over the Lowry
technique (see Background Information).
Materials
Calibration standard: 1 mg/ml BSA
Buffer or solvent used to prepare the protein-containing sample
Sample containing protein
BCA reagent A/reagent B mix (see recipe)
Spectrophotometer and cuvettes
1. Prepare a dilution series of calibration standard in the buffer or solvent used to
prepare the sample to cover the range 0.2 to 1.0 mg/ml. Also include a reference
standard (blank) containing no protein.
It is recommended to make triplicate calibration standards to obtain reliable values.
2a. For a 2.1-ml final assay volume: Mix 100 l protein-containing sample, calibration
standard, or reference standard with 2 ml BCA reagent A/reagent B mix in separate
tubes.
Figure A.3A.1 Bicinchoninic acid assay calibration plot using bovine serum albumin. Slope =
1.5 102 A562 (g protein/ml f.a.v.)1 cm1 .
Commonly Used
Techniques
A.3A.3
Commonly Used Techniques
2b. For a 4.2-ml final assay volume: Mix 200 l protein-containing sample, calibration
standard, or buffer used to prepare the sample (reference standard) with 4 ml BCA
reagent A/reagent B mix in separate test tubes.
The final assay volume depends on the size of the available cuvettes.
3. Incubate the samples and standards 30 min at 37 C, then cool to room temperature.
4. Measure the absorbance of the sample, calibration standards, and reference standard
at 562 nm (A562 ).
5. If the spectrophotometer does not automatically give net absorbance readings, subtract the values for the reference standard from those obtained for the sample and
calibration standards.
6. Prepare a calibration plot by graphing the net A650 values for the standards versus
protein concentration (micrograms protein/milliliter f.a.v; see Fig. A.3A.1).
7. Determine the protein concentration of the sample by interpolation from the plot.
Use only the linear portion of the plot. The absorbance plateaus at high protein concentrations, resulting in erroneous calculations.
BASIC
PROTOCOL 3
Equation A.3A.1
Molar absorption coefficients can be calculated from amino acid composition based
on tyrosine and tryptophan contributions to the total molecule. The 275-280 values for
tyrosine (in neutral or acidic solution) and for tryptophan are close to 1470 and 5700
absorbance units M1 cm1 , respectively. There are minor variations in these values
depending on the peptide and the solvent (Bencze and Schmid, 1957).
For example, a 0.04% to 0.12% solution of protein having four to twelve tyrosine groups,
two to six tryptophans, and an 280 value of 8, should have an A280 of 0.2 to 1 with a
1-cm path length cuvette. Hence UV absorption around the 280-nm band provides a fairly
sensitive, convenient means for detecting and quantitating pure proteins or mixtures of
pure proteins having concentrations in the range of 0.1 to 1 mg/ml.
Assays for Total
Protein
A.3A.4
Commonly Used Techniques
Materials
Protein sample, pH <8
Spectrophotometer and fused-silica or quartz cuvettes
1. Turn on the spectrophotometer, set the wavelength to 280 nm, and allow it to warm
up 30 min.
2. If necessary, dilute the protein sample to give an A280 between 0.2 and 1.
Some spectrophotometers may give precise measurements of A280 up to 2 absorption units,
but, in general, conditions and concentrations should be adjusted so the instrument is not
forced to read absorbances >1.0.
3. Read the absorbance of sample and reference standard (buffer or solvent) at 280 nm
in fused-silica or quartz cuvettes.
Glass and plastic cuvettes are opaque below the near UV range, 350 mm.
A complete UV absorption spectrum, 230 or 240 nm to >310 nm, may be very helpful
for detecting interfering compounds. For a solution of one or more pure proteins at acid
pH, there should be a deep trough at 250 nm, a peak around 275 to 280 nm, and very little
absorption above 310 nm, unless the proteins contain cofactors or prosthetic groups.
BASIC
PROTOCOL 4
Coomassie dye (brilliant blue G250) binds to protein molecules in acid pH by two
means: the triphenylmethane group binds to nonpolar structures in proteins and the anion
sulfonate groups interact with protein cationic side chains (e.g., arginine, lysine) in acid
pH (Lovrien et al., 1995). The color change produced when the dye binds to proteins
provides a measure of total protein, which is quite sensitive in the case of albumin and
certain globular proteins (Bradford, 1976; Sedmak and Grossberg, 1977). The Coomassie
dye-binding assay, or Bradford assay, also responds to some interfering substances which
are generally unknown unless the experimenter specifically tests for them (Van Kley and
Hale, 1977; Kirazov et al., 1993). Nevertheless, because of its apparent simplicity and
sensitivity towards many proteins, the Bradford assay is popular and widely used. Using
bovine serum albumin (ideally well behaved) to calibrate the Bradford assay produces a
calibration plot with a slope of 4.5 to 5.5 102 (g protein/ml f.a.v.)1 cm1 . Hence, it
is somewhat more sensitive, by a factor of roughly two or three, than the values generally
quoted for Lowry, Hartree-Lowry, or BCA assays (see Table A.3A.2).
Materials
Calibration standards: 1.5 mg/ml BSA and 1.5 mg/ml lysozyme
Buffer or solvent used to prepare the protein-containing sample
Sample containing protein
Coomassie dye reagent (see recipe) or commercial Coomassie reagent (Bio-Rad,
Pierce)
1. Prepare a dilution series of calibration standards in the buffer or solvent used to
prepare the sample to cover the range 150 to 750 g protein/ml. Also include a
reference standard (blank) containing no protein.
Depending on the kind of protein being measured, it may be useful to calibrate with
various proteins including ones related to (or even a purified preparation of) the protein
being analyzed. For example, if collagen is the analyte, various collagens, high-glycinehydroxyproline polymers, should be used as calibrating standards. Bovine serum albumin
Commonly Used
Techniques
A.3A.5
Commonly Used Techniques
Assay
Calibrating compound
Measured slope
Dinitrosalicylate (DNS)
Glucose
Glucose
Mannose
Biuret protein
BSA
Hartree-Lowry protein
BSA
BSA
Ammonium sulfate
(BSA) is often used as a calibration standard, but it has greater general dye-binding
capacity than most proteins.
3. Measure the absorbance of the sample, calibration standards, and reference standard
at 595 nm (A595 ).
4. If the spectrophotometer does not automatically give net absorbance readings, subtract the values for the reference standard from those obtained for the sample and
calibration standards.
5. Prepare a calibration plot by plotting the net A595 values for the standards versus
protein concentration (microgram protein/milliliter f.a.v.).
6. Determine the protein concentration of the sample by interpolation from the plot.
At maximal sensitivity (with BSA as a calibration standard) the Coomassie dye-binding
assay produces a calibration plot with a slope (sensitivity) of 4 102 A595 (g/ml
f.a.v.)1 cm1 and falls to a third or half of that value with less responsive proteins.
BASIC
PROTOCOL 5
A.3A.6
Commonly Used Techniques
BCA reagent A
1 g 4,4 -dicarboxy-2,2 -biquinoline, disodium salt (Na2 BCA; Pierce or Sigma; 26
mM final)
2 g Na2 CO3 H2 O (0.16 M final)
160 mg disodium sodium tartrate (7 mM final)
0.4 g NaOH (0.1 M final)
0.95 g NaHCO3 (0.11 M final)
100 ml H2 O
After mixing and dissolving the components, adjust the pH to 11.3 0.2, using
solid NaOH to increase pH or NaHCO3 to decrease pH. Store this alkaline reagent
in a plastic container 1 to 3 weeks at room temperature or longer at 4 C.
Only the disodium salt of Na2 BCA is soluble at neutral pH; the free acid is not readily
soluble, even in alkali.
BCA reagent B
4 g CuSO4 5H2 O (16 mM final)
100 ml H2 O
Store up to a few months at room temperature
BCA reagent A/reagent B mix
Mix 50 vol BCA reagent A with 1 vol BCA reagent B (see recipes). Prepare only
sufficient mix for a few hours work because the mixture is not stable.
BCA reagent A/reagent B mix is light apple-green in color.
Hartree-Lowry reagent A
2 g sodium potassium tartrate4 H2 O (Rochelle salt; 7 mM final)
100 g Na2 CO3 (0.81 M final)
500 ml 1 N NaOH (0.5 N final)
H2 O to 1 liter
Store 2 to 3 months at room temperature in a plastic container
Hartree-Lowry reagent B
2 g sodium potassium tartrate4 H2 O (0.07 M final)
1 g CuSO4 5H2 O (0.04 M final)
90 ml H2 O
10 ml 1 N NaOH
Store 2 to 3 months at room temperature in a plastic container
Commonly Used
Techniques
A.3A.7
Commonly Used Techniques
Hartree-Lowry reagent C
Dilute 1 vol Folin-Ciocalteau reagent (Sigma) with 15 vol water.
Prepare this solution daily in 16-ml quantities or multiples thereof. Do not adjust the pH.
COMMENTARY
Background Information
Equation A.3A.2
A.3A.8
Commonly Used Techniques
Figure A.3A.2
Commonly Used
Techniques
A.3A.9
Commonly Used Techniques
Assay
Interfering compounds
Hartree-Lowry
EDTA
GuanidineHCl
Triton X-100
SDS Brij 35
>0.1 M Tris
Ammonium sulfate
1 M sodium acetate
1 M sodium phosphate
Bicinchoninic acid
EDTA
>10 mM sucrose or glucose
1.0 M glycine
>5% ammonium sulfate
2 M sodium acetate
1 M sodium phosphate
UV absorption
Pigments
Phenolic compounds
Organic cofactors
Bradford
UV spectrum analysis
The ultraviolet (UV) absorption spectrum
for a sample protein can be used to quantitate the protein and to evaluate the purity of a
sample. Figure A.3A.3 shows the UV absorption spectra for glycyl-L-tyrosine. The molar
absorption coefficient as a function of wave-
length is dependent on pH. The glycyl moiety has negligible effect on the spectra, so they
are close in general shape to those of tyrosine side chains in proteins. Tryptophan side
chains (indole groups) have similar spectra at
acid pH. However, tryptophan absorption is
considerably more intense at 275 to 280 nm,
by a factor of nearly four. Thus, although different proteins have very variable tyrosinetryptophan compositions, the general shape
of their UV absorption spectra in acid are
fairly close to those of tyrosine at neutral and
acid pH.
Proteins in neutral and acid solutions exhibit a deep trough in absorption at 245 to
250 nm. Most proteins, except heme proteins
and proteins with cofactors such as NADH, do
not absorb from either tyrosine or tryptophan
above 300 to 310 nm. Increased absorption
at 250 nm and/or 300 nm is a strong indication of the presence of compounds that
interfere with A280 measurements and their interpretation. The effectiveness of techniques
such as dialysis and size-exclusion chromatography for removing interfering UV-absorbing
compounds can be monitored by analyzing the
sample for absorption at 250 nm (trough), 280
nm (peak), and 300 to 310 nm (residual). Ratios of these absorbances are often used to determine whether the UV spectrum is that of
A.3A.10
Commonly Used Techniques
Figure A.3A.3
gests of proteins (Bewley, 1982). It is necessary to remove oxygen from the spectrophotometer by nitrogen purging for measurements
at very low wavelengths (hard UV, <220
nm).
All proteins absorb strongly at 205 nm,
so at first glance this seems an attractive
means for quantitating protein. However, most
published methods describing hard UV absorbance are based on the behavior of pure,
mostly single-subunit proteins. Buffers with
carbonyl groups and many other impurities
also become strongly absorbing in the hard
UV range, and the problems of conventional
UV absorbance are magnified.
Coomassie dye binding assay (Bradford
assay)
Coomassie blue dye (see Basic Protocol 4)
produces a rather large absorption spectrum
shift in the visible-light range when it binds
to many globular proteins. The strong binding
forces are partly, but not entirely, due to electrostatic attraction between dye molecule sulfonate groups (two on the G250 dye molecule)
and cationic groups of proteins when they
are made quite acidic. The pH of the reaction mixture is low enough to thoroughly
convert proteins to cations, but not so low
that any sulfonate groups can become neutralized. The low pH of 0.8 M phosphoric acid
also causes most proteins to expand so that
they become exposed to the triphenylmethane,
Commonly Used
Techniques
A.3A.11
organic, and nonpolar sectors of the dye to enhance binding (Craig, 1967). Proteins that do
not bind anionic dye, even in very low pH,
may not bind Coomassie dyes. Early research
on Coomassie dyebased protein assays used
globular proteins, which are known to avidly
bind dye and other similar anions such as detergent anions (Edsall and Wyman, 1958). Albumin, hemoglobin, chymotrypsinogen, and
cytochrome c, which Bradford used to calibrate the method (Bradford, 1976), are particularly able proteins in this regard. However,
other classes of proteins are quite unlike these
globular, organic anionbinding proteins and
these differences may be at the root of some of
the failures reported for the Bradford method
(Pierce and Suelter, 1977). At any rate, the
Bradford method is especially simple and has
good sensitivity when it is calibrated with proteins to which it is sensitive (e.g., albumin).
Jernejc et al. (1986) compared four
different methods for determining total
proteinKjeldahl nitrogen, biuret, Lowry,
and Coomassie dye bindingfor each of
eight stages in the isolation of proteins from
mycelia. They found that the Coomassie dye
binding method often grossly underestimated
protein content, by factors >2-fold, relative to
the other methods tested. Although Coomassie
dye binding is an easy and convenient assay,
its reliability must be verified for each experimental system.
A similar method is the Udy dye binding
method, which uses acid orange 12, a sulfonated azobenzene dye. The technique was
originally developed for food proteins, and it
apparently compares well with Kjeldahl nitrogen analytic criteria (Udy, 1956).
Critical Parameters
A.3A.12
Commonly Used Techniques
Sometimes it is useful to precipitate the interfering compounds leaving the proteins in solution. Polyethyleneimines (PEI; Sigma) can be
used to precipitate nucleic acids, which have
a UV absorption peak at 260 nm (Jendrisak,
1987). PEI does not absorb in the ultraviolet
range. However, neither TCA nor PEI is free of
cross-precipitation, so it may be necessary to
experiment with the precipitant concentration,
pH, and temperature to optimize a particular
precipitation step.
Anticipated Results
Table A.3A.1 presents a summary of the
detection ranges to expect for the assays described here.
Time Considerations
Basic Protocols 1 and 2 (Hartree-Lowry and
BCA assays) require 1 hr for measuring sample, mixing, incubation, and reading the absorbance. Basic Protocol 3 (UV absorption) is
the simplest of the methods described in this
unit, taking only about 30 min to make dilutions, warm up the spectrophotometer, and
measure the absorbance curve. Basic Protocol 4 (Coomassie dye binding) is also fast, requiring 20 to 30 min.
Acknowledgement
This unit is dedicated to Rex Lovrien, one
of its original authors, who passed away prior
to its publication in this manual. His insight
into the use and advancement of this technology should benefit scientists for many years to
come.
Literature Cited
Annand, R. and Romeo, P.L. 1976. Protein in foodstuffs. Amer. Lab. 8(10):37-46.
Bencze, W.L. and Schmid, K. 1957. Determination of tyrosine and tryptophan in proteins. Anal.
Chem. 29:1193-1196.
Bewley, T.A. 1982. A novel procedure for determining protein concentrations from absorption spectra of enzyme digests. Anal. Biochem. 123:5562.
Beyer, R.E. 1983. A rapid biuret assay for protein
of whole fatty tissues. Anal. Biochem. 129:483485.
Bradford, M.M. 1976. A rapid and sensitive method
for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Chang, Y.-C. 1992. Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins. Anal. Biochem. 205:22-26.
Commonly Used
Techniques
A.3A.13
Commonly Used Techniques
Craig, L.C. 1967. Techniques for the study of peptides and proteins by dialysis and diffusion.
Methods Enzymol. 11:870-884.
Goldschmidt, R.C. and Kimelberg, H.K. 1989. Protein analysis of mammalian cells in monolayer
culture using the bicinchoninic acid assay. Anal.
Biochem. 177:41-45.
Sober, H.A. (ed.) 1970. Molar extinction coefficients and E1% values for proteins at selected
wavelengths of the ultraviolet and visible region.
In Handbook of Biochemistry, Selected Data
for Molecular Biology, pp. C71-C98. Chemical
Rubber Co. Press, Cleveland.
Daumantas Matulis
Institute of Biotechnology
Vilnius, Lithuania
A.3A.14
Commonly Used Techniques
APPENDIX 3B
Mycoplasma contamination is a serious and frequent problem in the cell culture laboratory. Although one may suspect mycoplasma contamination when cells fail to thrive,
formal diagnosis rests on the polymerase chain reaction (PCR; see Basic Protocol 1)
or on the detection of adenosine phosphorylase secretion by infected cell lines (see
Alternate Protocol 1). Basic Protocol 2 and Alternate Protocol 2 in this appendix present
methods for antibiotic treatment of infected cultures.
NOTE: It is recommended that any positive test for mycoplasma be validated using a
second assay.
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aseptic technique should be used accordingly.
BASIC
PROTOCOL 1
Materials
Cell cultures to be tested for contamination
Phosphate-buffered saline (PBS; APPENDIX 2A) or culture medium for cells
H2 O, sterile
Roche Mycoplasma PCR ELISA kit (cat. no. 1663925) including:
Lysis reagent
Mycoplasma positive control DNA
Neutralization reagent
PCR ready-to-go mix
Taq DNA polymerase
Tabletop centrifuge
Thin-walled PCR tubes
Thermal cycler
Additional reagents and equipment for agarose gel electrophoresis (Sambrook and
Russell, 2001; Voytas, 2000)
Commonly Used
Techniques
Contributed by Helena Harlin and Thomas F. Gajewski
Current Protocols in Microbiology (2005) A.3B.1-A.3B.6
C 2005 by John Wiley & Sons, Inc.
Copyright
A.3B.1
Perform PCR
8. For each sample and control to be tested, transfer 25 l PCR ready-to-go mix and
15 l sterile water into a thin-walled PCR tube. Add 10 l control or sample to the
corresponding tubes. Add 0.5 l Taq DNA polymerase to each tube.
Taq DNA polymerase is included in the PCR ready-to-go mix, but after repeated freezethawing the enzyme may lose its activity.
9. Transfer each tube to the thermal cycler and perform PCR using the following cycling
conditions:
1 cycle:
40 cycles:
1 cycle:
5 min
30 sec
30 sec
1 min
10 min
95 C
94 C
62 C
72 C
72 C
(initial denaturation)
(denaturation)
(annealing)
(extension)
(final extension).
10. Following amplification, resolve the PCR products on a 1% agarose gel (see, e.g.,
Sambrook and Russell, 2001; Voytas, 2000).
Positive bands are 500 to 600 kb. A prominent band will be present in the positive control
if the PCR reaction was successful, and will also be present in any samples contaminated
with mycoplasma.
ALTERNATE
PROTOCOL 1
Diagnosis and
Treatment of
MycoplasmaContaminated
Cell Cultures
A.3B.2
Current Protocols in Microbiology
This method can also be used as an alternative to PCR (see Basic Protocol 1) to evaluate
conditioned medium for the possible presence of mycoplasma. In the instructions accompanying the MycoTect kit obtained from Life Technologies, 3T6 cells are suggested for
use as the indicator cell. However, for many immunology laboratories the SP2/0-Ag14
hybridoma cell linea widely used fusion partner for generating antibody-secreting
hybridomas (Shulman et al., 1978)is more convenient to use.
In this protocol, a potential problem of differential susceptibility of cells to the toxic
effects of 6-MP is avoided by culturing a highly susceptible cell line overnight with
culture supernatants from the cells to be tested. Contamination is detected after addition
of 6-MPDRif growth is not observed, mycoplasma is present.
Materials
SP2/0-Ag14 hybridoma cells (ATCC #CRL 1581)
Cells to be tested in appropriate medium without antibiotics
MycoTect (Life Technologies) containing 6-methylpurine deoxyriboside
(6-MPDR)
6-methylpurine (6-MP; Sigma)
96-well flat-bottom microtiter plates (Costar, Falcon, or equivalent)
1. Prepare 50-l triplicate cultures containing 2 103 SP2/0-Ag14 cells (4
104 cells/ml) in 96-well flat-bottom microtiter plates.
2. Culture cells to be tested in medium without antibiotics in a humidified 37 C, 5%
CO2 incubator for 24 hr. Allow cells to overgrow, remove supernatant, and add
50 l culture supernatant to each well of the microtiter plate (from step 1).
Routinely, each cell line is tested in triplicatei.e., cells are cultured in nine microtiter
wellsalthough this may be unnecessary. Each cell line should be tested every 2 months
to be certain that contamination has not been inadvertently introduced.
3. Add 100 l medium to first well (negative control), 100 l 6-MPDR (40 M final) to
second well, and 100 l 6-MP (6 M final) to third well (positive control). Culture
cells 2 to 3 days in a humidified 37 C, 5% CO2 incubator.
The MycoTect kit also includes adenosine phosphorylase to be used as an additional
positive control; use according to the instructions provided.
4. Screen cultures visually using an inverted microscope for growth of indicator SP2/0Ag14 cells. If mycoplasma is not present, cell growth is observed in the presence of
medium and 6-MPDR but not in the presence of 6-MP. If mycoplasma is present, cell
growth is observed in the presence of medium but not in the presence of 6-MPDR
or 6-MP (see Basic Protocol 2 and Alternate Protocol 2 for treatment).
Staining cells with crystal violet is recommended in the instructions included in the
MycoTect kit, but is usually unnecessary.
BASIC
PROTOCOL 2
Commonly Used
Techniques
A.3B.3
Current Protocols in Microbiology
more toxic to cells. Nevertheless, there is significant experience indicating the efficacy
of BM-cyclin, so use of Alternate Protocol 2 should not be ruled out.
Materials
10 mg/ml ciprofloxacinHCl (see recipe), sterile
Contaminated cells (see Basic Protocol 1 and Alternate Protocol 1)
1. Add 10 mg/ml ciprofloxacinHCl to contaminated cells to 10 g/ml final (a 1:1000
dilution). Keep ciprofloxacinHCl in the cultures for 12 days in a humidified, 37 C
5% CO2 incubator.
2. After completion of the treatment, freeze aliquots of the mycoplasma-free cells.
3. Test cell line for presence of mycoplasma infection (see Basic Protocol 1 and
Alternate Protocol 1).
4. Culture cells in absence of ciprofloxacinHCl and watch for recurrence of mycoplasma contamination (see Basic Protocol 1 and Alternate Protocol 1).
ALTERNATE
PROTOCOL 2
Materials
BM-cyclin solutions 1 and 2 (see recipe), sterile
Contaminated cells (see Basic Protocol 1 and Alternate Protocol 1)
1. Determine the maximum amount of BM-cyclin solutions 1 and 2 that can be tolerated
by the contaminated cells by setting up a titration curve for each solution in the
following manner.
a. Prepare several different concentrations of each BM-cyclin solution beginning at
20 l of each BM-cyclin solution per 10 ml cell culture and then halving the concentration for several dilutions (e.g., use 10 l/10 ml, 5 l/10 ml, and 2.5 l/10 ml).
b. Culture cells at density appropriate for that cell line for 3 days in BM-cyclin
solution 1 and 4 days in BM-cyclin solution 2 in a humidified 37 C, 5% CO2
incubator.
2. Screen cultures visually using an inverted microscope to identify the BM-cyclin
solution dose that does not kill cells and allows cell growth.
Alternatively, an aliquot of cells can be counted using the trypan blue exclusion assay
(Phelan, 1996).
Diagnosis and
Treatment of
MycoplasmaContaminated
Cell Cultures
3. Culture cells 3 days in the maximum tolerable dose of BM-cyclin solution 1, then
4 days in BM-cyclin solution 2 at the same dose in a humidified 37 C, 5% CO2
incubator.
This represents one cycle of treatment. If the cell density has plateaued, split into fresh
growth medium plus the appropriate BM-cyclin solution at the maximal tolerated dose.
A.3B.4
Current Protocols in Microbiology
5. Repeat steps 3 and 4 twice. Screen cultures visually using an inverted microscope
after each cycle for signs of improved cell viability and growth. Split cells as their
growth rate and density dictate.
Alternatively, count cells using the trypan blue exclusion assay. In most cases, three cycles
are sufficient to eradicate the mycoplasma. Occasionally, additional cycles are necessary.
If so, treat cells with one cycle of antibiotics beyond eradication. Chronic administration
(>4 weeks) of the antibiotics is not recommended since resistant organisms may develop.
Treated cells should grow more vigorously and lose symptoms of mycoplasma contamination (see Troubleshooting).
CiprofloxacinHCl, 10 mg/ml
Dissolve ciprofloxacinHCl (Sigma) in water to 10 mg/ml. Filter sterilize through
a 0.45-m filter, aliquot, and store in sterile tubes at 20 C.
COMMENTARY
Critical Parameters
When treating cultures with BM-cyclin, the
major critical parameter is that the correct dose
of the drug be used (see Basic Protocol 2 and
Alternate Protocol 2). In general, toxicity of
ciprofloxacin has not been a problem.
Another consideration is whether the desired phenotype of the cell will change during the BM-cyclin or ciprofloxacin treatment.
It is possible that a monoclonal antibody
producing hybridoma will lose its secretory
capacity during the relatively prolonged treatment and may need to be recloned by limiting
dilution (Yokoyama, 1991) during treatment.
Troubleshooting
Sources of Contamination
The most common sources of mycoplasma
contamination are previously contaminated
cell lines, bovine sources (e.g., FBS), and
other animal sources. The best method of eradication is prevention. FBS should be heatinactivated before use in tissue culture (see
APPENDIX 2A). Any animals that will be used
to generate cell lines or bioactive products
should be purchased from specific-pathogenfree (SPF) suppliers and housed in pathogenfree facilities. In general, newly derived cell
lines and cell lines from other laboratories
Commonly Used
Techniques
A.3B.5
Current Protocols in Microbiology
Time Considerations
BM-cyclin treatment requires one week for
each cycle. At least three cycles are required
but sometimes more are necessary.
Literature Cited
McGarrity, G.J. and Carson, D.A. 1982. Adenosine phosphorylase-mediated nucleoside toxicity: Application towards the detection of mycoplasmal infection in mammalian cell cultures.
Exp. Cell Res. 139:199-205.
Phelan, K. 1996. Techniques for mammalian cell
tissue culture. In Current Protocols in Molecular
Biology (F.M. Ausubel, R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K.
Struhl, eds.) pp. A.3F.1-A.3F.14. John Wiley &
Sons, Hoboken, N.J.
Sambrook, J. and Russell, D. 2001. Molecular
Cloning: A Laboratory Manual, 3rd ed. Cold
Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
Schmitt, K., Daubeaner, W., Bitter-Suermann, D.,
and Hadding, U. 1988. A safe and efficient
method for elimination of cell culture mycoplasmas using ciprofloxacin. J. Immunol. Methods
109:17-25.
Shulman, M., Wilde, C.D., and Kohler, G. 1978.
A better cell line for making hybridomas secreting specific antibodies. Nature (Lond.) 276:269270.
Voytas, D. 2000. Agarose gel electrophoresis.
In Current Protocols in Molecular Biology
(F.M. Ausubel, R. Brent, R.E. Kingston, D.D.
Moore, J.G. Seidman, J.A. Smith, and K. Struhl,
eds.), pp. 2.5A.1-2.5A.9. John Wiley & Sons,
Hoboken, N.J.
Wirth, M., Grashoff, M., Schumacher, L., and
Hauser, H. 1995. Mycoplasma detection by the
Mycoplasma PCR ELISA. Biochemica 3:33-35.
Yokoyama, W. 1991. Production of monoclonal antibodies. In Current Protocols in Immunology.
(J.E. Coligan, A.M. Kruisbeek, D.M. Margulies,
E.M. Shevach, and W. Strober, eds.) pp. 2.5.12.5.17. John Wiley & Sons, New York.
Key Reference
McGarrity, G.J., Sarama, J., and Vanaman, V. 1979.
Factors influencing microbiological assay of
cell-culture Mycoplasmas. In Vitro (Rockville)
15:73-81.
Classic review of mycoplasma contamination and
its evaluation.
Anticipated Results
Diagnosis and
Treatment of
MycoplasmaContaminated
Cell Cultures
Most cultures will be relatively free of mycoplasma contamination after the antibiotic
treatment. Whether complete eradication has
been achieved should be viewed with caution
as long as the cell line is used.
A.3B.6
Current Protocols in Microbiology
Gram Staining
The gram stain ranks as one of the most important stains for bacteria. Named after
Hans Christian Gram who developed the method in 1884, the Gram stain allows one to
distinguish between Gram-positive and Gram-negative bacteria on the basis of differential
staining with a crystal violetiodine complex and a safranin counterstain. The cell walls
of Gram-positive organisms retain this complex after treatment with alcohol and appear
purple, whereas gram-negative organisms decolorize following such treatment and appear
pink. The method described is useful for assessing bacterial contamination of tissue
culture samples or for examining the Gram stain status and morphological features of
bacteria isolated from mixed or isolated bacterial cultures.
APPENDIX 3C
BASIC
PROTOCOL
Materials
Sample to be stained
Crystal violet solution (see recipe)
Grams iodine solution (see recipe)
Decolorizing solution (see recipe)
Counterstain (see recipe)
Bibulous paper (optional)
Microscope with oil immersion objective (UNIT 2A.1)
NOTE: A Gram stain kit (e.g., Fisher) may be used in lieu of the staining reagents
described above.
Prepare smear
1. Hold an inoculating loop in a flame until red-hot and then allow to cool 30 sec.
Using this sterilized loop, prepare a thin film of the sample to be stained on a glass
slide.
If checking a tissue culture sample for possible bacterial contamination, or a suspension
of bacteria growing in culture medium, remove a loopful of sample and apply this directly
to the slide. If examining bacteria growing as colonies on plates, pick one colony and
resuspend bacteria in a drop of sterile water previously applied to the slide.
2. Air dry the smear then heat-fix by passing the slide over a flame two or three times.
Do not leave the slide over the flame, as this will overheat the sample.
Stain smear
3. Cover the heat-fixed smear with crystal violet solution. Let stand 30 sec.
4. Wash the stain off gently with flowing water for 5 sec. Shake off excess water.
Gently dripping tap water can be used.
5. Cover the smear with Grams iodine solution for 1 min. Wash with water as in step
4 and shake off excess water.
Commonly Used
Techniques
A.3C.1
7. Wash with water as in step 4 for 5 sec. Shake off excess water.
8. Cover the slide with counterstain for 30 sec.
9. Wash with water as in step 4 then shake off excess water. Allow to air dry, or carefully
blot with bibulous paper.
A well-prepared smear should be barely visible to the unaided eye.
Examine slide
10. Examine the bacteria under oil immersion (900 to 1000) to distinguish between
Gram-positive and Gram-negative bacteria.
Do not use a cover slip. Simply add 1 drop of oil to the center of the slide and observe
bacteria. If necessary, slides can be stored in a slide box for many months and reexamined
under oil immersion.
Gram-positive bacteria appear purple as compared with Gram-negatives which appear
pink.
Counterstain
Mix 2.5 g safranin O with 100 ml of 95% ethanol. Store up to 1 year at room
temperature.
Crystal violet solution
Crystal violet stock (solution A)
Dissolve 20 g crystal violet (85% dye) in 100 ml of 95% ethanol. Store up to 1 year
at room temperature.
Oxalate stock (solution B)
Dissolve 1 g ammonium oxalate in 100 ml water. Store up to 1 year at room
temperature.
Working solution
Dilute solution A 1:10 with distilled water and mix with 4 vol solution B. Store up
to 6 months in a glass bottle at room temperature.
Decolorizing solution
Mix equal volumes of 95% ethanol and acetone. Store up to 1 year in a glass bottle
at room temperature.
Grams iodine solution
Dissolve 1 g iodine crystals (Sigma) and 2 g potassium iodide (Sigma) in 5 ml
water, then add 240 ml water and 60 ml of 5% (w/v) sodium bicarbonate solution.
Mix well and store up to 6 months in an amber glass or foil-covered bottle at room
temperature.
Contributed by Richard Coico
Weill Medical College of Cornell University
New York, New York
Gram Staining
A.3C.2
Current Protocols in Microbiology
APPENDIX 3D
Michael E. Woodman1
1
ABSTRACT
This protocol describes an efficient method for screening intact bacteria for the
presence of desired DNA sequences using polymerase chain reaction (PCR). This
method is commonly referred to as colony PCR. Curr. Protoc. Microbiol. 9:A.3D.1C 2008 by John Wiley & Sons, Inc.
A.3D.6.
Keywords: colony PCR r bacteria r screen
BASIC
PROTOCOL
This unit describes a method for screening individual bacterial colonies for specific DNA
sequences using the polymerase chain reaction (PCR; see Kramer and Coen, 2001). This
procedure, commonly referred to as colony PCR, is advantageous because it is a very
quick and easy way to screen a large number of bacteria to determine which bacteria
contain a particular DNA sequence, without first having to purify DNA from all of them.
This is especially useful when screening colonies after transformation with recombinant
plasmids or after targeted mutagenesis. Colony PCR can be effectively used not only to
identify clones with an insertion or deletion, but also to determine the orientation of a DNA
insertion, which may be important for proper transcription and translation of the insert.
Furthermore, the method can be used to amplify a desired DNA fragment for subsequent
sequencing or cloning. While colony PCR is most commonly used in the laboratory to
screen transformed E. coli, DNA sequences from virtually any bacterial species can be
detected and/or isolated regardless of location on the chromosome or a plasmid.
Materials
10 PCR buffer: supplied with DNA polymerase or see APPENDIX 2A
2.5 mM (each) dNTP mix: supplied with DNA polymerase or see APPENDIX 2A
30 M oligonucleotide primer 1 stock (see Critical Parameters)
30 M oligonucleotide primer 2 stock (see Critical Parameters)
5 U/l Taq DNA polymerase, heat-stable (e.g., Takara Taq, Fisher)
Distilled H2 O, sterile
MgCl2 (if required)
Gel electrophoresis loading buffer (see recipe)
200-l thin-walled PCR tubes (or other appropriate for thermal cycler)
Toothpicks, sterile
Thermal cycler
Additional reagents and equipment for performing agarose (Voytas, 2000) or
polyacrylamide (Chory and Pollard, 1999) gel electrophoresis
Procedure
1. Prepare the master mix for PCR amplification (e.g., 100 l for ten tubes of 10 l
each):
Current Protocols in Microbiology A.3D.1-A.3D.6, May 2008
Published online May 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mca03ds9
C 2008 John Wiley & Sons, Inc.
Copyright
Commonly Used
Techniques
A.3D.1
Supplement 9
4. Carry out PCR using conditions appropriate for the DNA being tested, with the
following amplification cycles as a guideline:
Initial step:
5 min
94 C
25 cycles:
30 sec
30 sec
1 min
5 min
94 C
50 C-55 C
68 C-72 C
72 C.
Final extension:
(bacterial lysis/
denaturation)
(denaturation)
(annealing)
(extension)
A thermal cycler with a heated lid is recommended for convenience. Otherwise, overlaying
the reaction mixture with mineral oil will be required (see thermal cycler instruction
manual and Kramer and Coen, 2001).
Twenty five cycles should be sufficient for production of a detectable amplicon.
It is important to note that the ideal annealing temperature will vary depending on the GC
content of the primers being used. Greater primer GC content requires higher annealing
temperatures for specificity.
Direct PCR of
Intact Bacteria
(Colony PCR)
Additionally, a general rule of thumb for PCR is to allow 1 min of extension time for every
1 kb of amplified DNA product. For a detailed description of the stages included in the
PCR reaction see Kramer and Coen (2001).
A.3D.2
Supplement 9
Figure A.3D.1 Representative results of colony PCR. Lane 1: 1-kb ladder (New England BioLabs); Lane 2: 1.3-kb insert; Lane 3: 400-bp insert; Lane 4: No insert. The faint bands at the
bottom of the gel are unused primers from the PCR reaction.
6. Electrophorese the reaction mixture on an agarose gel (Voytas, 2000) or a nondenaturing, continuous polyacrylamide gel (Chory and Pollard, 1999), depending on the
size of the PCR product.
The optimal percentage of agarose used in gels can vary depending on the size of the
DNA product. A 0.8% agarose gel can be used for most PCR products, but if the product
is large, a lower percentage gel can be used, and vice versa.
For PCR products under 500 bp, nondenaturing polyacrylamide gels provide better
resolution than agarose gels.
7. Stain the gel with ethidium bromide and use ultraviolet (UV) light to visualize the
PCR products (see Voytas, 2000).
CAUTION: Gloves should be worn when handling ethidium bromide (a carcinogen) and
acrylamide (a neurotoxin).
CAUTION: UV-protectant eyewear should be worn when viewing the gel with UV light.
Representative results are shown in Figure A.3D.1.
Commonly Used
Techniques
A.3D.3
Supplement 9
COMMENTARY
Background Information
The detection or isolation of a specific DNA
sequence directly from a colony of E. coli or
most other bacterial species of interest can
be performed using this simple PCR-based
method. Most commonly, colony PCR is used
to screen E. coli transformants for a specific
DNA sequence of interest. Screening is important because not all colonies that arise following transformation may contain a plasmid
with the desired DNA insert. Additionally, the
DNA insert may be in the wrong orientation for
proper transcription and translation. Colony
PCR is an efficient method for easily identifying the bacteria with the desired DNA, as well
as the desired orientation of the insert.
A major advantage of colony PCR is that
this procedure does not require DNA purification. Simply, a small aliquot of whole bacteria
(e.g., a colony) is added to a PCR mix (specific primers, heat-stable DNA polymerase,
dNTPs, and buffer) and then subjected to PCR.
The initial PCR step, heating to 94 C, is sufficient to lyse bacteria and liberate their DNA
contents.
This procedure is not exclusively for use
with bacterial colonies; a small aliquot of
bacteria from an actively growing liquid culture, centrifuged and washed with phosphatebuffered saline (PBS; APPENDIX 2A), can be
used just as easily. A small sample of bacteria
from a frozen stock may also be used.
Although, colony PCR is most commonly
used to screen E. coli transformants, the detection and isolation of a specific DNA sequence
from most bacterial species can be performed
using this same procedure. The DNA sequence
of interest does not have to be located on a recombinant plasmid, but can be located on a
chromosome or a native plasmid in the bacterial genome.
Critical Parameters
Direct PCR of
Intact Bacteria
(Colony PCR)
A.3D.4
Supplement 9
Researchers commonly use previously constructed plasmid vectors to insert the desired
DNA fragment into a specific site on the plasmid, often a multiple cloning site (MCS). Plasmid vectors can be purchased commercially
(e.g., the pUC series) or are readily available
from other researchers. Cloning of PCR products is generally simplified by use of TA vectors (e.g., pCR2.1; Invitrogen). The MCS or
TA-cloning sites of widely used cloning vectors are very often bordered on each side with
conserved DNA sequences. Commonly used
flanking DNA includes sequences based on
the bacteriophages M13 and/or T7.
One can take advantage of conserved vector sequences to minimize the expense of PCR
screening by using the same primers for amplification of an insert in any plasmid that contains these common flanking DNA sequences.
To determine the oligonucleotide primers that
can be used for the plasmid, product information sheets are provided with commercially
obtained cloning vector plasmids and are also
available at no cost on the manufacturers Web
sites. These Web sites are freely accessible to
researchers even if the plasmid was not purchased from the commercial vendor, but obtained from a fellow scientist.
If the DNA insert is being introduced into
an expression vector, the proper orientation
is required for production of the protein. The
orientation can be determined using the same
approach as described above, but with a different primer set, as shown in Figure A.3D.2.
Two primer sets can be designed, each with
one primer complementary to a flanking DNA
sequence and the other to a DNA sequence in
the inserted fragment (primer sets should be
designed to amplify in opposite directions).
Colony PCR using the described methods
will allow you to determine if a DNA insert
or deletion is in the proper location. However,
sequencing the DNA is strongly recommended
to ensure no errors were introduced during the
cloning processes.
Troubleshooting
Common problems arising during execution of this technique, as well as their potential causes and solutions, are listed in Table
A.3D.1.
Anticipated Results
Colony PCR using primers specific for
DNA sequences flanking the insertion site will
allow you to determine if you have an insertion
in the appropriate location and if it is the expected size. PCR amplification with primers
specific for DNA on each side of the insert
(see Critical Parameters and Fig. A.3D.2) will
yield a product the size of the insert plus the
distance between the two primer locations and
the insertion. Vectors without an insert will
yield a small product representing only the
distance between the two primers. In general,
inserts that are not the desired DNA fragment
will yield amplicons of different sizes than the
insert.
After performing PCR reactions with either primers 1 + 3 or primers 2 + 4 as diagrammed in Figure A.3D.2, you will obtain a
product only if the DNA insert is in the proper
Current Protocols in Microbiology
orientation. If the insert is in the wrong orientation, the primers will be aligned in the same
direction, yielding no product. If primers 1 +
2 or primers 3 + 4 are used, a PCR product
will be produced only if the insert is in the
opposite orientation.
Time Considerations
A PCR reaction mix can be prepared in under 10 minutes, once reagents are thawed. For
the amplification of a 1-kb DNA fragment,
2 hr should be allotted for the reaction to run
in the thermal cycler. If the amplicon is larger
than 1 kb, the PCR will require additional time,
accordingly.
An agarose gel can be prepared in approximately 20 min, with a few minutes required
to measure and melt the agarose in the microwave and the rest of the time to allow for
the gel to cool and solidify.
Electrophoresis of the PCR products on
an agarose gel requires 1 hr, as well as
20 to 30 min to stain the gel in ethidium
bromide.
Overnight incubation of bacteria such as
E. coli is typically required for formation of
colonies on plates, but this may vary depending on the specific bacterial species and strain
being examined, as well as the growth medium
and conditions.
Figure A.3D.2 Diagram of oligonucleotide primer locations for colony PCR. Primers 1 + 4 determine if a DNA insert size is correct. Primers 1 + 3 or primers 2 + 4 can be used to determine
if there is a DNA insertion as well as its orientation. For additional conrmation of the orientation
of the insert, PCR reactions can be performed with primers 1 + 2 or primers 3 + 4 as well (see
Anticipated Results).
Table A.3D.1 Troubleshooting Guide for Colony PCR
Problem
Possible cause
Solution
No PCR product
Multiple PCR
products
PCR product of
undesired size
Commonly Used
Techniques
A.3D.5
Current Protocols in Microbiology
Supplement 9
Literature Cited
Chory, J. and Pollard, J.D.Jr. 1999. Separation of
small DNA fragments by conventional gel electrophoresis. Curr. Protoc. Mol. Biol. 47:2.7.12.7.8.
Kramer, M.F. and Coen, D.M. 2001. Enzymatic
amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol.
56:15.1.1-15.1.14.
Riley, S.P., Woodman, M.E., and Stevenson, B.
2008. Culture of Escherichia coli and related bacteria. In Current Protocols Essential
Laboratory Techniques (S.R. Gallagher and
E.A. Wiley, eds.) pp. 4.2.1-4.2.25. John Wiley
& Sons, Hoboken, N.J.
Voytas, D. 2000. Agarose gel electrophoresis. Curr.
Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.
Direct PCR of
Intact Bacteria
(Colony PCR)
A.3D.6
Supplement 9
APPENDIX 4
APPENDIX 4A
Bacterial species differ from each other in their metabolic capabilities, requiring researchers to use specific media and culture conditions. As those requirements often vary
widely between species, readers should refer to the units in this manual focusing on their
particular organism for details on cultivation and media. However, a number of common
techniques are used in the study of a wide variety of bacteria, and these are presented in
this appendix. In addition, since many strains of Escherichia coli serve as important tools
for almost all modern biologists, commonly used techniques and media for that species
are also included.
ASEPTIC TECHNIQUE
This phrase refers to all of the measures that are taken to prevent the introduction of
sepsis (contamination) into cultures. Aseptic technique is sometimes mistakenly called
sterile technique, but this phrase is inappropriate since the goal of bacterial culture is
to grow a microorganism rather than maintain a sterile environment devoid of bacteria.
It is imperative that the microbiologist practice aseptic technique to ensure that cultures
remain uncontaminated; otherwise, resulting studies will be meaningless. Examples of
aseptic technique include cleaning and disinfecting laboratory surfaces prior to use,
limiting the amount of time during which cultures or media are left uncapped and thus
exposed to the air, keeping petri dishes closed whenever possible, effectively sterilizing
inoculating loops and other equipment that come into contact with cultures or media, and
avoiding breathing on cultures or sterile instruments.
Commonly Used
Methods for Cell
Culture
A.4A.1
Figure A.4A.1
Making a spreader.
Culture Spreader
A spreader is used to evenly distribute bacterial cells over the surface of a plate. This will
yield either a lawn on a nonselective medium or isolated colonies on a selective medium.
A spreader can be easily made by heating and bending a piece of 4- to 5-mm diameter
glass rod, as illustrated in Figure A.4A.1. In a pinch, a much less durable spreader can
be quickly made by substituting a Pasteur pipet for the glass rod. For safety in handling,
melt away sharp ends of the rod as a first step, i.e., prior to bending. A pair of needle-nose
pliers or similar tool is useful for forming bends in glass.
Before each use, sterilize the spreader by immersing the triangular end in a beaker of
95% ethanol, passing the spreader through a flame, then allowing the flame to burn out.
Be careful not to let the burning ethanol drip into the beaker or onto flammable objects.
Cool the spreader by touching to an unused portion of the agar surface. Sterilize again
after use by immersing in ethanol.
Common
Bacterial
Culture
Techniques and
Media
Presterilized, disposable plastic culture spreaders are also available from a variety of
sources. As with plastic inoculating loops (see above), disposable spreaders are expensive
over the long run and generate considerable waste, but may be preferable in certain
situations.
A.4A.2
Current Protocols in Microbiology
CULTURE TECHNIQUES
Oxygen Environment
Bacteria differ in their ability to grow in the presence of oxygen. Not all species can
utilize oxygen, and many are unable to survive exposure to it. Be sure to follow guidelines
described in this book for each species.
Growth of E. coli is generally enhanced by the presence of oxygen, with the Krebs
cycle and oxidative phosphorylation facilitating complete catabolism of complex carbon
sources to carbon dioxide. For this reason, E. coli cultures on solid media should be incubated in standard (aerobic, not CO2 ) incubators. Liquid cultures should be oxygenated
by shaking during incubation. Special Erlenmeyer flasks with baffles on the bottom are
ideal, as the baffles perturb liquid flow and help dissolve air into the medium. Caps should
be loosely attached, or vented caps or other enclosures used. To maximize exposure of
liquid to air, do not fill culture containers more than one-third full.
Commonly Used
Methods for Cell
Culture
A.4A.3
Current Protocols in Microbiology
Figure A.4A.2 Example illustrating a gridded counting chamber, a hemacytometer slide (Improved Neubauer), and a coverslip. The coverslip is applied to the slide and the cell suspension
is added to the counting chamber using a mechanical pipettor or a Pasteur pipet. Each counting
chamber has a 3 3mm grid (enlarged). The four corner squares (1, 2, 4, and 5) and the central
square (3) are counted on each side of the hemacytometer (numbers added).
volume of culture (50 to 500 l) in the middle of the agar surface. Sterilize the spreader
by immersing the hooked end into 95% ethanol in a small beaker, then burn off the
ethanol by passing the spreader through a Bunsen burner flame. Cool the spreader by
touching it lightly to a sterile agar surface, then spread the bacterial culture uniformly
around the plate. Return the spreader to the beaker with the ethanol and incubate the
plate at the appropriate temperature.
Common
Bacterial
Culture
Techniques and
Media
Rotating lazy Susan devices, which spin the plate, are readily available from many
sources, and can aid uniform spreading of cultures. To use such a device, place the petri
dish, agar side down, in the center of the rotating device. Flame and cool the spreader,
lift the lid off the dish, spin the device with a free hand, and touch the spreader to the
agar surface. After a dozen or so rotations, stop the spinning by hand and replace the lid.
A.4A.4
Current Protocols in Microbiology
Figure A.4A.3
Liquid Media
LB (Luria-Bertani) medium
10 g tryptone
5 g yeast extract
5 g NaCl
Adjust volume to 1 liter with H2 O
Commonly Used
Methods for Cell
Culture
A.4A.5
Current Protocols in Microbiology
Medium
Comments
LB medium
M9 minimal salts
SOC medium
A rich medium with high osmolarity; ideal for the recovery step
following transformation of E. coli and similar bacteria
Sterilize by autoclaving
Store indefinitely at room temperature
Some researchers adjust the pH to 7 by titrating with 1N NaOH, but this is not necessary.
M9 minimal salts, 5
30 g Na2 HPO4
15 g KH2 PO4
5 g NH4 Cl
2.5 g NaCl
15 mg CaCl2 (optional)
Adjust volume to 1 liter with H2 O
Add 50 ml chloroform to 5 stock solution as a preservative
Store 5 concentrate up to many months at 4 C
Just before use, dilute 1:5 with water, and sterilize by autoclaving. Cool to <50 C
and add the following:
SOC medium
20 g Bacto tryptone
5 g Bacto yeast extract
10 ml 1 M NaCl
2.5 ml 1 M KCl
Adjust volume to 980 ml with H2 O
Sterilize by autoclaving
Cool to <50 C and add the following:
10 ml 2 M MgCl2
20 ml 20% (w/v) glucose
Store indefinitely at room temperature
Common
Bacterial
Culture
Techniques and
Media
TB (Terrific broth)
12 g Bacto tryptone
24 g Bacto yeast extract
4 ml glycerol
A.4A.6
Current Protocols in Microbiology
Table A.4A.2 Antibiotic Stock Solutions for Use with E. coli Cultures
Stock concentration
Working concentration
for E. coli
10 mg/ml in H2 O
50 g/ml
Carbenicillin
10 mg/ml in H2 O
50 g/ml
Chloramphenicol
30 g/ml
Gentamicin
3 mg/ml in H2 O
15 g/ml
Kanamycin
10 mg/ml in H2 O
50 g/ml
Rifampicin
100 g/ml
Spectinomycin
20 mg/ml in H2 O
100 g/ml
Streptomycin
10 mg/ml in H2 O
50 g/ml
TetracyclineHCl
12.5 g/ml
Antibiotic
Ampicillina (Na salt)
a
Commonly Used
Methods for Cell
Culture
A.4A.7
Current Protocols in Microbiology
If not to be used immediately for pouring plates, solid media can be stored indefinitely
at room temperature in tightly capped bottles. To use, loosen cap, melt by autoclaving or
by heating in a microwave oven at a high setting (microwave oven times vary according
to oven and volume of medium, and must be determined empirically), then proceed as
described above.
Common
Bacterial
Culture
Techniques and
Media
A.4A.8
Current Protocols in Microbiology
APPENDIX 4B
Cultured human endothelial cells have provided great insights into the biology of coagulation and endothelial activation in various disease states including inflammatory disorders,
atherosclerosis, and cancers (Nachman and Jaffe, 2004). However, recent studies suggest
that the endothelial cells are also the targets for microbial adhesion and invasion. In some
cases, the adhesion is direct (Prasadarao et al., 2003), while in others it may be mediated
via bridging molecules such as fibronectin (Peacock et al., 1999; Massey et al., 2001) or
fibrinogen (Cheung et al., 1991; Bayer et al., 1995). Activation of endothelial cells would
ensue as a result of bacterial adhesion (Grimminger et al., 1997). In some cases, invasion
of endothelial cells may ensue after adhesion (Drevets et al., 1995; Prasadarao et al.,
1996; Menzies and Kourteva, 1998). Cultured HUVEC have also been used to study
viral and fungal infections (Sahni, 2006). Many of these studies on microbe-endothelial
cell interaction are conducted with endothelial cells isolated and cultured from source
tissue that yields a relatively pure population of cells. The authenticity of these cells can
be verified by the presence of a immunohistochemical and functional marker such as
von Willebrand factor (Jaffe et al., 1974). Among host tissues, human umbilical vein is
by far the most common source of cultured human endothelial cells (Jaffe et al., 1973a;
Folkman et al., 1979; Kubota et al., 1988; Fukuda et al., 1989). Due to the success of
this procedure, studies on the interaction of microbes with human endothelial cells have
received increasing attention. These studies are now the subject of intensive investigation
in the field of bacterial pathogenesis and cell biology.
CAUTION: Follow all appropriate guidelines and regulations for the use and handling
of human-derived materials. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
NOTE: Institutional Review Board (IRB) approval is often required for permission to
use human umbilical vein as a source for harvest of HUVEC.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.
NOTE: All cell culture incubations should be carried out in a 37 C, 5% CO2 humidified
incubator.
BASIC
PROTOCOL
Materials
Human umbilical cord (cords held for more than 3 hr should be discarded)
HEPES-buffered saline (HBS; see recipe), 37 C
1 (0.1% w/v) collagenase, type I (lyophilized; 150 to 200 U/mg; Invitrogen);
store in 10-ml aliquots at 20 C
M199 medium (Bio-Whittaker) supplemented with 20% (v/v) FBS
Phosphate-buffered saline (PBS; APPENDIX 2A)
Complete HUVEC culture medium/20% FBS (see recipe)
0.02% (w/v) EDTA/0.5% (w/v) bovine serum albumin
Contributed by Ambrose L. Cheung
Current Protocols in Microbiology (2007) A.4B.1-A.4B.8
C 2007 by John Wiley & Sons, Inc.
Copyright
Commonly Used
Methods for Cell
Culture
A.4B.1
Supplement 4
400-ml beakers
Gauze
Nylon tie, 20 size, 6-in. (15-cm) length
Cannulas: blunt, hubless 10-G needles
Hemostat
1-, 30- and 50-ml syringes
10-G needles, blunt and hubless
No. 10 surgical blade
Surgical gloves
Polyethylene tubing, 1/8 in. (0.3 cm) outer diameter
50-ml conical polypropylene centrifuge tubes (e.g., Falcon 2070)
Tabletop centrifuge
25-cm2 gelatin-coated tissue culture flasks (see recipe) and other gelatin-coated
tissue culture vessels as needed for passaging
Additional reagents and equipment for phase-contrast microscopy (UNIT 2A.1) and
cryopreservation of HUVEC (Support Protocol)
NOTE: Utilize aseptic technique in all manipulations of human umbilical cords.
Gauze
6-in., 20 nylon tie
Two cannulas (blunt, hubless 10-G needles)
One hemostat
Two 50-ml syringes
One 30-ml syringe
One no. 10 surgical blade.
3. Pour 150 ml HBS into a beaker. Put on sterile surgical gloves and fill two 50-ml
syringes and one 30-ml syringe with HBS from the beaker.
4. Using a hemostat, remove the cord from the container. Wipe clean with sterile gauze.
Cut away any clamp marks on the cord.
9. Briefly massage the clamped umbilical cord, using a gloved hand, to loosen up
the cells. Release the distal hemostat. Flush the collagenase solution from the
A.4B.2
Supplement 4
Split into
cord with the 30 ml HBS in a syringe (prepared at step 3), and collect the liquid
that is flushed out in a 50-ml conical polypropylene tube containing 10 ml M199
medium/20% FBS.
10. Sediment the cells by centrifuging 10 min at 250 g, room temperature. Decant
supernatant and resuspend pellet in 4 ml complete HUVEC culture medium/20%
FBS.
Culture HUVEC
11. Plate the 4 ml of medium with the cells (from step 10) in a 25-cm2 flask coated with
gelatin.
12. Incubate the flasks, feeding the cells twice a week, each time with a complete change
of culture medium, until they reach confluence (which usually takes 4 to 5 days).
Required volume
Vessel
medium
25-cm2 flask
4 ml
10 ml
75-cm flask
96well microtiter plate
100 lb
16-mm dish
800 l
35-mm dish
2 ml
60-mm dish
8 ml
30 ml
Commonly Used
Methods for Cell
Culture
A.4B.3
Current Protocols in Microbiology
Supplement 4
Vessel
Cells plated
Cell yield
96-well platea
0.52 104
25 104
24-well platea
46 104
13 105
6-well platea
0.72 105
36 105
25-cm2 flask
48 105
12 106
Figure A.4B.1 Culture of human umbilical vein endothelial cells 2 days after passaging.
Passage-3 cells at 70% conuence were xed with Diff-Quik Fix and stained with Diff-Quik
Solution II (Baxter). Cells were viewed using a Zeiss inverted microscope. Originally published in
Kleinman and Cid (1998).
17. Perform experiments, generally, with second to fourth-passage cells that have reached
confluence as determined by phase-contrast microscopy (UNIT 2A.1). If required, cryopreserve HUVEC as in the Support Protocol.
Refer to Tables A.4B.2 and A.4B.3 for more information.
Figure A.4B.1 demonstrates passage-3 cells. Results obtained with HUVEC beyond the
fifth passage can be less consistent
SUPPORT
PROTOCOL
Isolation and
Culture of
HUVECs
A.4B.4
Supplement 4
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Culture
A.4B.5
Current Protocols in Microbiology
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COMMENTARY
Background Information
Isolation and
Culture of
HUVECs
many bacteria. An important step in the pathogenesis is the translocation of circulating bacteria across the endothelium to tissues. This
interaction has attracted significant interest as
determinants of host defense and pathogenic
mechanisms.
The signaling pathways and homeostatic
mechanism affected by infection of vascular
endothelial cells have been studied with cultured vascular endothelial cells. Jaffe et al. first
isolated endothelial cells derived from human
umbilical veins in 1973 (Jaffe et al., 1973a).
The criterion that has been used to identify the
correct cell type is the expression of von Willebrand factor antigen (Jaffe et al., 1973a,b). Human umbilical vein is an ideal source because
it is easy to obtain, grows well in pure culture, and is available in abundant quantities.
A disadvantage to this procedure is the cost of
the medium (e.g., FBS, endothelial cell growth
factor) needed to propagate cells. Nonetheless,
the ease with which pure cultures of endothelial cells can be obtained with this method
has ushered in the rapid growth of studies in
microbe-endothelium interactions.
A.4B.6
Supplement 4
Anticipated Results
The harvest of endothelial cells from human umbilical vein has become a standard
and well accepted technique for acquisition
of HUVEC. Provided that sterile techniques
are strictly observed and the umbilical cord is
fresh (less than 3 hr old), success in acquisition
and propagation of HUVEC is expected. The
yield of HUVEC as described in Table A.4B.3
is highly achievable.
Time Considerations
It generally takes 4 to 5 days for the
HUVEC to propagate from the harvest of umbilical cord endothelial cells to confluence in a
T25 flask. The cells can then be passaged every
3 to 4 days upon confluence. Once confluence
from the 2nd to 4th passage is reached, the experiment can be performed. In some cases, the
experiments can also be performed with the
5th-passage cells. Cells beyond the 6th passage should be discarded.
Literature Cited
Commonly Used
Methods for Cell
Culture
A.4B.7
Current Protocols in Microbiology
Supplement 4
Oh, P., Li, Y., Yu, J., Durr, E., Krasinska, K.M.,
Carver, L.A., Testa, J.E., and Schnitzer, J.E.
2004. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for
tissue-specific therapy. Nature 429:629-635.
Peacock, S.J., Foster, T.J., Cameron, B.J., and
Berendt, A.R. 1999. Bacterial fibronectinbinding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus
aureus to resting human endothelial cells. Microbiology 145:3477-3486.
Prasadarao, N.V., Wass, C.A., and Kim, K.S. 1996.
Endothelial cell GlcNAc beta 1-4GlcNAc epitopes for outer membrane protein A enhance
traversal of Escherichia coli across the bloodbrain barrier. Infect. Immun. 64:154-160.
Isolation and
Culture of
HUVECs
A.4B.8
Supplement 4
APPENDIX 4C
ABSTRACT
In this appendix, several basic methods are described for preparation of primary B
C 2007 by John Wiley & Sons,
lymphocyte. Curr. Protoc. Microbiol. 6:A.4C.1-A.4C.9.
Inc.
Keywords: B lymphocyte r sheep blood cell r T-cell rosetting r CD19
INTRODUCTION
In the protocol for isolating total white blood cells, red blood cells are usually present
in the white blood cells. These red blood cells need to be removed as they can otherwise interfere with further experiments in that they can lyse, resulting in binding and
sequestering the viral particles nonspecifically hindering infection. This can be achieved
by incubating for 5 min with 1 ml NH4 Cl/Tris (0.16 M NH4 Cl, 0.17 M TrisCl, pH 7.65)
at 37 C. The buffer has been developed for the preferential lysis of red blood cells from
human whole blood. This process can be repeated two or three times until the red cells
are completely removed.
CAUTION: Follow all appropriate guidelines and regulations for the use and handling
of human-derived materials. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for
more information.
BASIC
PROTOCOL 1
Peripheral blood is the primary source of lymphoid cells for investigations of the human
immune system. This protocol is a simple and rapid method of purifying peripheral
blood mononuclear cells (PBMC) that takes advantage of the density differences between
mononuclear cells and other elements found in the blood sample. Mononuclear cells and
platelets collect on top of the Ficoll-Hypaque layer because they have a lower density;
in contrast, red blood cells (RBC) and granulocytes have a higher density than FicollHypaque and collect at the bottom of the Ficoll-Hypaque layer. Platelets are separated
from the mononuclear cells by subsequent washing or by centrifugation through a fetal
bovine serum (FBS) cushion gradient that allows penetration of mononuclear cells but
not platelets.
Materials
1000 U/ml heparin
Ficoll or Lymphoprep solution (Amersham Biosciences)
Phosphate-buffered saline (PBS; APPENDIX 2A)
RPMI-1640 supplemented with 2 mM glutamine and 5% FBS
Alcohol swabs
60-ml syringe
Tourniquet
18-G needle
Current Protocols in Microbiology A.4C.1-A.4C.9, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mca04cs06
C 2007 John Wiley & Sons, Inc.
Copyright
Commonly Used
Methods for Cell
Culture
A.4C.1
Supplement 6
6. Transfer the lymphocyte-containing band into a new centrifuge tube using a 10-ml
sterile pipet (Fig. A.4C.2).
The washing steps described above usually remove most of the platelets from mononuclear cell suspension. There are certain disease states associated with increased platelet
concentrations (mononuclear cell to platelet cell ratio >10:1) in the peripheral blood,
and additional steps are needed to remove the extra platelets in these cases. Add 3 ml FBS
to a centrifuge tube for each milliliter of mononuclear cells. Layer the cell suspension (1
to 2 107 cells/ml) over the FBS (alternatively, carefully layer the FBS under the cell
suspension, which will rise as FBS is added). Centrifuge 15 min at 800 rpm (200 g),
18 to 20 C. Discard the supernatant containing the platelets. Resuspend cell pellet in
complete RPMI-1640 containing 5% FBS and proceed.
Isolation of
Human PBMCs
Figure A.4C.1 Components including syringe, vacutainer, tourniquet, heparin, alcohol swab,
band aid, and gauze for bloodletting.
A.4C.2
Supplement 6
Figure A.4C.2 (A) Scheme showing blood separation by Ficoll gradient. (B) An actual separation
of blood after Ficoll gradient centrifuge.
7. Wash the total lymphocyte population with RPMI 1640 containing 5% FBS three
times. Between washes, to pellet down the cells, centrifuge 10 min at 1200 g,
room temperature.
8. Dilute the lymphocyte pellet in RPMI 1640 to 107 cells/ml and briefly spin the cells
5 min at 200 g, room temperature.
BASIC
PROTOCOL 2
Materials
Staining buffer (see recipe), ice-cold
Anti-CD19 antibody: Purified mouse antihuman CD19 antibody (BD Pharmingen)
Human Fc-receptor blocking antibody
Paramagnetic microbeads conjugated to appropriate secondary antibody
30-m nylon mesh
Commonly Used
Methods for Cell
Culture
A.4C.3
Current Protocols in Microbiology
Supplement 6
Figure A.4C.3
Separation tube
Powerful magnet
Fluorescent-activated cell sorter (FACS)
Additional reagents and equipment for isolating lymphocytes from whole blood
(see Basic Protocol 1) and counting cells using a hemacytometer (APPENDIX 4A).
Prepare lymphocytes
1. Isolate lymphocytes from whole blood (see Basic Protocol 1).
2. Wash cells once in ice-cold staining buffer.
3. Centrifuge 10 min at 300 g, 4 C to pellet.
4. Count cells in a hemacytometer (APPENDIX 4A) and allocate 107 cells into a microtube.
Centrifuge 10 min at 300 g, 4 C to pellet cells and resuspend cells in 100 l
staining buffer.
5. Filter cells through a 30-m nylon mesh to get rid of clumps that could result in an
impure population.
A.4C.4
Supplement 6
8. Wash once with staining buffer and resuspend in 100 l staining buffer containing
anti-CD19 antibody.
9. Mix well and incubate for 10 min on ice for.
10. Wash cells 2 to 3 times in ice-cold staining buffer (use at least 10 the labeling
volume).
11. Resuspend cells in 100 l staining buffer.
14. Wash cells once in ice-cold staining buffer (use at least 10 the labeling volume).
15. Centrifuge 10 min at 300 g, 4 C to pellet.
16. Resuspend cells in 1 ml staining buffer.
17. Place tube horizontally on a magnet (tube is placed horizontally to prevent gravity
contamination of the purified population).
Be sure to keep the separation tube attached to the magnet until step 21.
18. Allow cells attached to paramagnetic microbeads to separate out for 10 min.
19. Discard supernatant (or pellet and save for negative selection).
20. Carefully wash microbead/cell pellet with appropriate medium.
21. Remove magnet and resuspend in appropriate medium.
Purified cells can be used straight for culturing and/or other downstream application as
the magnetic particles are nontoxic and biodegradable. Alternatively, the cells can be
trypsinized to remove the attached microbeads.
BASIC
PROTOCOL 3
This protocol is simpler, and can be used if CD19 is the only antigen for positive selection.
Basic Protocol 2 is slightly more laborious, but it is more versatile. The same reagents,
with the exception of the primary antibody, can be used for positive selection of cells
expressing an array of different antigens. If working primarily with one antigen, however,
this protocol has the advantage of less noise due to one less antibody incubation.
Materials
Staining buffer (see recipe)
30-m nylon mesh
Microbeads mix (Miltenyi Biotech GmbH)
Magnetic separator (Miltenyi Biotech GmbH)
Magnetic separation column (Miltenyi Biotech GmbH)
Plunger (Miltenyi Biotech GmbH)
Fluorescence-activated cell sorter (FACS)
Additional reagents and equipment for isolating lymphocytes from whole blood
(see Basic Protocol 1)
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Culture
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Current Protocols in Microbiology
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Figure A.4C.4 Picture showing the assembled Miltenyi MACS positive-selection apparatus. The
column is vertically mounted in a strong magnet.
Prepare lymphocytes
1. Isolate lymphocytes from whole blood (Basic Protocol 1).
2. Wash cells once in ice-cold staining buffer.
3. Centrifuge 10 min at 300 g, 4 C to pellet.
4. Resuspend 107 -108 cells in 800 l staining buffer.
5. Filter cells through a 30-m nylon mesh to get rid of clumps that could result in an
impure population.
Isolation of
Human PBMCs
A.4C.6
Supplement 6
BASIC
PROTOCOL 4
Materials
Sheep red blood cells (SRBC; usually purchased from Biowhittaker)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Peripheral blood mononuclear cells (PBMC) in complete RPMI-10 medium (see
Basic Protocol 1)
RPMI-1640 supplemented with 2 mM glutamine and 5% FBS
0.14 M 2-aminoethylisothirouroniumuronium hydrobromide solution (AET; see
recipe)
Tris-buffered NH4 Cl, pH 7.5
Fetal bovine serum (FBS; APPENDIX 2A)
Centrifuge with a swinging-bucket rotor
37 C water bath
5-ml pipet, sterile
Additional reagents and solutions for counting cells (APPENDIX 4A)
NOTE: Prepare AET treated SRBC in a sterile manner.
NOTE: To prepare a large amount of AET treated SRBC, increase the number of tubes
in step one.
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Current Protocols in Microbiology
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19. Suspend the SRBC-T cell pellet. Centrifuge 10 min at 1000 g, room temperature.
20. Aspirate all of the supernatant. Break up the cell pellet by gently shaking.
AET
Dissolve 1.967 g 2-aminoethylisothirouroniumuronium hydrobromide (AET) in
35 ml sterile water. Adjust to pH 8.0 with 1.0 N NaOH. Bring volume to 50 ml with
sterile water. Filter through a 0.2-m filter system. Store up to 1 year at 2 to 8 C.
Check pH every 2 weeks.
Staining buffer
Isolation of
Human PBMCs
A.4C.8
Supplement 6
COMMENTARY
Background Information
Several methods for isolation of B lymphocytes from whole blood cells have been
developed. The T-cell rosetting is a classic
method which can separate T cells from B
cells and has been widely used. In addition,
a B cell magnetic isolation kit has also been
developed based on the B cell specific marker
CD19.
Anticipated Results
In general, 200 million B cells can be obtained from 500 ml of whole blood based on
the T-cell rosetting method. However, the yield
can vary using B cell isolation kits which depends on the manufacturer.
Time Considerations
Purification of B cells is a consuming
process. Firstly, isolation of total white blood
cells needs 3 to 4 hr since there is low-speed
and long-time spinning and a number of
washes. To further isolate B cells, first the
SRBC needs to be treated with AET followed
by a number of washes. This will take another
2 or 3 hr. The authors suggest that isolation
of total WBC and preparation of AET-treated
SRBC be initiated at the same time. In this
way, the total PBMCs and SRBC can be
ready for mixture almost at the same time.
In addition, the time for T-cell rosetting is
flexible and takes 1 hr to 12 hr based on the
schedule of the researcher. Ideally, it takes
24 hr to isolate B lymphocytes from whole
blood cell starting from blood letting.
Literature Cited
Fong, S., Tsoukas, C.D., Pasquali, J.L., Fox, R.I.,
Rose, J.E., Raiklen, D., Carson, D.A., and
Vaughan, J.H. 1981. Fractionation of human
lymphocyte subpopulations on immunoglobulin coated Petri dishes. J. Immunol. Methods
44:171-182.
Madsen, M. and Johnsen, H.E. 1979. A methodological study of E-rosette formation using
AET-treated sheep red blood cells. J. Immunol.
Methods 10:61-74.
Strelkauskas, A.J., Teodorescu, M., and Dray, S.
1975. Enumeration and isolation of human T
and B lymphocytes by rosette formation with
antibody-coated erythrocytes. Clin. Exp. Immunol. 22:62-71.
Commonly Used
Methods for Cell
Culture
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Current Protocols in Microbiology
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