Anda di halaman 1dari 128

Name: ____________________

Lab Section: __________

TA Name and Email: _____________________________

Lab Partners’ Name and Contact Info:

________________________________________

_______________________________________

_______________________________________

Cellular, Molecular and Microbial Biology 343

Microbiology

Laboratory Manual

2018

Department of Biological Sciences

The University of Calgary


W. Huddleston
F. Cusack

Copyright © 2018 The University of Calgary


ii
Table of Contents
Laboratory Schedule ......................................................................................................... v

Laboratory Policies ........................................................................................................... vi

Emergency and Safety Information ................................................................................ ix

Preface and Introduction to CMMB 343 Laboratories .................................................. xiii

Laboratory 1 - The Microbiology Laboratory ............................................................... 1


Laboratory 2 - Laboratory Techniques ........................................................................... 5
Laboratory 3 - Microbial Morphology and in vitro Culture .......................................... 9
Laboratory 4 - Effect of Environment on Bacterial Growth ........................................... 15
Laboratory 5 - Effect of Chemical Agents on Bacterial Growth ................................... 21
Class Presentations .......................................................................................................... 27
Lab Exam 1 ................................................................................................................ 29
Term Project - Identification of Bacterial Unknowns ...................................................... 31
Laboratory 6 - Microbial Metabolism ............................................................................ 35
Laboratory 7 - Transposon Mutagenesis........................................................................ 39
Laboratory 8 - Effect of Radiation on Viral Growth .................................................... 41
Lab Exam 2 ................................................................................................................ 45

Appendix 1- Laboratory Exercises .................................................................................... 49


Appendix 2 - Microscopy ............................................................................................... 51
Appendix 3 - Media and Methods Used for Cultivating and Isolating Bacteria ............. 53
Appendix 4 - Review of Microbiological Techniques ..................................................... 59
Appendix 5 - Bacterial Culture Media ............................................................................ 71
Appendix 6 - WHMIS ..................................................................................................... 77
Appendix 7 - Biohazard Safety Level Designations ....................................................... 79
Appendix 8 - Image Reference Sheet ........................................................................ 81
Appendix 9 - TA Evaluation Form .................................................................................. 83

iii
iv
LABORATORY SCHEDULE
B01/B03/B05/B07 - EEEL 369
B02/B04/B06/B08 - EEEL 303

WARNING: this course requires time in the laboratory outside of scheduled class time
(see page xiv)
Date Exercise Assignment

January 9/11 LABORATORY 1 Exercise 1 due January 16/18


Complete Laboratory Quiz 1 before your next scheduled lab

January 16/18 LABORATORY 2 Exercise 2 due January 19/22

January 23/25 LABORATORY 3 Exercise 3 due January 26/29


Complete Laboratory Quiz 2 before your next scheduled lab

Jan. 30/Feb. 1 LABORATORY 4 Exercise 4 due February 2/5


Complete Laboratory Quiz 3 before your next scheduled lab

February 6/8 LABORATORY 5 Ames Test Exercise due February 6/8


Exercise 5 due February 9/12

February 13/15 CLASS PRESENTATIONS

February 20/22 NO LABS (READING WEEK)

Feb 27/March 1 MIDTERM LAB EXAM Winogradsky Exercise due March 2/5
BACTERIAL UNKNOWN Unknown Exercise due March 2/5

March 6/8 LABORATORY 6 Exercise 6 due March 9/12


BACTERIAL UNKNOWN
Complete Laboratory Quiz 4 before your next scheduled lab
March 13/15 LABORATORY 7 Exercise 7 due March 20/22
BACTERIAL UNKNOWN
Complete Laboratory Quiz 5 before your next scheduled lab
March 20/22 LABORATORY 8 Exercise 8 due March 27/29
BACTERIAL UNKNOWN Journal due March 20/22

March 27/29 NO LABS

April 3/5 FINAL LAB EXAM

v
LABORATORY POLICIES

Composition of Laboratory Mark:


Component: Weight: Both individual and group work will be used
to evaluate your understanding of concepts
Submissions 10 % and techniques. Lab exam questions will be
short answer and will cover both practical and
Quizzes 5%
theoretical aspects of the laboratories.
Presentation 5%
* You must complete this course component
Unknown Journal 4%
in order to pass the course
Midterm Lab Exam* 8%
Final Lab Exam* 8% ** You must pass (>50%) the lab component
in order to pass the course
Total** 40 %

The Final Lab Exam will take place in the scheduled laboratory the week of April 2

Re-evaluation of Laboratory Work


The course policy on re-evaluation of student work is that students must first take the time to read
over their TA’s comments; for this reason, requests for re-evaluation cannot be made until the
day after graded work has been returned. If you would like a piece of term work re-evaluated,
prepare a written summary of your concerns related to the grading of your work using the Re-
Evaluation Request Form available on D2L, and make an appointment to meet with your TA.
This re-evaluation must be requested within 15 DAYS of the date when the graded work
was originally handed back or when your mark was posted. If you still have concerns, you
should submit the work to the Lab Coordinator for an independent reappraisal. See the University
of Calgary Calendar for more information on re-evaluation of term work (http://www.ucalgary.ca/
pubs/calendar/current/i-2.html).

Policy on Late Assignments


Assignments are due at 12 pm in the submission box (outside EEEL 369) on the day indicated on
the laboratory schedule (see page v) unless indicated otherwise. Late work will not be accepted
and will be graded as a zero, unless you provide the Lab Coordinator with documentation to
support a valid reason for handing in an assignment late. Valid reasons (and documentation)
include:
• personal illness (doctor’s note on the University of Calgary‘s official stationery available
at: http://www.ucalgary.ca/UofC/departments/UHS/PDFs/deferred_exam_form.pdf);
• family emergency (doctor’s note, death notice, etc., on official stationery);
• professional school interview (letter of invitation);
• team absences (coach’s letter)
Once marked assignments have been returned to students, late assignments will not be accepted
for any reason.

vi
Academic Misconduct
Common forms of academic misconduct are cheating and plagiarism. Cheating is receiving or
giving information on an assignment, exam or quiz. Plagiarism is the inclusion of someone else’s
ideas or information in a written or oral assignment without giving credit or acknowledging the
source. This is considered to be the theft of another’s ideas. Copying all or part of someone else’s
written assignment is one example of plagiarism. In addition, plagiarism includes word for word
copying of text from a textbook, a journal article or a document posted on the world wide web,
even if the source is referenced.
Cooperation in the carrying out of laboratory experiments is often necessary and both formal
and informal discussions on the interpretation of data is encouraged. HOWEVER, lab reports
and other assignments must be written in your own words (i.e. produced independently). There
are instances in which students are instructed to prepare joint reports. When this is specifically
requested, this is, of course, acceptable.
Penalties for academic misconduct range from reduced or failing grades on a course component
to expulsion from the University. See the University Calendar (http://www.ucalgary.ca/pubs/
calendar/current/k.html) for more information.

Attendance
Attendance is required at all laboratory classes. You may attend only the lab section in which you
are registered, due to space and equipment limitations. If you miss a lab, you must contact the Lab
Coordinator within 48 hours of the absence. The documentation must explicitly state the day(s)
missed, the reason for the absence, and when you are able to return to classes. Please note that lab
exercises run for one week at a time and are not repeated in subsequent weeks, so if you miss an
entire week of classes, you will not be able to make up that lab. You are still responsible for all
the material covered in that lab.
Assignments will not be accepted from a student who was absent from the lab in which the data
were collected, unless you have a valid excuse and supporting documentation.
If you miss a lab in which an assignment was due or an exam was written and have a valid excuse,
you must present the Lab Coordinator with supporting documentation. Students who miss a
substantial number of labs may not be permitted to write the lab final exam. Depending on
the circumstances, if you cannot make up the lab, you may either be given an excused absence for
the assignment, or other arrangements will be made.

Required Laboratory Components


Attendance is required at all laboratory classes. Students who miss a substantial number of labs
will not be permitted to take the lab exams.
You must take the lab exams in order to pass the course. Students who do not show competency
with the laboratory material will not receive a passing grade in the course.
You must pass (>50%) the laboratory component of the course in order to pass the course.

vii
viii
Emergency and Safety Information
Emergency Numbers
Campus Security 220-5333

Safety Office 220-6345


University Health Services 220-5765/8
Poison Control Center 403-670-1414

Emergency Procedures
Fire*
• Pull nearest fire alarm.
• Phone 220-5333 (24 hour Campus Security number)
• Give name, telephone number, location and nature of fire.
• Use fire extinguisher if small fire.
• If possible, students must turn off all sources of heat, gas or open flames, e.g. hot plates,
Bunsen burners.
• All personnel must leave the lab; ensure lab doors are closed.
• Exit the building via closest emergency exit.
• Meet fire fighters outside door closest to fire alarm panel and inform them of fire loca-
tion and hazards.
Serious Personal Injury*
• Do not move seriously injured persons unless there is a danger to their safety, taking
precautions for your own safety.
• Phone Campus Security: 220- 5333 (24 hours) (give name, telephone number, location
and type of injury). If possible have someone go to main entrance and flag down ambu-
lance.
Minor Personal Injury*
• Make injured person comfortable.
• Telephone Campus Security: 220-5333
• Phone University Health Services: 220-5765/5768
* Notify one of the laboratory coordinators as soon as possible after the incident.
Building Damage, Floods, Utility Failure*
• Inform the coordinator of the teaching laboratories and telephone the Safety Office at
220-6345 and describe the problem.
• After 4:30 phone Campus Security at 220-5333. Give your name, telephone number,
location and describe the problem.

ix
Guidelines for Safety Procedures in Teaching Laboratories in the Department of
Biological Sciences
Objectives:
Students in laboratories in the Biological Sciences should be aware that there are risks of personal in-
jury through accidents, e.g. fire or explosion, exposure to biohazardous materials, corrosive chemicals,
fumes, cuts. These guidelines are meant to minimize the risk of injury by emphasizing safety precau-
tions and to clarify emergency procedures in the event of an accident.

Emergency Equipment:
You should know the locations of the following equipment; your instructor will inform you of at the
first lab:
• Eyewash facilities and explanation of operation • Closest emergency exit
• Fire extinguisher and explanation of use. • Closest fire alarm and pull station.
• Safety showers and explanation of operation. • First aid kit.
• Closest emergency telephone and emergency phone #s.

General Safety Regulations:


1) Eating, drinking and smoking are prohibited in all laboratories. Cell phones are not allowed
in the laboratories.
2) Always wash your hands prior to leaving the laboratory.
3) Laboratory Coats and Safety Glasses (splash-proof glasses recommended) and appropriate
attire are required for all laboratories unless otherwise indicated by the coordinator of the
course.
4) Students are not allowed to work in a laboratory unless a lab instructor is present.
5) Before starting an experiment, establish a plan of action and list of required materials.
Compile all materials before starting. Confusion can cause accidents.
6) Report equipment problems to the lab instructor immediately.
7) Report all spills to the lab instructor immediately.
8) Long hair must be tied back to keep from being caught in equipment, Bunsen burners,
chemicals, etc.
9) It is recommended that contact lenses not be worn in laboratories. Use chemical splash gog-
gles over normal eyeglasses.
10) Enthusiasm is encouraged but pranks and horseplay can be hazardous.

x
Spills:
Minor spill of SOLUTION/CHEMICAL: Put on gloves and wipe up the spill using paper towels
and a sponge as indicated by the lab instructor.
Major spill of ACID/BASE/TOXIN: Immediately contact your lab instructor. DO NOT TOUCH!
Radioactive material:
Small volume/little activity: Contact your lab instructor. While wearing lab coats/gloves, clean up
the fluid and deposit all materials in the solid waste container.
Large volume/unsure of total activity: Contact your lab instructor and the Safety Office will be
called.
In EITHER SITUATION, contact the coordinator of the teaching laboratories and notify them of
the time, location and person(s) involved in the accident.
All spills of radioactive material on individuals or on lab coats shall be reported to the Safety Of-
fice as stated in the Radiation Policies and Procedure Manual.

Blood/Bacterial/Fungal/Virus spills:
If you spill a container of a biohazardous material, immediately tell your lab instructor and stu-
dents around the spill area. If necessary, remove any contaminated protective and personal cloth-
ing. Prevent anyone from going near the spill. Wash your hands and face. The lab instructor and
laboratory coordinator are required to supervise the clean up at this point.

Health concerns:
1) Allergies:
Students with allergies (chemical, plant, animal or antibiotic) should inform the laboratory coor-
dinator if the allergy may be relevant to any laboratory exercises in the course. The student should
then receive a note from his/her doctor to indicate if the student should be allowed to carry out the
experiment(s) using appropriate precautions arranged through the Safety Office or if the student
should not be present during the experiment.

2) Pregnancy
Some chemicals used in laboratory teaching are teratogens, which cause fetal deformities. It is
important that students who are pregnant inform the laboratory coordinator so that the appropriate
precautions are taken in consultation with the Safety Office.

3) Other health concerns:


a) Animal Bites must be reported to the lab instructor immediately, the student will then
be taken to Health Services for a tetanus shot.
b) The student should make the laboratory coordinator aware of any serious health condi-
tions which may be exacerbated by work in the laboratory.

xi
Disposal of Wastes
Most waste produced by laboratories cannot be disposed of in the sink or regular garbage cans.
Please pay close attention to the following procedures.

Radioactive liquid: All radioactive LIQUID must be poured into a radioactive liquid waste con-
tainer from the Safety Office. Do not pour anything down the designated sink without first check-
ing with your lab instructor or the laboratory coordinator. All SOLID radioactive waste such as
pipette tips, gloves, paper towels, microfuge tubes, etc., is placed in the solid radioactive waste
container (Safety Office).

Yellow Container Blue Container

All sharps: needles, scalpels, syringes, razor Clean Lab Glassware: broken glass, Pasteur pipettes,
blades and other sharp items. Broken glassware broken pipettes Clean Plastic Labware
and materials such as pipette tips, microtitre
plates, gloves, paper towels CONTAMINATED no chemical, biological, or
with BLOOD, BODY FLUIDS or other BIO-
HAZARDOUS MATERIAL. radioactive materials

no chemical or radioactive
materials

Petri plates: Place in clear plastic biohazard bags for autoclaving.


Bacterial or fungal liquid: Tubes and flasks containing liquid cultures are placed in marked trays
or on carts for autoclaving.
Viruses: Pour liquid and place materials into a bleach solution. (This is followed by exposure to
UV light and autoclaving before disposal).
Animal Tissues/Carcasses: Place in plastic bags for disposal through the Animal Resources
Center (Main Campus).
Liquid Chemicals/Fixatives: Place in 20 L containers provided by the Safety Office. Other la-
belled screw-cap bottles may be provided to keep certain chemicals separate.
Solid Chemicals: Place in containers for disposal by Safety Office.
In case of any uncertainty in waste disposal please contact the lab instructor

xii
Preface

Welcome to CMMB 343!

This course is intended for students wishing to major in the Cellular, Molecular and Microbial
Biology Program in the Department of Biological Sciences. The study of microorganisms is
a dynamic and fast-paced field, with advances and discoveries occurring almost every day.
This course focuses on the bacteria, but consideration will be made for the archaea, eukaryotic
microorganisms and viruses.

Microbiology is a relevant science that impacts our daily lives in numerous ways. By performing
the exercises in this manual you will develop an appreciation of the variability of microbes that
are involved in areas such as microbial genetics, ecology, medicine and biotechnology.

It is hoped that the scope of the experiments in this laboratory manual challenge, interest and
inspire you to further your studies in this interesting field.

INTRODUCTION TO THE CMMB 343 LABORATORY
A. Objectives of the Laboratory

The main purpose of laboratory manipulations is to put into practice concepts you are learning
in your lectures, textbooks and the scientific literature. It is in the laboratory where the tools and
techniques necessary to develop new findings and ultimately new concepts are incorporated.
Science, by nature, is a product of laboratory effort. Therefore one can say that real science is
found in the laboratory.

The overall objectives of the CMMB 343 laboratories are to:


1. reinforce aseptic technique and basic microbiological methodology
2. provide practical information on heterotrophic, chemotrophic and phototrophic
microorganisms
3. provide information that demonstrates the importance of microorganisms in the environment,
in the food industry and in medicine

A primary feature of the microbiology laboratory is that living organisms are employed as part
of the experiment. Most of the microorganisms are harmless. However, whether they are non-
pathogenic or pathogenic, the microorganisms are treated with the same respect to ensure that
personal safety in the laboratory is maintained. Careful attention to technique is essential at all
times.

Your attitude to laboratory study is an important factor in determining how well the session will
go. If you are interested in learning and willing to put in both time and effort, it will be a most
pleasant and rewarding experience.

xiii
B. Open Laboratory
Due to the nature of microbial growth, it is necessary to incubate the cultures prepared in the labs
for 24 - 48 hours at the appropriate temperature to allow time for the microorganisms to grow.
As a result, it will be necessary for you to return to the lab the day following the scheduled
lab to observe your results.
The lab submissions following each exercise are scheduled to be handed in with enough time to
properly observe the results. References will be available in the open labs and on reserve in the
library during the week. Use these resources to help you answer the submission questions and
interpret your results.
OPEN LAB SCHEDULE
Monday: 0900-1200
Wednesday: 0900-1500
Friday: 0900-1500
If your class schedule conflicts with the open lab schedule, contact Mr. Huddleston
(wrhuddle@ucalgary.ca) to make alternate arrangements.

Laboratory Dress Code


You can’t put a covering over the entire surface of the Earth, so instead, wear sandals
-Buddhist wisdom

Throughout this term, you will be working directly with microorganisms. Although the
organisms used in this course have been approved for use in an undergraduate laboratory,
care must be taken to ensure safe handling practices. We will therefore treat all organisms
as if they were classified as Biosafety Level 2 (BSL2 - see Appendix 7).

We will be maintaining a strict regulation of dress code in the lab that is appropriate for
working with BSL2 organisms. In addition to using aseptic technique when working in the
laboratory, your dress must meet the following guidelines at all times:

1. No open-toed footwear (e.g. sandals, etc.)


2. A laboratory jacket must be worn properly at all times
3. No exposed skin is allowed that is not covered by the laboratory jacket (e.g.
no shorts, skirts, etc.)
4. Long hair must be tied back and tucked into the laboratory jacket

Failure to meet these criteria will result in denied admittance


to both the scheduled and open laboratory

xiv
Laboratory 1
Objectives:
1. To become familiar with the microbiology laboratory environment
Recommended Laboratory Resources:
Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology
of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 2; 18
Lab Manual Appendices 1, 4, and 7

A. Ubiquity of Microorganisms
Unless special precautions are taken to exclude them from an environment, microorganisms are always present
(ubiquitous) in the air, on surfaces, and on your skin, clothing, hair, etc. Microbiologists must be acutely
aware of the ubiquity of microorganisms because their presence is a potential source of contamination.
Aseptic (sterile) technique is essential in order to protect you (and your colleagues!) from the organisms with
which you are working and to prevent contamination of the cultures you are studying.

Importance of Aseptic Technique


To demonstrate the importance of thorough hand-washing and workspace sterilization, you will inoculate
nutrient agar plates before and after the use of soap and disinfectant. Your TA will demonstrate how to handle
agar plates and how to work aseptically in order to minimize the risk of contamination.

Procedure: (work individually)

1. Hand-washing
1. Obtain 1 nutrient agar (NA) plate from the side bench.
2. Ensure that you keep the lid on the plate until you are ready for inoculation.
3. Without washing your hands, remove the lid and gently roll each finger and thumb from one
hand across the surface of the agar of the plate - press gently so you do not gouge the agar. Replace
the lid as soon as you are finished.
4. Label the plate properly (page 63) as demonstrated by your TA.
5. For now, place the plate on your bench where it will not be disturbed.

2. Environmental survey
1. Obtain 1 nutrient agar (NA) plate from the side bench. Use a marker to “divide” the plate in half as
demonstrated by the TA.
2. Swab a surface (e.g. floor, bench, shoe, etc.) of your choice with a moist applicator and gently wipe
it across the surface of the agar on one side of the plate. Ensure that the lid is only removed during
the inoculation of the plate.
3. Label the plate properly (page 63) and place it with your hand-washing plate.

1
Aseptic Technique and Laboratory Standard Operating Procedures
Read these guidelines carefully. They must be strictly adhered to so they become routine.
Remember, the microbiology laboratory is considered a BIOHAZARDOUS area.

1. Thoroughly read each day’s experiment(s) before coming to the laboratory. When you are prepared, the
lab runs more efficiently and accurately. As Pasteur stated, “chance favours the prepared mind”.
2. Laboratory coats must always be worn properly. Do not come to the laboratory with long loose hair,
loose clothing or open-toed footwear. Avoid the use of hair spray before coming to the lab.
3. Do not bring anything that is not required (coats, bags, cell phones, etc.) into the laboratory. Smoking,
eating, or drinking are not permitted. Do not place anything (pencils, fingers, etc.) in your mouth.
4. Place your lab manual to the side of your work area. Work over the lab bench - not over your book.
5. Clean the bench top properly with disinfectant before and after your laboratory work.
6. Wash your hands properly with soap and water before and after your laboratory work.
7. Know the location of the fire extinguisher, eye wash, shower and first-aid kit.
8. Lab benches must be completely cleared and reset after each laboratory session. E.g. microscopes are
cleaned and stored properly, inoculating loops are placed in their correct holders, etc.
9. It will often be necessary to incubate cultures overnight. Cultures should always be labelled properly
(Ap4-5). Instructions will be provided regarding where to place each culture and for how long each
culture needs incubation. Any plates left on the student bench will be discarded.
10. Any spillage of culture must be cleaned up immediately by covering with paper towel and pouring
disinfectant onto the covered spill. Wait 5 minutes before wiping. Immediately report any spill.
11. Always use the appropriate equipment properly when handling chemicals, reagents or bacterial
cultures. Instructions will be given for each exercise as to which piece of equipment to use and how to
use it.
12. Used micropipette tips must be ejected directly into the red biohazard disposal bag located on the
student bench. Used tips are NEVER to be placed on the bench.
13. Used glass pipettes must be placed immediately in the pipette bucket located on the side bench. Used
pipettes are NEVER to be placed on the bench.
14. Agar plates to be discarded are placed in the autoclave bucket at the front of the lab. Culture tubes to
be discarded are placed in the test tube rack at the front of the lab. Microscope slides to be discarded
are placed in the used slide container at the front of the lab.
15. All broken, but sterile glassware and plastic is placed in the blue bucket at the front of the lab.
16. All broken, contaminated glassware and plastic is placed in the yellow bucket at the front of the lab.
17. Never remove cultures or any other materials or equipment from the laboratory.
IF YOU ARE UNSURE OF ANY PROCEDURES, USE OF EQUIPMENT, OR TECHNIQUES, ASK
THE LAB INSTRUCTOR FOR HELP

2
LEVEL II LABORATORY:
Assume that you are always working with BSL II microorganisms in the laboratory (page 79). Although all
organisms have been approved for use in the laboratory, proper microbiology techniques should be used in all
experiments.
Now that your TA has discussed lab safety and aseptic technique, you will follow these guidelines and
inoculate a second set of hand-washing and environmental survey plates.

Procedure: (work individually)

1. Hand-washing
1. Obtain 1 nutrient agar (NA) plate from the side bench.
2. Wash your hands and then use aseptic technique to inoculate the plate by gently rolling each finger
and thumb from one hand across the surface of the agar of the plate - press gently so you do not
gouge the agar. Use the same hand you used to inoculate the first agar plate.
3. Label the plate properly, stack both hand-washing plates together and place them in the correct box
on the side bench. They will be incubated and returned to you next week.

2. Environmental survey
1. Use the environmental survey nutrient agar (NA) plate you inoculated earlier. Remember, you
divided the plate in half.
2. Use the supplied detergent to clean the surface you sampled earlier.
4. Use a new moist applicator to swab the surface again to inoculate the second half of the agar plate.
5. Place it in the correct box on the side bench. It will be incubated and returned to you next week.

B. Lab Quiz 1
You must complete Lab Quiz 1 (posted on D2L) before your next scheduled lab.

Checklist for Laboratory 1:


q Each student should inoculate two hand-washing plates and one environmental survey plate
q Each student should complete Lab Quiz 1 before their next scheduled lab

3
4
EXERCISE 1 SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

SCORE: __________

A. Ubiquity of Microorganisms
1. Note that part of the grade for this submission is from the assessment of the three plates you submit. Ensure
you have labelled and stacked them properly as demonstrated by your TA. (4)

2. Record your observations of the two hand washing plates and the environmental survey plate. Note the
relative abundance and diversity of organisms on the plates. (4)
Hand-washing plates
Before soap After soap

Environmental survey plate


Before detergent After detergent
Laboratory 2
Objectives:

1. To gain competence with the use of common laboratory techniques used in


microbiology
2. To describe the colony and cellular morphology characteristics of microorganisms

Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology
of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 11-14; 35-36; 42-
45; 74-75; 144-150.

Lab Manual Appendices 3 and 4

A. Ubiquity of Microorganisms

Importance of aseptic technique

Procedure: (work individually)


1. Collect the two hand washing plates and one environmental survey plate you prepared in Lab 1.
2. Compare the abundance and diversity of bacteria on each plate. Record your observations on the
Exercise 1 submission sheet.
3. Discard the plates properly in the autoclave bucket at the front of the lab.
4. Submit the Exercise 1 submission sheet before you leave the lab.

B. Nutrition and Culture of Microorganisms


In order for microorganisms to grow and divide, they must be able to make the complex macromolecules
needed for life. Organisms make these macromolecules by obtaining nutrients from the environment,
transforming them into the precursor molecules they need, and than building the specific polymers they
require. Microbial nutrition involves nutrient acquisition, enzymatic transformation and polymerization of
these complex macromolecules.
Due to the diversity of microbial life, the amounts and types of nutrients required for life can vary greatly
depending on the species. However, all organisms must acquire a source of macronutrients (nutrients required
in large amounts) such as: oxygen (H2O, O2, etc.), carbon (CO2, glucose, etc.), nitrogen (NH3, NO3-, etc.), and
minerals (P, S, K, Mg). Organisms also require micronutrients (nutrients required in trace amounts) such as:
metal ions (Fe, Cu, Ni, etc.) and organic growth factors such as vitamins (thiamine, biotin, etc.).

5
Culture Media for Bacteria
To study microorganisms in the laboratory, we must be able to culture them. Microbiologists refer to the
nutrient solutions used to culture organisms as media (singular: medium).
Chemically defined media consist of a basal (basic) salt solution, which supplies the inorganic requirements
of the organism, and organic components that provide a source of carbon and energy. The exact chemical
composition of each component is known.
Complex media contain substances that are rich in both inorganic and organic nutrients such as meat or
vegetable infusions, blood and hormones. The exact chemical composition of the media is not known.
Enriched media are loaded with nutrients and macromolecular monomers to promote the growth of even the
most fastidious organisms. Minimal media contain only the most basic nutrients.
Microorganisms are ubiquitous, therefore culture media must be sterilized in a giant pressure cooker called
an autoclave. The media is prepared and autoclaved at 15 pounds pressure (psi) at a temperature of 121°C.
After autoclaving, the media is cooled (~50°C) and poured into sterile petri dishes or test tubes using aseptic
technique.
Media may be prepared as a liquid (broth), or have agar added to provide a semisolid support for growth.
Agar liquefies at high temperatures (100°C) and chemically defined or complex media can be added. As media
containing agar continues to cool, it solidifies (42°C) forming a firm transparent gel.
Broth or agar media can then be inoculated with a sample and incubated under the desired environmental
conditions. On agar media, microbial cells are immobilized, allowing them to grow in aggregates of cells
that eventually form visible colonies. In broth, microbial cells grow in solution in a planktonic (free-living),
unattached phase.

Colony Morphology of Bacteria


Robert Koch was the first to notice that different species of bacteria could be distinguished from one another
when grown on agar media. Today we know that individual microbial cells placed on agar media grow and
divide, eventually forming a mass of cells visible with the naked eye. These masses of cells (colonies) have
physical characteristics that differ depending on the species of bacteria growing on the agar. We refer to these
characteristics as the organism’s colony morphology (page 62). For many years, colony morphology was a
major distinguishing feature used by microbiologists to isolate and describe species of bacteria and is still used
extensively today. When only one colony morphology (and therefore only one microbial species) is present on
an agar plate, we say that we have a pure culture.

Procedure: (work individually)


1. Observations of microbial cultures (may be completed in the open lab)
1. Observe the sample agar plates on the side bench that demonstrate various colony morphologies. These
can be used to help you recognize the morphologies on your plates.
2. Observe the streak and spread plates on the side bench that demonstrate commonly observed errors.
These can help you identify how to improve your technique.

6
2. Streak plate method to obtain single colonies (page 62):
1. Obtain one stock plate, one NA plate and one minimal glucose (MG) plate from the side bench.
2. Observe the TA demonstration for preparing a streak plate.
3. Prepare a complex media streak plate on the NA plate.
4. Prepare a chemically-defined media streak plate on the MG plate.
5. Label both plates properly and place them in the correct box on the side bench. Return the stock plate
to the side bench.
6. In the open lab, collect your plates from the box on the side bench, note the pattern of growth, confirm
the presence of single colonies and record the colony morphology for each plate on the Exercise 2
submission sheet.
7. Submit your best streak plate to the correct box on the side bench and discard the other plate properly.

3. Spread plate method to enumerate bacteria (pages 64-66)


1. Obtain one stock broth culture tube and three NA plates from the side bench.
2. Compare your broth culture tube with the turbidity standards on the side bench and record your
estimate of the cellular concentration on the Exercise 2 submission sheet.
3. Observe the TA demonstration for preparing serial dilutions and a spread plate.
4. Prepare seven serial 10-fold dilutions (10-1 to 10-7) of the stock broth culture.
5. Prepare a spread plate for the 10-5, 10-6, and 10-7 dilutions on separate NA plates.
6. Label the plates properly and place in the correct box on the side bench. Return the stock broth culture
tube to the side bench.
7. In the open lab, collect your plates, determine which one has between 30 and 300 colonies (page 66),
calculate the concentration of the original stock broth culture and record your answer on the Exercise 2
submission sheet.
8. Discard the plates properly.

7
C. Microscopy and Staining
Your TA will review the parts, function and proper handling of the microscope (Appendix 2). Make sure you
have working knowledge of the microscope before you proceed. Gloves will be available to reduce the risk of
staining your hands.

Cellular Morphology of Bacteria


Under a light microscope, most bacterial cells do not provide enough contrast to be detected without the aid
of stains. Once stained, microbial cells can only be visualized under the oil-immersion lens at the very limits
of a light microscope’s resolving ability. We refer to the characteristics observed under a microscope as the
organism’s cellular morphology. Many techniques have been developed that stain specific structures such
that we can observe a variety of physiological characteristics of bacteria. Differential stains can be used to
highlight differences between types of bacteria because they do not stain all cells the same. The major features
recorded when describing cellular morphology are listed on page 60.

Procedure: (work individually)


1. Gram stain preparation and observation (pages 59-60):
1. Obtain a stock plate from the side bench
2. Your TA will demonstrate how to prepare a bacterial smear and how to carry out the Gram stain.
3. Focus on a patch of cells under the 10x objective and then follow the procedure for using the oil
immersion lens (page 52).
4. Use the space on the Exercise 2 submission sheet to record the cellular morphology and prepare a
diagram of a representative microbial cell.
5. Look at your colleague’s slides: some of the samples are Gram-positive and some are Gram-negative.
You should be able to identify both of these Gram reaction morphologies.
6. Discard the slide properly in the slide bucket at the front of the lab. Return the stock plate to the side
bench.
2. Maintaining the microscope:
1. Clean the lenses and put the microscope away properly as demonstrated by your TA.

D. Class Presentation
You (in a group of students) will give a 20 minute class presentation in the lab the week of February 12. Your
TA will assign a topic before you leave the lab this week. More information can be found on page 27 and will
also be provided by your TA in the lab.

Checklist for Laboratory 2:


q Each student complete and submit the Exercise 1 submission sheet
q Each student should prepare a NA and MG streak plate
q Each student should prepare 3 NA spread plates
q Each student should prepare a Gram stain and diagram
q Each student should record their results in the open lab and discard their plates properly
q Each student should submit the Exercise 2 submission before noon on January 19/22
q Each student group should start preparing for the class presentation

8
EXERCISE 2 SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

SCORE: __________
Nutrition and Culture of Microorganisms
1. Note that part of the grade for this submission is from the assessment of the streak plate you submit. (2)

2. Record the colony morphology of the organism from the NA plate and the MG plate. (2)
NA MG

3. Using the turbidity standards, what do you estimate the cellular concentration of your unknown broth
culture tube to be? (1)

4. What is the cellular concentration of your unknown broth culture tube based on the spread plate method of
enumeration (page 66)? Show your calculation and use proper scientific notation. (3)
5. Use the space below to prepare a diagram of a representative microbial cell from your Gram stain (page 61).
Ensure your caption indicates the cellular morphology of the organism. (5)
Laboratory 3
Objectives:
1. To identify an organism by growth and morphology on selective and differential media
2. To use staining techniques to describe the cellular morphology characteristics of microorganisms
3. To identify an organism based on differential staining of morphological characteristics
4. To prepare a Winogradsky column for weekly observations of metabolic diversity and ecosystem
stratification

Recommended Laboratory Resources:


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock:
Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 24;
48; 53; 56-57; 413-315; 585-588.
Lab Manual Appendices 2, 3 and 4
A. Use of selective and differential media
Compounds can be added to defined or complex media that help select for or differentiate between species
of bacteria in a mixed culture. Selective media contain compounds that selectively enrich and/or selectively
repress the growth of certain organisms while not affecting the growth of others. In selective enrichment, the
nutritional or environmental conditions are controlled to favor the growth of a sought-after species. Selective
repression involves inhibiting the growth of interfering species while permitting the growth of a sought-after
species.
Some selective media are also differential. Differential media contain an indicator that differentiates the
occurrence of specific chemical reactions such that groups of bacteria can be differentiated from each other
based on their ability to carry out that reaction (page 57).
Procedure: (work individually)
You will be given a pure broth culture containing any one unknown bacterial species: A, B, C or D.

Record your unknown code on the Exercise 3 Submission sheet


1. Prepare a streak plate on Brain-Heart Infusion (BHI), Phenylethyl Alcohol (PEA), Desoxycholate
(DOC), and Mannitol Salts (MSA) agar plates.
2. Label each plate properly and place them in the correct box on the side bench.
3. Your TA will demonstrate how to use the DifcoTM & BBLTM Manual. It contains detailed information
about the media used in many microbiological applications. Using the DifcoTM & BBLTM Manual and
Appendix 3, list, in point form, on the Exercise 3 submission sheet, the ingredient(s) in each medium
which make that medium either selective, differential or both. For the selective ingredients, indicate
which organisms are either inhibited or enhanced and explain why. For the differential ingredients,
explain how they are differential. Your TA will work through an example with you. A link to the
DifcoTM & BBLTM Manual is posted on D2L.
4. In the open lab, note the presence of growth and record the colony morphology on the Exercise 3
submission sheet. Discard the plates properly.

9
B. Microscopy and Differential Staining
Last week you reviewed the parts, function and proper handling of the microscope (Appendix 2). Make sure
you have working knowledge of the microscope before you proceed. Your TA will demonstrate how to prepare
the acid-fast, capsule, endospore and flagella stains (pages 59-60).

Procedure: (work in a group of 4)


You will be given a pure agar streak plate containing any one unknown bacterial species: a, b, g, d.

Record your unknown code on the Exercise 3 Submission sheet


1. Look at the demonstration Gram, acid-fast, capsule, endospore, and flagella stains of the positive
controls located on the side bench. Use these as a guide to help you interpret the results of your stains.
Ensure you can explain how each stain works and how to interpret the results for each.
2. Prepare a Gram, acid-fast, capsule and endospore stain (i.e. one stain per student) of your unknown
organism and record the results on the Exercise 3 submission sheet.
3. Use the chart (page 12) to identify your unknown.
4. Discard all slides properly and return the unknown agar plate to the side bench.

C. Winogradsky Column
In the 1880s, Sergei Winogradsky devised a procedure to study soil microorganisms. The Winogradsky
column models an anaerobic ecosystem that can be used for the isolation of bacteria and archaea and is
an effective means to study some of the most numerous organisms on Earth. These microorganisms show
extreme metabolic diversity, including oxygenic and anoxygenic photoautotrophy, chemolithoautotrophy, and
photoheterotrophy. They are also descendants of the first organisms on the planet, with a history that goes back
4 billion years.
In this exercise, you will use an environmental slurry (a mixture of soil and pond water from Calgary) to
observe and study some of the different types of metabolism illustrated by this diverse group of microbes.
Each student bench will prepare four Winogradsky columns: a control column (A), a column containing
added carbon (B), a column that contains added sulfur (C), and a column that contains both added carbon and
sulfur (D). The columns will be incubated for several weeks and you will make a series of observations as the
microorganisms develop within the column.
Procedure: (Please work as a lab bench)
Each lab bench will prepare four Winogradsky columns, one of each treatment:
A: control B: +carbon C: +sulfur D: +carbon/+sulfur
Work with a partner to prepare one of the above Winogradsky columns
1. Label a Winogradsky column tube with your initials, lab section, and the treatment (A, B, C or D)
Mark the tube 10 cm and 13 cm up from the base of the tube as demonstrated by your TA
2. Fill your column with the appropriate soil (A, B, C or D) up to the 10 cm line
3. Gently tap the tube to remove as much air from the slurry as possible without compacting the soil
4. Add water to the tube until it has been filled to the 13 cm line

10
5. Place the lid on the tube and turn until it is halfway closed. Do NOT tighten!
6. Use the space below to record your initial observations of all four columns. This should include the
colour of each layer, changes to the sediment, development of layers within the sediment and water,
changes in the thickness of sediment layers, and differences among the columns and anything else that
becomes evident (don’t forget that smell is a great sense!)
You may take a photograph of your columns as long as you maintain aseptic technique.
7. Place your column in the appropriate rack on the light cart
8. Clean up any mess that you made while assembling the column. Your entire bench will be penalized if
your workspace is not cleaned up properly.
Winogradsky Column Observations (each pair should observe all four columns)

You will make weekly observations of all four columns throughout this 5 week exercise, so you will need
to prepare a table to organize your work. You may use the space on page 13 for your table (or using your
own resources). Make sure you include your Week 0 observations! You should also start thinking about the
questions on page 14 as the Winogradsky columns develop over the course of the exercise.
Suggested Resources (links provided on D2L)
1. Microbial Life Educational Resources-Winogradsky Column.
http://serc.carleton.edu/microbelife/topics/special_collections/winogradsky.html
2. The Winogradsky Column.
http://www.sumanasinc.com/webcontent/animations/content/winogradsky.html
Note: This exercise has been adapted from a Howard Hughes Medical Institute Laboratory exercise with
many thanks.

11
Table 3.1: Table of Staining Results for Known Organisms

Gram Cell Cell Acid-Fast Capsule Spore Flagella


Organism
Stain Morphology Arrangement Stain Stain Stain Stain

Bacillus coli - rod single cells - + + +

Actinobacterium
+ rod single cells - + + +
aureus

single cells,
Lactobacter
- rod pairs or short - + - -
caseus
chains

Seratia
+ rod pairs or chains - + - -
pneumoniae

Micrococcus
+ coccus pairs or tetrads + - - -
citrus

single cells
Pseudomonas
weak + rod or in dense + - - -
catarrhalis
clusters

Mycobacterium
- coccus pairs - + - -
shmegmatis

single cells,
Escherichia
+ coccus pairs, tetrads or - + - -
boydii
clusters

D. Lab Quiz 2
You must complete Lab Quiz 2 (posted on D2L) before your next scheduled lab.

Checklist for Laboratory 3:


q Each student should prepare 3 selective/differential streak plates of their unknown (A, B, C, or D)
q Each student group should complete the differential stains (4) on their unknown (a, b, g, or d)
q Each student pair should prepare a Winogradsky column
q Each student should record their results in the open lab and discard their plates properly
q Each student should submit the Exercise 3 submission before noon on January 26/29
q Each student should complete Lab Quiz 2 before their next scheduled lab

12
Winogradsky Column Observations

13
Winogradsky Column Questions
1. How do your columns differ? How are they the same? Explain the differences.

2. Did you observe changes in the control column? If so, explain why they occurred.

3. Winogradsky columns form oxygen concentration gradients. Predict the distribution of oxygen
throughout the column (sediment, water, air).

4. Winogradsky columns form sulfide concentration gradients as well. In the columns that contain egg
yolk, predict how sulfide will be distributed throughout the column (sediment, water, air).

5. Desulfovibrio are an example of bacteria that grow in Winogradsky columns. What type of metabolism
do they carry out? What substrates are required? What products are formed? Where in the column would
you expect to find them?

6. Chromatiaceae, Rhodospirillaceae, and Chlorobacteriaceae are examples of bacteria that grow in


Winogradsky columns. What type of metabolism do they carry out? What substrates are required? What
products are formed? Where in the column would you expect to find each of them?

7. Cyanobacteria and algae are examples of organisms that grow in Winogradsky columns. What type of
metabolism do they carry out? What substrates are required? What products are formed? Where in the
column would you expect to find each of them?

8. Explain how Winogradsky columns illustrate the diversity of microorganisms found on Earth today in
terms of the diversity of niches they occupy.

9. Explain what the Winogradsky columns illustrate about life on early Earth.

14
EXERCISE 3 SUBMISSION NAME: _____________________________

SCORE: __________ LAB SECTION: ______________________

A. Use of Selective and Differential Media


SELECTIVE/DIFFERENTIAL PLATES UNKNOWN CODE: _________
1. For the following media, use the DifcoTM & BBLTM Manual to: (8)
a. Indicate the selective and/or differential components and explain how each component is selective and/
or differential
b. Indicate which component provides a source of macronutrients
Phenylethyl Alcohol (PEA) agar
Selective:

Differential:

Macronutrients:

Desoxycholate (DOC) agar


Selective:

Differential:

Macronutrients:

Mannitol Salts (MSA) agar


Selective:

Differential:

Macronutrients:
2. Record the colony morphology (if available) of your unknown organism on each of the 4 plates. (4)
Brain-Heart Infusion (BHI) agar

Phenylethyl Alcohol (DOC) agar

Desoxycholate (DOC) agar

Mannitol Salts (MSA) agar


3a. Identification of unknown organism: ________________________________________________ (1)

3b. Provide a rationale for your identification. (3)

B. Microscopy and Differential Staining


Differential Staining Unknown Code: __________
4a. Record the results of the Gram, acid-fast, capsule, and endospore stains for your group’s unknown. (4)

4b. Identification of unknown organism: _______________________________________________ (1)

5. What is one advantage, to your unknown organism, of possessing the morphological property above? (1)
Laboratory 4
Objectives:

1. To become familiar with the effects of various environmental parameters on the growth
of microorganisms
2. To observe the effect of heat on bacterial cell viability in spore-forming and non-spore-
forming organisms
3. To screen potentially carcinogenic chemicals using the Ames test

Recommended Laboratory Resources:


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock:
Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp.
53-56; 152-159; 310-313.

A. Factors Affecting the Growth of Bacteria


We have considered the nutrient requirements of microorganisms grown in culture and the ability to
selectively culture organisms based on the nutrient composition of media. However, microbial growth is also
influenced by the physical and chemical characteristics of the environment. Understanding the environmental
requirements of an organism helps us understand the diversity of life on Earth and allows us to study a greater
range of organisms in the laboratory.

1. The Effect of Temperature


Temperature has one of the greatest influences on the growth of microorganisms since all the processes of
growth are dependent on chemical reactions that are affected by temperature. Growth occurs at temperatures
that range between a minimum, the temperature below which growth cannot occur, an optimum at which
they grow most rapidly, and a maximum above which the organism cannot grow. We refer to these three
temperatures as the cardinal temperatures.
The cardinal temperatures are unique to each organism and allow us to classify microbes into four groups
defined by the range of temperatures between their minimum and maximum growth requirements.

Research Applications
A clear understanding of the cardinal temperatures of an organism is essential for expression of recombinant
proteins. The temperature a bacterial culture is grown at can have a significant impact on the expression
of soluble recombinant protein. When a recombinant protein is expressed at high levels in E. coli, for
example, the protein may end up in an insoluble protein fraction, called inclusion bodies. These bodies
contain misfolded, inactive proteins that are targeted for degradation by intracellular mechanisms. Thus, if
the recombinant protein ends up in inclusion bodies, the protein is difficult to purify and is often unusable
for further experiments. The optimum temperature for the expression of each recombinant protein must be
determined experimentally and must also fit within the temperature range at which the host organism will
grow.
15
Procedure: (work in a group of 4)
1. Divide 4 BHI agar plates into quadrants. Label one plate 15°C, the second 25°C, the third 37°C and
the fourth 56°C.
2. Collect one broth culture tube of each of the four organisms on the side bench.
3. Aseptically spread a loopful of each culture (remember to vortex each tube well before using) within
one quadrant of each plate (you are not interested in obtaining single colonies!).
4. Place each properly labeled plate in the appropriate box (i.e. there are four boxes!) on the side bench
and incubate for 24 hours.
5. In the open lab, rank the relative abundance of growth for each organism on each plate on the Exercise
4 submission sheet. The TA can help you if you require assistance.

2. The Effect of Hydrogen-Ion Concentration


The hydrogen ion concentration (measured by pH) of an organism’s environment affects the growth of most
bacteria. The pH range of an organism is the difference between the maximum and minimum pH at which the
organism will grow. The optimum pH is the pH at which the organism grows best. It is important to note that
these pH ranges refer to the organism’s external environment. The internal pH of most organisms can only
tolerate very minor adjustments before life is no longer possible.

Procedure: (work in a group of 4)


1. Each pair of students needs to aseptically prepare one set of the following solutions using
micropipettes (as demonstrated by your TA).

Tube # nutrient broth 0.2 M K2HPO4 0.1 M citric acid total pH

1 8 ml 0.10 ml (100 µl) 1.90 ml (2 x 950 µl) 10 ml 2.8

2 8 ml 0.30 ml (300 µl) 1.70 ml (2 x 850 µl) 10 ml 3.6

3 8 ml 0.60 ml (600 µl) 1.40 ml (2 x 700 µl) 10 ml 4.4

4 8 ml 1.00 ml (1000 µl) 1.00 ml (1000 µl) 10 ml 5.2

5 8 ml 1.30 ml (2 x 650 µl) 0.70 ml (700 µl) 10 ml 6.0

6 8 ml 1.50 ml (2 x 750 µl) 0.50 ml (500 µl) 10 ml 6.8

7 8 ml 1.90 ml (2 x 950 µl) 0.10 ml (100 µl) 10 ml 7.6

0.2 M boric acid 0.2 M NaOH

8 8 ml 1.30 ml (2 x 650 µl) 0.70 ml (700 µl) 10 ml 8.4

9 8 ml 1.00 ml (1000 µl) 1.00 ml (1000 µl) 10 ml 9.2

10 8 ml 0.20 ml (200 µl) 1.80 ml (2 x 900 µl) 10 ml 10.0

16
2. One pair will aseptically inoculate 1 set of tubes with 100 µl from a broth culture of organism A. One
pair will aseptically inoculate the other set of tubes with 100 µl from a broth culture of organism B.
3. Place the properly labeled tubes in the appropriate box on the side bench and incubate for 24 hours.
4. In the open lab, gently resuspend the cells that may have settled to the bottom of each tube. Rank the
relative turbidity for each tube on the Exercise 4 submission sheet. The TA can help you if you require
assistance.
Tubes 1-3 may form a precipitate because of the strong acidity of the solution. Be sure to compare your
tubes with the control tubes which will be available in the open lab.

3. The Effect of Heavy Metals


Heavy metals (Cu, Ag, Hg, etc.) are capable of exerting a lethal effect upon bacteria at high enough
concentrations even though some may be required in trace amounts. Resistance to heavy metals is facilitated
by plasmids that enable bacteria to pump the ions out of the cell or alter the oxidation state of the ion.
Procedure (work in a group of 4)
1. Remove a tube of molten NA from the water bath and inoculate with 100 µl of E. coli broth culture.
Gently rotate the tube between the palms to obtain a uniform distribution of organisms and then pour
the agar into the empty sterile petri dish. Allow the agar to harden.
2. Using sterile forceps, dip a sterile paper disc into a solution of 1% silver nitrate. Place the disc in the
centre of the plate.
3. Place the properly labelled plate in the appropriate box on the side bench and incubate for 24 hours.
4. In the open lab, observe the growth pattern of the organism. Measure the diameter of the inhibition
zone and record your results on the Exercise 4 submission sheet.

B. Effect of Spore Formation on Culture Viability


Early microbiologists noted that at temperatures above the maximum cardinal temperature, most microbial
cells are killed. Consequently, the use of heat (autoclaving, boiling, and flaming) became (and still is) a
standard part of aseptic technique used to kill microorganisms to sterilize environments.
However, it was also noted that heat was not always an effective means of destroying all microbial life.
Ferdinand Cohn studied heat-resistant bacteria and eventually characterized the genus Bacillus. It was
Cohn who first discovered and described endospore formation and brought to light why some methods of
sterilization were not always successful.
Endospores are differentiated bacterial cells that form due to changes in the nutritional and physical
environment of the organism. The highly resistant nature of endospores allow spore forming bacteria to
survive extremes of heat, dessication (drying), UV radiation, pH, and chemical disinfectants compared
with vegetative (undifferentiated) cells. Endospores can remain dormant for long periods of time - until
environmental conditions become favorable again - and will then germinate into a fully viable vegetative cell.
Endospore formation is limited to a few microbial species, but forms part of the normal life cycle of the spore-
forming bacteria.
The location of the endospore in the cell is usually characteristic of the species. For example, the location and
shape of Bacillus endospores are different from the location and shape of Clostridium endospores. Therefore,
the presence or absence of endospores and the description of the endospore is useful to a microbiologist as an
aid in identification.
17
Procedure: (work in pairs)
1. Collect a mixed broth culture tube from the side bench. Vortex the tube well to resuspend the culture.
2. Prepare 2 streak plates on BHI agar for single colony isolation. Place each properly labeled plate in the
appropriate box on the side bench and incubate for 24 hours (one at 37°C and one at 55°C).
3. Place the broth culture in the 80°C water bath for 10 minutes. Prepare a streak plate on BHI agar for
single colony isolation. Place the properly labeled plate in the appropriate box on the side bench and
incubate for 24 hours at 37°C.
4. Place the broth culture in the 100°C water bath for 15 minutes. Prepare a streak plate on BHI agar for
single colony isolation. Place the properly labeled plate in the appropriate box on the side bench and
incubate for 24 hours at 37°C.
5. In the open lab, note the presence of growth on each plate and observe the demo Gram stains. Record
the colony and cellular morphologies on the Exercise 4 submission sheet.

C. The Use of Bacteria to Screen for Carcinogenic Chemicals Using the Ames Test
The conventional way to determine whether or not a chemical substance is carcinogenic is to inject the
material into animals and look for the development of tumours. If tumours develop, it is presumed that the
substance can cause cancer. Although this method works well, it is costly and time consuming.
The fact that carcinogenic compounds induce increased rates of mutation in bacteria has led to the use of
bacteria for screening chemical compounds for possible carcinogenesis. The Ames Test, developed by Dr.
Bruce Ames at the University of California, Berkley, has been widely used for this purpose. The correlation
between carcinogenesis and mutagenicity is between 85% and 90%. Scientists are now using the Ames test to
screen many chemicals quickly and inexpensively to determine which are mutagenic and therefore, potentially
carcinogenic.
The standard way to test chemicals for mutagenesis has been to measure the rate of back mutations
(reversions) in strains of auxotrophic bacteria. In the Ames test, a strain of Salmonella enterica that is
auxotrophic for the amino acid histidine and lacks DNA repair enzymes (to prevent the correction of DNA
injury) is exposed to a chemical agent. After chemical exposure and incubation on histidine‑deficient medium,
the rate of reversion to prototrophy is determined by counting the number of colonies that are seen on the
histidine‑deficient medium. Each colony represents a his‑ —> his+ revertant. A positive result, indicating
mutagenicity, is obtained when an obvious increase in the number of colonies is evident when compared to the
number of spontaneous revertants in the negative control.
Keep in mind, when making your observations and conclusions, that this is a simplified version of the Ames
test. Only one tester strain of Salmonella enterica is being used in this experiment. Normally, several other
strains would be used in order to accommodate different kinds of chemical compounds. While one chemical
agent may be mutagenic on one tester strain, it may produce a negative result on another strain. Also, the test
chemical agents are normally treated with a mammalian liver enzyme preparation. There is evidence that liver
enzymes (mixed-function oxygenases) activate many carcinogenic chemical agents once they are in the body
that are not active otherwise. This reagent will not be used in this experiment since the experiment will work
well without it.

18
Procedure: (work in pairs)

The first three steps must be performed very quickly, before the top agar cools (about 20 seconds).

1. Pipette 100 μl of Salmonella mutant TA1538 into 1 tube of soft agar (from the hot water bath on the
side bench).
2. Gently mix the contents of the tube by rotating between the palms of both hands - do not shake!
3. Pour the contents of the tube over a plate of minimal glucose (MG) agar. Gently swirl the plate around
to spread the agar evenly over the entire surface. Allow to harden.
4. Repeat the steps 1-3 twice more using two new soft agar tubes and MG agar plates.
5. Label the first plate “negative control”. Dip a sterile disc, using sterile forceps, into sterile water. Drain
any excess water by touching to the side of the container. Place the disc in the centre of the plate and
press gently so it will adhere to the surface of the agar.
6. Label the second plate “positive control”. Aseptically moisten a disc in a sodium azide solution, place
the disc in the centre of the plate and press gently so it will adhere to the surface of the agar.
Sodium azide is a carcinogenic substance and should be handled with extreme care
7. Label the third plate “unknown”. Using the same technique as above, moisten a disc in an unknown
solution, place the disc in the centre of the plate, and press gently so it will adhere to the surface of the
agar.
8. Place the plates in the appropriate box on the side bench. The plates will be incubated for 72 hours and
stored in the refrigerator. They will be returned in Lab 5.

D. Lab Quiz 3
You must complete Lab Quiz 3 before your next scheduled lab.

Checklist for Laboratory 4:


q Each student group should inoculate 4 temperature plates, 20 pH tubes and 1 heavy metal plate
q Each student pair should inoculate 4 endospore BHI plates
q Each student pair should inoculate 3 Ames test plates
q Each student should record their results in the open lab and discard their plates and tubes properly
q Each student should submit the Exercise 4 submission before noon on February 2/5
q Each student should complete Lab Quiz 3 before their next scheduled lab

19
20
EXERCISE 4 SUBMISSION NAME: __________________________

LAB SECTION: ___________________

SCORE: __________
A. Factors Affecting the Growth of Bacteria
1. What are the four classes used to distinguish microorganisms in relation to their temperature optima?
Indicate the approximate optimum temperatures for each class. (4)

2. Indicate the relative abundance of growth (ranked from 0 to 3) of each organism on the temperature
plates. Use the standards on the side bench (and the TA!) to help you gauge the level of growth. Classify
each of the organisms into the appropriate temperature class. (4)
1 2 3 4
15°C

25°C

37°C

56°C

Classification:

3. Briefly explain why the optimum cardinal temperature of an organism is closer to the maximum cardinal
temperature than to the minimum cardinal temperature. (2)

4. What is one molecular adaptation that allow organisms that grow in hot environments (greater than 45°C)
to survive? (1)
5. What three classes are used to distinguish microorganisms in relation to their pH tolerances? (3)

6. Use the table below to indicate the relative turbidity (ranked from 0 to 3) for each of the pH tubes. Use the
standards on the side bench (and the TA!) to help you gauge the level of growth. Classify the organism
into the appropriate pH class. (4)

A B
Tube pH Turbidity Tube pH Turbidity
1 2.8 1 2.8
2 3.6 2 3.6
3 4.4 3 4.4
4 5.2 4 5.2
5 6.0 5 6.0
6 6.8 6 6.8
7 7.6 7 7.6
8 8.4 8 8.4
9 9.2 9 9.2
10 10.0 10 10.0
Classification Classification

7. Explain how microorganisms maintain a constant internal pH despite changes in the external pH. (2)

8. What is the zone of inhibition (diameter) around the heavy metal disc? Based on your results, is the
organism resistant or susceptible to silver? (2)
9. How does silver inhibit microbial growth? (2)
B. Effect of Spore Formation on Culture Viability
10. In the space below, note the presence of growth and record the colony and cellular morphology (if
available) of any organisms that grow on the four BHI streak plates. (4)

11. Explain your results, indicating the purpose of each plate and what information they told you about the
organisms in the mixed culture. (4)
Laboratory 5
Objectives:
1. To determine the phenol coefficient of a test disinfectant
2. To evaluate the effectiveness of samples of commercially prepared antibacterial agents
3. To observe the effect of several antibiotics using the Kirby-Bauer disk diffusion
method

Recommended Laboratory Resources:


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology
of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 167-170; 839-
840; 852-858.

A. The Effect of Chemical Agents on Bacterial Growth


Antimicrobial substances are either cidal (bacteriocidal, fungicidal, etc.), concerned with the killing of
microorganisms; or static (bacteriostatic, etc.), growth-inhibiting. Antiseptics are cidal or static against
microorganisms, but are still safe enough for use on living tissues. Disinfectants are more potent cidal agents
that destroy nearly all microorganisms, but can be applied only to inanimate material because of their toxicity.
The effectiveness of a chemical agent is measured by determining the minimum inhibitory concentration
(MIC), which is the lowest concentration of agent that completely inhibits the growth of the test organism.

1. Phenol coefficient
In the 1800s Joseph Lister introduced dilute phenol, formerly called “carbolic acid,” as a disinfectant for
surgical dressings. Because his disinfection procedure dramatically reduced postsurgical infection and death,
other surgeons soon accepted it. Phenol, in various dilutions was the first disinfectant and antiseptic to gain
favour in western medicine. For this reason, other solutions are now compared with it.
The phenol coefficient is the ratio of the test agent’s MIC to the MIC of phenol. The test agent and phenol are
tested against the same organism under the same conditions in order to generate the MICs. If the coefficient is
greater than 1, the test disinfectant is better than phenol; if the coefficient is less than 1, then it is not as effective
as phenol.

reciprocal dilution of test disinfectant that kills at 10 minutes but not at 5 minutes
Phenol coefficient = reciprocal dilution of phenol that kills at 10 minutes but not at 5 minutes

Research Applications
E. coli is one of the primary workhorses of modern molecular biology. It is used routinely for producing copies
of cloned genes and for the production of recombinant heterologous proteins (produced from a cloned gene
from a different species or organism). E. coli has been used for production of human insulin, bioremediation
and industrial scale fermentative production of specific proteins. In the lab today, you will use E. coli to test the
effectiveness of chemical agents on microbial growth and survival.

21
Procedure: (work in pairs)
1. One partner will use the 5% phenol and follow steps 2a through 5a. The other partner will use the test
disinfectant (1% Savlon) and follow steps 2b through 5b.
USE THIS PROCEDURE IF YOU ARE USING PHENOL
2a. Divide 2 BHI plates into quadrants and label with the appropriate duration (5 or 10 minutes),
disinfectant (phenol), and the 4 dilutions (see below).
3a. Label 4 sterile 16 mm test tubes “1”, “2”, “3”, and “4”. Use a micropipette and sterile tip to dispense
the following into the sterile tubes. Pipette E. coli last, at 1 minute intervals and immediately vortex the
tubes.
TUBE 1 2 3 4

distilled water 3.5 ml 3.6 ml 3.7 ml 3.8 ml


5% phenol 1000 µl 900 µl 800 µl 700 µl
final dilution of phenol 1/100 1/110 1/125 1/150

E. coli 500 µl 500 µl 500 µl 500 µl

4a. Exactly 5 minutes after the addition of E. coli to the tubes, use a sterile loop to spread the appropriate
quadrant of the 5 minute BHI plate.
5a. After a second interval of 5 minutes, repeat step 4a on the 10 minute BHI plate.
USE THIS PROCEDURE IF YOU ARE USING SAVLON
2b. Divide 2 BHI plates into quadrants and label with the appropriate duration (5 or 10 minutes),
disinfectant (Savlon), and the 4 dilutions (see below).
3b. Label 4 sterile 16 mm test tubes “1”, “2”, “3”, and “4”. Use a micropipette and sterile tip to dispense
the following into the sterile tubes. Pipette E. coli last, at 1 minute intervals and immediately vortex the
tubes.
TUBE 1 2 3 4

distilled water 4.2 ml 4.4 ml 4.45 ml 4.49 ml


1% Savlon 300 µl 100 µl 50 µl 10 µl
final dilution of Savlon 1/1500 1/5000 1/10,000 1/50,000

E. coli 500 µl 500 µl 500 µl 500 µl

4b. Exactly 5 minutes after the addition of E. coli to the tubes, use a sterile loop to spread the appropriate
quadrant of the 5 minute BHI plate.
5b. After a second interval of 5 minutes, repeat step 4b on the 10 minute BHI plate.
--------------------------------
6. Place all four plates in the appropriate box on the side bench and incubate for 24 hours.
7. In the open lab, determine the MIC of phenol and Savlon that kills E. coli at 10 minutes but not at 5
minutes and calculate the phenol coefficient. Record your results on the Exercise 5 submission sheet.

22
2. Evaluation of commercially prepared antibacterial agents
Many commercial preparations claim to be antibacterial. The purpose of this investigation is to make a limited
evaluation of these claims and to compare the effectiveness of competitive products.

Procedure: (work in pairs)


1. One partner will use a Gram-positive organism and the other partner will use a Gram-negative organism.
2. Prepare a microbial lawn by placing 100 µl of your organism onto a BHI plate. Spread the culture
evenly over the plate using a glass spreader sterilized in alcohol and flamed as demonstrated by your TA.
When dry, divide the plate into quadrants and label with the organism and names of the test solutions.
3. Obtain a sample of the 4 antiseptic/disinfectant test solutions from the side bench.
4. Using sterile forceps (see TA demonstration), dip a filter paper disc into a test solution and place it
on one of the quadrants on the BHI plate. With the tip of the forceps, touch the surface of the disc to
improve its contact with, and adhesion to, the medium. Be sure to sterilize the forceps before picking up
the next disc.
5. Place both plates in the appropriate box on the side bench and incubate for 24 hours.
6. In the open lab, observe and measure the zone of inhibition (i.e. diameter in mm) around the discs for
each organism and record your results on the Exercise 5 submission sheet.

B. The Effect of Chemotherapeutic Agents on Bacterial Growth


The previous exercise dealt with chemicals that control the growth of microorganisms outside the human body,
but most of them are too toxic to be taken internally. Chemotherapeutic agents are antimicrobial compounds that
can be administered internally and are important in human and veterinary medicine. Paul Ehrlich began to look
for chemotherapeutic agents that had selective toxicity: the ability to kill or inhibit microorganisms, but have no
adverse effects in the host.
Antibiotics are compounds naturally produced by bacteria and fungi that kill or inhibit other microorganisms
and provide a competitive advantage for the producing organism. Most antibiotics are produced by the two
bacterial genera Streptomyces and Bacillus and the fungal genus Penicillium.
Synthetic agents are compounds developed in laboratories that have antimicrobial action and also display
selective toxicity. Antibiotics that have been structurally modified in the laboratory to improve activity and
minimize toxicity are referred to as semisynthetic antibiotics.
Different chemotherapeutic agents have a wide range of effects on different microbes. Some agents are broad
spectrum: effective against a wide range of microorganisms (Gram-positive, Gram-negative, fungi, etc.). Others
have a narrow spectrum of activity: only a few species are killed or inhibited by these agents.

23
1. Kirby-Bauer antimicrobial susceptibility test
The Kirby-Bauer antibiotic disc diffusion test is a rapid, inexpensive simple test used in diagnostic laboratories
to determine the effectiveness of an antibiotic on a particular strain of bacteria. The procedure is designed to
evaluate one variable, the sensitivity (susceptibility) of a pathogen to assorted antibiotics, all other variables are
held constant. Variables that can alter results of an antibiotic disc diffusion test include concentration and rate
of diffusion of the antibiotic in each disc, density of bacterial growth, thickness of medium, and temperature
and length of incubation. The discs are prepared commercially by adding known amounts of the antibiotic to
the disc. During incubation, the antibiotic diffuses from the filter paper into the agar, the further it gets from the
filter paper, the weaker the concentration of the antibiotic.

Procedure: (work in pairs)


1. Prepare a lawn of a Gram-positive organism and a Gram-negative organism on different BHI plates.
2. Obtain the 3 antibiotics from the side bench and aseptically place the discs evenly around both BHI plates.
The discs must not be closer than 3 cm from each other and not closer than 2 cm from the edge of the
plate. Ensure that the disc has made firm contact with the agar. Label both plates appropriately so that a
comparison between the effectiveness of the antibiotics on Gram-positive and Gram-negative organisms can
be made.
3. Place the plates in the appropriate box on the side bench and incubate for 24 hours.
4. In the open lab, observe and measure the zone of inhibition (in mm) around the discs. Use the standards
to determine the susceptibility of each organism to the antibiotics. Record your results on the Exercise 5
submission sheet.

C. Ames Test Results


Collect the three Ames test plates you prepared in Lab 4. Record your results on the Ames Test submission
sheet, which is due before you leave the lab.
a) Count the number of colonies present on each plate. Ignore the barely visible background lawn of bacterial
growth.
b) Determine and record the number of chemically induced mutations by subtracting the number of colonies on
the negative control plate, representative of spontaneous mutations, from the number of colonies on:
i. The positive control plate, and
ii. The unknown test plate
c) Determine and record the relative mutagenicity of the test compound on the basis of the number of induced
mutations: If below 10, (‑); if greater than 10 (1+); if greater than 100 (2+); and if greater than 500 (3+). The
TA can help you with this classification.

24
Checklist for Laboratory 5:
q Each student pair should inoculate 4 phenol coefficient plates
q Each student pair should inoculate 2 antiseptic/disinfectant BHI plates
q Each student pair should inoculate 2 Kirby-Bauer plates
q Each student should complete and submit the Ames Test submission
q Each student should record their results in the open lab and discard their plates properly
q Each student should submit the Exercise 5 submission before 1500 (3:00pm) on February 9/12

25
26
EXERCISE 5 SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

SCORE: __________

A. The Effect of Chemical Agents on Bacterial Growth


1a. Calculate the phenol coefficient of Savlon. Show your calculations. (3)
MIC of phenol that inhibits growth at 10 minutes, but not at 5 minutes:

MIC of Savlon inibits growth at 10 minutes, but not at 5 minutes:

Phenol coefficient of Savlon:

1b. Based on your results, which is the better disinfectant: 5% phenol or 1% Savlon? Explain. (1)

2a. Complete the table below indicating the zone of inhibition on the bacterial lawn of the Gram-positive
organism and the Gram-negative organism for each of the antiseptics/disinfectants you used. (4)

Zone of Zone of
Antiseptic OR inhibition (mm) inhibition (mm)
Name of Agent
Disinfectant? for Gram- for Gram-
positive negative

2b. Based on your results, are there any differences in the effectiveness of antiseptics or disinfectants, or in
their effectiveness against Gram-positive organisms or Gram-negative organisms? (2)
B. The Effect of Chemotherapeutic Agents on Bacterial Growth
3a. Complete the table below indicating the zone of inhibition on the bacterial lawn of Staphylococcus for
each Complete the table below indicating the zone of inhibition on the bacterial lawn of the Gram-positive
and Gram-negative organisms for each of the antibiotic discs you used. Indicate whether the organism is
susceptible (S), resistant (R), or intermediate (I) to the agent. (3)

Agent

Organism Zone Zone Zone


S/R/I S/R/I S/R/I
(mm) (mm) (mm)

Gram-positive

Gram-negative

3b. Based on your results, classify the antibiotics that you used as either broad or narrow spectrum. (1)
AMES TEST SUBMISSION NAME: _____________________________

LAB SECTION: ______________________



SCORE: __________

Screening for Carcinogenic Chemicals Using the Ames Test

1. Complete the table below indicating the number of colonies on the three Ames test plates. Determine the
number of induced mutations for each plate and rank the relative mutagenicity for each plate. (4)

Number of induced
Test Number of colonies Degree of mutagenicity
mutations

Negative control

Positive control

Unknown

2. The MG plates you used for this test contained trace amounts of histidine, which may have resulted in a
faint background lawn of growth on the plates. Why was histidine added, why did the background growth
not develop into a complete lawn, and why did some colonies develop fully? (4)

3. Why are the positive and negative controls important for the Ames test? (2)
Class Presentations
Now that you are well on your way to becoming a world-renowned microbiologist, it is
time to share your knowledge and familiarity of everything microbial with the world!

You (in a group of students) will be assigned either a microorganism, a microbiologist,


or an everyday/global problem with a microbial solution, and give a short presentation to
your colleagues in lab the week of February 12.

Your presentation should outline any pertinent information relevant to your topic with a
focus on the properties of the microorganism involved, major scientific contribution of
the biologist, and any other relevant practical information you discover.

The presentation must be between 15 and 20 minutes in duration. You will be graded on
your oral presentation by your TA and will be expected to answer any questions that arise.

There will be a small whiteboard, digital projector and a PC (with Microsoft PowerPoint)
available for your use. You must bring your presentation on a flash drive or bring your
own computer (with appropriate cables for connecting to the projector).

Computer/file issues are NOT a valid reason for not being able to complete your
presentation. The 20-minute time limit includes time for set-up, presenting, answering
questions and wrap-up.

In addition to giving the presentation, you will be expected to participate in the


presentation of your peers by paying attention and asking questions. There will be
questions on the lab midterm exam pertaining to the presentations.

Any student identified as being disrespectful during a colleague’s presentation will


receive a zero for this component of the course.

Presentation topics will be assigned the week of January 15. You must present the topic
assigned by your TA and you must provide your classmates with an introduction to the
topic and current research being carried out for that area. You must present as thorough a
review of the material as possible without exceeding the time limit.

27
EXPECTATIONS FOR ORAL PRESENTATIONS
Your presentation will be graded on 4 main criteria. Make sure you consider these criteria
as you plan and practice your presentation. There will also be a peer evaluation mark. Your
presentation should provide a suitable introduction for your colleagues and outline any current
and relevant information regarding your topic.
Have fun and make sure you ask your TA if you have any questions.
Grading Components:
1. Information (3% of course grade)
Content (7 marks):
Your group will be graded on how well you introduce your topic, how well you use appropriate
terminology, how well you demonstrate understanding of the material and how comfortable you
are presenting the information.
Answering Questions (2 marks):
Your group will be graded on your ability to understand and answer simple questions based on
your presentation.
2. Presentation (0.5% of course grade)
Presentation Skills (7 marks):
Each individual will be graded on their speaking skills, timing, confidence, organization, and
engagement with the audience.
3. Citations (0.5% of course grade)
Literature Cited (2 marks):
Your group will be graded on your use of in-text citations and the literature-cited section that
you hand in prior to beginning your presentation.
4. Collaboration (1% of course grade)
Peer Evaluation (7 marks):
Each member of your group will assess the other members on their participation and
cooperation within the group dynamic. This is a required component of the presentation
- if you do not submit a peer evaluation form (posted on D2L), you will get a zero on all
Presentation components (5% of course grade).

28
Lab Exam 1
and Inoculations
Lab Exam 1
You will write Lab Exam 1 in your scheduled section on February 27/March 1. The exam will cover material
learned in Exercises 1-5 (plus the presentations) and will be a mix of factual recall, practical and application
questions. The exam will be 2 hours in length and consist of ~10 short answer/practical quesions. You may use a
pen or pencil and a non-programmable calculator. You may not bring personal electronic devices (PDAs, MP3s,
cell phones, etc.)

What should you study?


Terminology - know how to use the proper terms at the proper times. I.e. a bacterial cell is cocci, not
circular
Procedures and theory - know how to do a Gram stain, a streak plate, etc. and how to classify an organism
based on test results.
Results and calculations - know how to interpret any of the tests we have done, how to determine phenol
coefficients, etc.
Conclusions and study questions - know how to draw conclusions from results. Study the questions in the
lab manual and your submissions.
Experimental design - understand how/why an experimental procedure works and be able to design an
experiment to answer a question
Aseptic technique - you should follow proper safety guidelines and know how to maintain a safe, aseptic
work environment

What should you NOT study?


Quantities and lists - you will not be asked how many grams of agar need to be added to 200ml water or
list the ingredients of BHI agar, etc.
Specific results - you will not be asked to recall the specific results of the hand-washing plates, etc.

Preparation for Exercise 6


Homolactic Fermentation Procedure: (work in pairs)
1. Using the sample provided, inoculate one tube of litmus milk medium with a loop of yogurt.
2. Place the properly labeled tube in the appropriate box on the side bench and incubate for one week.

29
Winogradsky Columns
Procedure: (work individually):
1. Make your final observations of the four Winogradsky columns.
2. Submit your observation chart (page 13 or the one you generated) before noon on March 2/5

Preparation for Bacterial Unknown Project


Procedure: (work individually)
1. Collect two pure unknown culture plates from the side bench and record your unknown number on page
29 and on the Unknown submission sheet.
2. Prepare streak plates for single colony isolation and incubate the plates according to the culture
conditions “hint” provided by the TA.
3. Return the original plates to your TA.

Before next week’s lab:


1. Maintain a pure culture of each organism (if you need to replace any of your plates throughout this
project, you will be penalized on your journal grade).
2. Perform a Gram stain on each unknown from your plates.
3. Complete the Unknown submission before noon on March 2/5

30
Term Project
Identication of Bacterial Unknowns
Objectives:
1. To identify two unknown organisms using classic bacteriological techniques and a
dichotomous key

Identification of Heterotrophic Bacteria


Heterotrophic bacteria have been more extensively studied than the autotrophic bacteria since all animal and
plant pathogens are heterotrophic and they form the greater part of our immediate environment.
Since heterotrophic bacteria require an organic form of carbon, they are most easily cultivated on
complex media such as nutrient agar or brain heart infusion agar or broth. Under normal conditions, most
environmental samples are a heterogeneous mixture of microbial species that first need to be separated before
individual organisms can be identified. In order to separate and identify organisms in a mixed culture the
streak plate technique may be used. Once pure cultures have been prepared, then differential media and a
variety of biochemical and staining tests are employed in order to identify the organisms. A bacteriological
dichotomous key simplifies the procedure since only certain characteristics are specific to a particular genus or
species.

Isolation and Identification of an Unknown Bacterial Species


Last week, you received two pure culture plates containing unknown bacterial species.

Unknown # ___________________

Make sure you collect the Unknown submission sheet and the pure culture streak plates of each organism.
Remember, you will be responsible for properly maintaining both of your organisms. You will need to plate,
incubate and refrigerate your cultures until you have successfully identified both of them.
Over the next few weeks (in the lab and open lab), you will collect data about the environmental,
physiological, enzymatic and nutritional characteristics of the unknowns and make tentative identifications
using classic taxonomic methods.
To assist you in this effort, you were given culture hints and you will have access to a dichotomous key
(posted on D2L and in the lab). Your TA will give a brief description on its use.
As you collect information about your unknowns, you need to rationalize the important steps you take during
the identification process, provide a flow chart indicating how you moved through the dichotomous key, record
the results of the tests that you perform, illustrate your understanding of each test and what information each
test provides about your organism, and answer a discussion question once you have identified both organisms.
To facilitate this, you will maintain an “Unknown Journal”.

31
UNKNOWN JOURNAL
You may use any format you feel is appropriate for your journal (e.g. a hard-cover lab notebook, loose leaf,
computer document, etc.). To properly record and present the information you collect, please organize your
journal following these guidelines:
1. Prepare a Table of Contents directing the reader to specific information
E.g. Results of Gram Stain – page 4
2. Number the pages of your journal (so that your TOC is useful!)
3. Prepare a flow chart
In order to increase efficiency and decrease waste, indicate how you used the dichotomous key to
complete your analysis, characterization and identification of both organisms.
4. Prepare Headings for each result
Please make these as detailed as possible. E.g. Growth on Mannitol Salt Agar.
5. Record your results accurately and completely
Your TA may not see the results that you obtain, so you must record a description of how your
organisms responded to each test. I.e. the colour, extent of growth, etc.
6. Write (discuss) your results
In a few sentences, explain the rational for performing each test and explain the interpretation of each
result, indicating what the results for each test tells you about your organisms. I.e. what you learned
about the physiology and biochemistry of your organisms.
7. Identify your unknown
Make sure you rationalize the identity of both unknown organisms.
8. Complete the discussion question
Once you know the identity of your unknown organisms, a discussion question for “Organism A” will
be made available to you. Provide a complete, accurate and concise answer to the question in your
journal. Ensure you use CSE citation rules for both the in-text citations and Literature Cited (see page
49)
You will submit your journal the week of March 19. Your TA will assess the format of your journal, the
results and appropriateness of the tests that you performed, the identification of your unknowns, and the final
discussion.
WARNING: You are responsible for the proper maintenance of your unknowns. You will need to keep a pure
culture that is fresh (less than 1 week old) before you inoculate each test. If you fail to maintain your culture
(contamination, desiccation, etc.) or if you fail to label, store or destroy your media and tests properly, you
will receive a penalty on your journal.
After you have completed your identifications, you need to destroy all plates and tests.

32
PROTOCOL FOR HANDLING UNKNOWNS
1. The identification of the organism is to be determined using the media and resources provided in the
lab. All of the media required will be in the shelves along the side bench. If you cannot find the media
you require, ask a TA or technician for assistance. Refer to Appendices 4 and 5 for a review of staining
procedures and biochemical tests.

The medium you use must be applicable to the identification of your organism. Media are not to be
randomly inoculated in the hope of achieving successful identification of the unknown. Remember,
you must present a rationale for the use of each test in your journal. Use of the dichotomous key will
facilitate your procedure and save valuable time.

2. Ensure that you are maintaining a fresh culture of your organisms throughout this exercise. You will
need to use single, isolated colonies from a fresh (less than 1 week old) plate to inoculate the tests
and media. The TAs will be looking at your test results and source of inoculum regularly throughout
this exercise. Make sure you are properly disposing old plates and tests when they are no longer
required.

3. Ensure that you are recording all of your results in your unknown journal and that it is kept up to
date regularly. This will help make sure that you do not forget anything and will spread the written
component of this exercise over a manageable amount of time.

4. Use the dichotomous key and references that are available in the library or in the lab. Some references
that may be used to facilitate identification of the organisms are:
a. Bergey’s Manual of Determinative Bacteriology or Bergey’s Manual of Systematic Bacteriology,
Vol. 1 & 2.
b. DifcoTM & BBLTM Manual - lists all the media made by Difco with a discussion of the media
including the purpose and application of each kind of medium.

5. TIDY your working space before you leave the lab. For example:
a. Refill staining bottles as necessary from the supply bottles on the shelf above the side bench
b. Put your microscope away properly
c. Put the stool back under the bench before you leave

If your work area is not properly maintained, you will receive a penalty on your journal

6. If you have any major problems with your unknowns, talk to your TA right away.

The journal will be due the week of March 19

33
34
UNKNOWN SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

UNKNOWN # ________________

SCORE: ________________

1. What is the colony morphology of your two unknown organisms? (4)

2. Submit a pure culture streak plate of both organisms. (4)

3. What is the cellular morphology of your two unknown organisms? Make sure the cellular morphology
for each organism is matched to the appropriate colony morphology from Question 1. (4)
4. Use the space below to diagram both of the unknown organisms (page 61). Make sure the diagram for each
organism is matched to the appropriate morphology from Questions 1 and 3. (6)
Laboratory 6
Objectives:
1. To become familiar with the effects and consequences of oxygen on microbial growth
and metabolism
2. To become familiar with fermentation pathways common to many microorganisms
3. To become familiar with respiratory pathways common to many microorganisms

Recommended Laboratory Resources:


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock:
Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp.
85-95; 160-163; 419-421; 434-438.
Lab Manual Appendices 3, 4, and 5
* Collect a broth culture tube and a streak plate of one of the unknown organisms (A, B, C, D, E or
F) from the side bench. Remember to vortex the tube well before each use. Record the unknown
code on the Exercise 6 submission sheet. You will need these cultures for parts A and B below.

A. Oxygen as a Nutrient
Although oxygen is a major component of macromolecules and is thus needed for life, molecular oxygen (O2) is
never used as a source of these atoms. The oxygen found in these compounds is derived from water and organic
molecules. Molecular oxygen is normally only required as the final electron acceptor in the electron transport
chain of respiring organisms and it is in this sense that we are considering oxygen a required nutrient.
1. Oxygen tolerance
Organisms that respire have to remove toxic oxygen by-products (O2-, H2O2, OH.) that form during the
reduction of O2 to H2O. If not removed, organic cellular components are oxidized and the cell dies. Not all
microorganisms require the same levels of oxygen for metabolism, and some organisms cannot tolerate any
oxygen at all.

Procedure: (work individually)


1. Record the colony morphology of your unknown organism on the Exercise 6 submission sheet.
2. Prepare one tube of each of the following media for the unknown culture (A, B, C, D, E, or F).
i. BHI slant - aseptically inoculate and spread a loopful across the surface of the agar.
ii. Deep shake culture (page 55) - aseptically inoculate 200 μl into a tube of molten tryptose agar. Mix
gently by rotating the tube between the palms of your hands.Allow to harden in an upright position.
iii. Thioglycollate medium (page 55) - aseptically inoculate 200 μl into a thioglycollate broth tube below
the upper, pink layer. Handle very carefully so the oxygenated layer is not disturbed.
3. Place both properly labeled tubes in the appropriate box on the side bench and incubate for 24 hours.
4. In the open lab, record your observations and interpretations on the Exercise 6 submission sheet.
35
2. Respiratory enzymes
The respiratory electron transport chain is a sequence of enzymes and electron carriers responsible for
transferring reducing equivalents from substrates to molecular oxygen. Oxygen acts as the final electron/
hydrogen acceptor in the respiratory chain producing either water or hydrogen peroxide, depending upon the
species and its enzyme system. Two enzymes which are part of the respiratory chain are cytochrome c oxidase
and catalase.

Procedure: (work individually)


1. Use the agar streak plate of the unknown organism (A, B, C, D, E, or F) to complete each of the following
tests.
i. Oxidase test (page 67) - Use an oxidase stick (hold the red end) to pick off a small mass of cells (with
the brown end) from a single colony. Examine the stick over a 3 minute period.
ii. Catalase test (page 67) - Add two drops of hydrogen peroxide to a colony on the agar plate.
2. Record the results and interpretations on the Exercise 6 submission sheet.

B. Utilization of Glucose (Fermentation or Respiration?)


Glucose, because of its wide distribution and importance in animal metabolism, has served as the
initial substrate for most studies of product formation and mechanism of carbohydrate metabolism in
microorganisms. Furthermore, glucose is assigned a major role in reactions leading from polysaccharides
and as a substrate for phosphorylation prior to rearrangement and cleavage in fermentation and oxidation
pathways.
Glucose may be utilized in a variety of ways. It may be respired aerobically when oxygen is present or it
may be anaerobically fermented. Facultative organisms may respire or ferment glucose depending upon the
presence or absence of oxygen.

1. Hugh and Leifson (H&L) glucose media (page 67)

Procedure: (work individually)


1. Aseptically stab inoculate 2 tubes of H&L glucose from an isolated colony on the agar streak plate (A, B,
C, D, E, or F). Gently overlay one tube with a layer of mineral oil as demonstrated by your TA.
2. Place both properly labeled tubes in the appropriate box on the side bench and incubate for 24 hours.
3. In the open lab, record your observations and interpretations on the Exercise 6 submission sheet.

36
C. Fermentation Pathways
Heterotrophic microorganisms may break down carbohydrates to obtain energy by fermentation. Fermentation
has four basic features: (1) it occurs in the absence of oxygen; (2) energy producing electron transport is
absent; (3) it is much less energy efficient than respiration; and (4) metabolic intermediates or fermentation
end products are produced.
Fermentation of glucose occurs in two stages. The first involves the splitting of glucose and the removal of
two pairs of hydrogen atoms, resulting in the formation of carbon compounds more oxidized than glucose (i.e.
pyruvate). In the second, or reductive part of fermentation, the oxidized compounds are reduced, recycling the
hydrogen atoms removed in the first stage. Since an oxidation cannot proceed without an equivalent reduction,
the number of hydrogen atoms removed in the first part of a fermentation is always equal to the number used
in the second part.
The two major pathways used for bacterial fermentations are the Embden-Meyerhof-Parnas (EMP) scheme
(glycolysis) and the hexose monophosphate (HMP) scheme. In either pathway, pyruvic acid is always one
of the compounds formed in the first stage of bacterial fermentation. The end products characteristic of the
various bacterial fermentations are derived from the pyruvic acid and the hydrogen atoms produced in the first
(or oxidation) stage of fermentation.
Among the bacteria, several different pathways for the fermentation of carbohydrates have been identified.
Each is associated with a specific set of end products and each is characteristic of a particular species.

1. Alcohol fermentation
Bacteria (i.e. Streptococcus sp. and some Lactobacillus sp.) first oxidize a carbohydrate (glucose, lactose, etc.)
to pyruvic acid via the Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced almost
entirely to lactic acid.
Last week, you inoculated a litmus milk tube with yogurt to observe the homolactic fermentation of milk.
Follow the procedure below to complete this observation.

Procedure: (work in pairs)


1. Observe the changes in the litmus milk tube (page 68) and record your results and interpretations on the
Exercise 6 submission sheet.

2. Mixed acid or Butanediol fermentation (page 69)


Bacteria (i.e. Escherichia coli) first oxidize a carbohydrate (glucose, lactose, etc.) to pyruvic acid via the
Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced to a variety of acids (lactic acid,
acetic acid, succinic acid, etc.), which accumulate in the media.
Bacteria (i.e. Enterobacter aerogenes) first oxidize a carbohydrate (glucose, lactose, etc.) to pyruvic acid via
the Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced to a variety of acids, which
are then converted to acetoin and 2,3-butanediol, which accumulate in the media.

Procedure: (work in pairs)


1. Aseptically inoculate two sterile MR-VP broth tubes with E1 and two more with E3.
2. Place the properly labeled tubes in the appropriate box on the side bench and incubate for 24 hours.
3. In the open lab, collect the tubes and vortex each tube well. Carry out the Methyl Red test on one set of
tubes and the Voges-Proskauer test on the other set. Record the results and interpretations on the Exercise 6
submission sheet.

37
D. Anaerobic Respiration
Respiration may occur with a final electron acceptor other than oxygen, which results in the reduction of
sulfates, nitrates, CO2, ferric iron, or several organic molecules such as fumarate.

1. Nitrate respiration
Nitrates may be produced in the soil by nitrifying bacteria through the oxidation of ammonia. Alternatively,
they may be applied to the soil in the form of commercial fertilizer. Nitrates are very soluble and if not
promptly assimilated by plants, may be lost from the soil either by leaching or by microbial reduction.
Many bacteria, as well as plants and some fungi, are capable of reducing nitrates to ammonia and subsequently
incorporating ammonia into amino acids and proteins. When NO3 is reduced for use as a nutrient source in
this way, it is called assimilative nitrate reduction. This may occur anaerobically as well as under aerobic
conditions. In general, assimilative nitrate reductases are soluble proteins which are ammonia repressed.
Dissimilative nitrate reduction is restricted to bacteria and occurs when NO3 is used as an electron acceptor
in energy metabolism (anaerobic respiration). Dissimilative nitrate reductases are membrane bound proteins
which are repressed by O2 and synthesized under anaerobic conditions only. These bacteria are able to use
nitrate instead of oxygen as the final electron acceptor in the electron transport chain.
The ability to reduce nitrate to nitrite in the absence of oxygen is possessed by many bacteria and the process
is called nitrate reduction:
anaerobic > NO - + H O
NO3- + 2H+ 2 2

However, the ability to reduce nitrate beyond nitrite is limited to a relatively small number of genera. The
formation of N2O, NO or N2 is called denitrification:

anaerobic > NO - anaerobic > N (NO or N O)


NO3- 2 2 2

Since these products are all gaseous, they can be easily lost into the atmosphere. This process is the main
means by which gaseous N2 is formed biologically, and since N2 is much less readily available to organisms
than nitrate as a source of nitrogen, denitrification is considered a detrimental process.

Procedure: (work in pairs)


1. Aseptically inoculate two sterile nitrate reduction broth tubes (page 71) with E3 and P9 as demonstrated by
your TA. Watch out for the Durham vial!
2. Place the properly labeled tubes in the appropriate box on the side bench and incubate for 24 hours.
3. In the open lab, collect the tubes and record the results on the Exercise 6 submission sheet.

E. Lab Quiz 4
You must complete Lab Quiz 4 before your next scheduled lab

38
EXERCISE 6 SUBMISSION NAME: _____________________________

SCORE: __________ LAB SECTION: _________________

A. Oxygen as a Nutrient Unknown Code: ____


1. Record your observations and interpretation of each media inoculation or the results of each of the tests
you performed on the unknown organism. (6)
BHI plate (colony morphology):

BHI Slant:

Deep Shake tube:

Thioglycollate broth:

Oxidase test:

Catalase test:
B. Utilization of Glucose (Fermentation or Respiration?)
2. Record your observations and interpretation of the H&L glucose test you performed on the unknown
organism. (2)

3. Based on the results (question 1), to which oxygen requirement class does the organism belong? What do
you know about the metabolic capabilities of the organism? Be specific. (4)

4. What was the purpose of the H&L glucose test tube that you covered with mineral oil? Explain. (2)
5. What were the results (and significance) of the litmus milk test for the yogurt sample? (2)

6. Record your observations and interpretation of the MR-VP media for both organisms inoculated. (4)

7. Why can you read the results of the methyl red test immediately after adding the methyl red reagent, but
you have to wait for the results of the Voges-Proskauer test? (2)
8. Record your observations and interpretation of the nitrate reduction test for both of the organisms tested.
(3)
Gas
Organism Interpretation
Y/N

E3

P9

9. Explain why gas production suggests, but does not confirm, nitrate reduction. (2)
Laboratory 7
Objectives:

1. To introduce transposons and techniques for generating transposon mutants

Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology
of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 307-308, 314-315,
320-321, 325-328.

Transposon Mutagenesis
Creation of mutations and subsequent genetic mapping can elucidate the identity, relative size, number
and organization of genes involved in a physiological process. Three general treatments can be used
to mutagenize microorganisms: electromagnetic radiation, chemical mutagens and transposons. A
transposon (Tn) is a genetic element which can move from one site on a DNA molecule to another site
either on the same or on a different DNA molecule. When the insertion site of a Tn is within a gene,
the linear continuity of that gene is disrupted, i.e., the Tn insertion is mutagenic. As well as encoding
transposition genes, Tn often encode for genes conferring resistance to antibiotics.

A modified transposable element, Tn5, encodes for resistance to the antibiotic gentamicin and will be used
to illustrate the use of Tn5 as a mutagen using Escherichia and Rhizobium
Research Applications

Scientists have taken advantage of the unique ability of transposons to jump from site to site in a genome,
using them to create large mutant collections. During cell division, transposons can become activated
and jump randomly in a genome. In doing so, the transposon has a chance of inserting into a region that
disrupts the expression of a gene, knocking the gene out in the process. Using these single knockouts,
researchers are able to study the effects of losing the function of specific genes on an organism.

However, transforming genes into a host organism is inefficient. Often only a small percentage of the
host cells successfully incorporate the target DNA (e.g. plasmid). To identify (select) the transformed
cells, antibiotic resistance genes are included in the plasmid, allowing transformants to grow in media
containing the selected antibiotic. Untransformed cells do not survive this treatment.

Plasmid Vector
Wild-type Rhizobium VF39 (recipient strain) will be mutagenized by conjugation with an E. coli S17-1
(donor) strain that harbours a plasmid (pSUP102::Tn5-B22) that carries the transposon, Tn5-B22. The
mutagenesis protocol relies upon the special properties of the plasmid, pSUP102::Tn5-B22 and the E. coli
strain that harbours it.

39
Plasmid pSUP102::Tn5-B22 contains three important properties:
1. A modified Tn5 transposon that has had the normal antibiotic resistance gene of Tn5 (kanamycin)
replaced with a gene coding for antibiotic resistance to gentamicin.
2. An origin of transfer. This sequence of DNA is recognized by the conjugation apparatus and will allow
transfer of the plasmid during conjugation between two cells.
3. An E. coli specific origin of replication. This plasmid is unable to replicate in Rhizobium and therefore
will be lost as the Rhizobium replicates. This makes the plasmid a “suicide vector”.
The E. coli strain contains the conjugal transfer genes on the chromosome. It is capable of taking any
plasmid that has an origin of transfer and mobilizing it to a suitable recipient. In this experiment, the
plasmid pSUP102::Tn5-B22 from the E. coli strain will be mobilized by conjugation to a Rhizobium wild
type stain that has a chromosomal gene coding for antibiotic resistance to streptomycin. After conjugation,
both the donor and recipient cells will be plated on TY +streptomycin + gentamicin (TY + Sm + Gn) plates.

Procedure: (work in pairs)


1. Pipette 1.0 ml of E. coli S17-1 pSUP102::Tn5-B22 (donor cells) into a sterile centrifuge tube. Pipette 2.0 ml
of Rhizobium (recipient cells) to the same centrifuge tube.
2. Centrifuge at 4,000 rpm for 10 minutes and decant the supernatant very carefully by pouring it into a waste
beaker. Resuspend the pellet in 200 μl of sterile TY broth. Mix well on the vortex.
3. Place 3 sterile nitrocellulose filters evenly around the surface of a dry TY plate (as demonstrated by your
TA). The filters must not be closer than 3 cm from each other and not closer than 2 cm from the edge of the
plate. Ensure the filters make firm contact with the agar.
4. Using a micropipette with a sterile yellow tip attached, aliquot 40 μl of resuspended cells onto 1
nitrocellulose filter. If necessary, spread the cells around on the filter using the tip. Do the same with the
remaining two filters. Allow the spots to dry on the filters at room temperature, place the properly labeled
plate in the appropriate box on the side bench and incubate for 24 hours.

In the open lab:


5. Aseptically pipette 1000 μl of sterile distilled water into 3 sterile test tubes. Use sterile forceps to carefully
pick up each filter and place each into a different test tube. You may have to push the filter gently into the
water using a sterile glass rod. Mix very well on the vortex to wash the cells from the filter into the water.
6. Pipette 100 μl from each tube onto separate TY + Sm + Gn plates. Spread each plate with a sterile glass
spreader. Place the properly labeled plates in the appropriate box on the side bench. They will be incubated
for 5 days and returned in Lab 8.

Lab Quiz 5
You must complete Lab Quiz 5 before your next scheduled lab (Lab 8).

40
Laboratory 8
Objectives:
1. To explain the effect of UV radiation on the replication of T4 and explore the use of sunscreens through UV-
induced mutation in T4
2. To perform a plaque assay with bacteriophage T4 and its host cell E. coli

Recommended Laboratory Resources:


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology
of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 166; 224; 227-
229; 312.

A. Effect of Radiation on Viral Growth


Bacteriophage
In 1917, F. d’Herelle, a Canadian microbiologist working in Paris and F. Twort, a British scientist,
discovered bacterial viruses, which they called bacteriophage, meaning “to eat bacteria”. All viruses are
considered to be noncellular infectious entities having either a DNA or RNA genome. They replicate only
in living cells, using the cell’s metabolic machinery to synthesize copies of themselves for transfer of their
own genomes to new hosts. Although a virus contains nucleic acid hereditary material and is capable of
reproducing itself, it has none of the other attributes of a living organism.
The virus used for this experiment is an E. coli T4 bacteriophage. It is a classical virulent virus that can go
through the lytic cycle but not the lysogenic cycle. It is one of largest of the bacterial viruses. The genome is
composed of one, linear, double stranded DNA molecule with a total size of 169 kbp (kilo base pairs).

The Effect of UV Radiation on DNA Replication


Biologically significant radiation, such as that used for photosynthesis, has wavelengths in the visible range
of the electromagnetic spectrum from 380-750 nm. Ultraviolet light is just below this visible range of
the spectrum and is divided into three types: UVA, UVB, and UVC. Known as the “tanning spectrum”,
UVA wavelengths are the longest. This energy causes increased melanin production by melanocytes found
just below the epidermis of the skin. Although still used in tanning salons, UVA radiation is now known to
penetrate the skin causing the skin to age 40 times faster than normal. As well, UVA light is associated with
cataract formation and with malignant melanomas, the most deadly form of skin cancer. UVB radiation, the
“burning spectrum”, is more energetic and is associated with sunburn and the most common form of cancer,
basal cell carcinoma. UVC radiation comprises cosmic rays which rarely reach the surface of Earth.
UV light is a powerful mutagen. It is now well established that the damage done to DNA due to UV light
is the induction of pyrimidine dimers (e.g. thymine dimers). This occurs when two adjacent pyrimidine
residues along the DNA backbone become “fused” to each other rendering them unable to hydrogen bond
with the complementary base on the opposite DNA strand.
Other changes to DNA structure which can occur as a result of exposure to UV light are strand breakages,
duplications, deletions, inversions and translocations. Any one of these alterations in DNA, if not repaired,
will ultimately disrupt DNA replication and protein synthesis.

41
The Role of Sunscreen
The term sunscreen refers to anything that blocks UV radiation. The first sunscreen lotions contained the
chemical para-amino-benzoic acid (PABA), which absorbs and blocks UVB radiation. Many people have
developed sensitivities to PABA and it is not widely used anymore. Current sunscreens are combinations
of chemicals that block a range of UV wavelengths. These chemicals include PABA-esters and cinnamates
which block UVB, as well as Parsol 1789 and benzophenones which block UVA.
SPF (sun protection factor) is a means of rating the effectiveness of a sunscreen based on numerical
classification of skin sensitivities to sun exposure (i.e. how effectively the cream blocks UVB radiation).
Skin types range from 1 (white skin/freckles) to 6 (brown/black skin). Redness occurs in 10-20 minutes in
type 1 and 40-75 minutes in Type 6 depending on the latitude where exposure occurs. By multiplying the
SPF by the amount of time it takes to cause your skin to burn, you can calculate how much protection is
offered by the sunscreen. For instance, if your skin burns in 15 minutes, and you apply a sunscreen with an
SPF of 8, it will take 120 minutes for your skin to start to burn (8 x 15 minutes).
To illustrate the effect of UV light on DNA, a model system using a T4 bacteriophage and its host bacterium
E. coli C will be used. The UV source will be a germicidal tube with a wavelength of 264 nm. On exposure
of phage to UV light, the types of DNA damage described earlier occur. This damage will ultimately affect
the phage’s ability to propagate successfully within the host cell and cause cell lysis. Therefore, UV damage
can be indirectly visualized by observing the number of plaques produced from UV exposed phage and
comparing this number with plaques produced by phage which have not been exposed to UV light.
In this exercise, the effectiveness of sunscreen with various degrees of protection factors will be tested.
However, instead of risking damage to human skin, the DNA of T4 phage will be exposed to the UV light.
So, relax, think about a hot day at the beach and get ready to “catch a few rays”.

The Plaque Assay


To determine phage titer (concentration) throughout this experiment, samples will be taken at intervals after
infection and a plaque assay will be performed. When a population of host bacteria are grown as a lawn on
an agar plate, individual cells infected with a virus will lyse, exposing adjacent cells to virions. Over time,
this will create an area of clearing on the agar plate called a plaque. It can be assumed that this area was
cleared of host cells by progeny of one original virion and represents a plaque-forming unit (PFU). By
counting the number of plaques on an agar plate, you are counting the number of virions (or infected host
cells) present in the culture at the time the plate was made. Viral titer has the units PFU/ml.

Note: This exercise has been adapted from a University of Lethbridge Biology Laboratory Manual with many
thanks to Ms. Laurie Pacarynuk.

42
Procedure: (work in pairs)
1. Prepare a cardboard template by spreading a piece of plastic wrap across the circular opening in the
cardboard frame and taping the wrap to the cardboard, pulling the wrap tight as you do so.
2. Your TA will assign you one sample of sunscreen with an unknown SPF.
* Controls will be prepared for you. For the first control, no sunscreen will be applied to the plastic wrap. The
phage will be UV treated and samples assayed in an identical manner to the phage tested with the lotions.
For the second control, the phage will not be treated with sunscreen or UV but samples will be assayed in
the same manner as the procedure indicates.
3. Weigh 0.2 g of the sunscreen assigned to you and rub the lotion over the plastic wrap to create a thin film
over the template. There should not be any visible white streaks on the plastic wrap. Use a small piece of
tape to label your template with the name of your sample sunscreen.
4. Obtain 6 sterile 13 mm test tubes and 6 H-agar plates. Label the tubes and plates with your name and the
time intervals of 0, 5, 10, 15, 20, and 30 minutes.
5. Aliquot 100 µl of E. coli (108 CFU/ml) to each of the 6 labelled test tubes.
6. Aliquot 2.0 ml of T4 bacteriophage (103 PFU/ml) to an empty sterile petri dish.
*Caution: UV light is damaging to the naked eye and exposed skin. Always cover bare skin and view
through safety glasses which absorb harmful wavelengths*
7. When you are ready to start the assay:
a. Aseptically aliquot 100 μl of phage from the petri dish to the test tube labelled “0”.
b. Place the petri dish in the UV hood (without the lid) as soon as you have removed your time 0 sample.
Place your cardboard template over the petri dish.
c. Quickly vortex the phage/E. coli suspension and incubate for 15 minutes at room temperature.
For the best results, keep your 15 minute incubations consistent! You may want to keep track of the time
you added the phage to the E. coli and the time when you need to add the overlay (terminate incubation).
8. Aliquot 100 μl of phage from your petri dish to the appropriately labelled tube of E. coli, vortex and
incubate for 15 minutes at each sample time (5, 10, 15, 20 and 30 minutes).
9. Upon completion of the 15 minute incubation for each tube:
a. Quickly remove a tube of soft overlay agar from the 55°C water bath and pour into the appropriately
labelled test tube containing the phage/E. coli suspension.
b. Mix well by rotating the tube between the palms of your hands, immediately pour over an H-agar plate,
and tip the plate to spread the overlay agar over the surface. Allow to harden.
10. Place the 6 plates in the appropriate box on the side bench to be incubated for 24 hours at 37°C. Remove
the plastic wrap and all tape from your template and discard the plastic wrap. Do not discard the template
(replace on the side bench).
11. In the open lab, count the plaques on each plate as demonstrated by your TA. Record your results in the
appropriate column in the table on the class record sheet posted in the open lab. Results must be recorded
by noon on March 23/26.
Submit your plates to the correct box on the side bench
12. The class results (and the controls) will be posted on D2L. The Exercise 8 submission is due, with your
Unknown Journal, on March 27/29.

43
B. Transposon Mutagenesis
1. Controls have been prepared for you.
Donor cells (E. coli S17-1) were plated alone on a TY + Sm + Gn plate. Record the number of cells that
grew on this plate on the Exercise 7 submission sheet (Question 1).
Recipient cells (wild-type Rhizobium VF39) were plated alone on a TY + Sm + Gn plate. Record the number
of cells that grew on this plate on the Exercise 7 submission sheet (Question 2).
Recipient cells (wild-type Rhizobium VF39) were also plated alone on a TY + Sm plate. Record the number
of cells that grew on this plate on the Exercise 7 submission sheet (Question 3).
2. Collect the three TY + Sm + Gn plates you prepared in Lab 7.
Count the number of streptomycin- and gentamicin-resistant colonies observed on the three plates. Record
the average number of transposed Rhizobium cells that cam from the nitrocellulose filters on the Exercise 7
submission sheet (Question 4).
3. To determine the frequency of transposition, we need to know the number of Rhizobium cells that underwent
both conjugation and transposition from the original culture.
From step 2 above, we know the average number of cells that successfully completed both events.
Determine the number of transposed cells in the 200 μl suspension (step 2 of the procedure) and record the
answer on the Exercise 7 submission sheet (Question 5).
4. The Rhizobium cells in the 200 μl suspension were from a pellet of cells that, before centrifugation, were
suspended in 2 ml of broth (we can ignore the 1 ml of broth that contained the E. coli).
Determine the number of transposed cells that would have come from 1 ml of the original Rhizobium culture
(step 1 of the procedure) and record the answer on the Exercise 7 submission sheet (Question 6).
5. Determine the frequency of transposition. This is defined as the number of transposon mutants per ml of
original culture divided by the total number of viable Rhizobium in the original culture. Record the answer
on the Exercise 7 submission sheet (Question 7).

The Exercise 7 submission sheet is due at the end of your scheduled lab (March 20/22).

Submit your plates to the correct box on the side bench

44
EXERCISE 7 SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

SCORE: __________

A. Plasmid Vector
1. Note that part of the grade for this submission is from the assessment of your results (plates). (2)
2. How many E. coli S17-1 cells grew on the TY + Sm + Gn plate? Explain this result. (2)

3. How many wild-type Rhizobium VF39 grew on the TY + Sm + Gn plate? Explain this result. (2)

4. How many wild-type Rhizobium VF39 grew on the TY + Sm plate? Explain this result. (2)

5. What was the average number of transposed Rhizobium on the nitrocellulose filters? (1)
6. What would be the number of transposed Rhizobium cells in the 200 μl suspension? Show your calculations
for full marks. (2)

7. What would be the number of transposed Rhizobium cells that would have come from 1 ml of the original
Rhizobium VF39 culture? Show your calculations for full marks. (2)

8. What was the frequency of transposition? What does this number tell you? (2)
EXERCISE 8 SUBMISSION NAME: _____________________________

LAB SECTION: ______________________

SCORE: __________

The Effect of UV Radiation on DNA Replication


1. Note that part of the grade for this submission is from the assessment of your results (plates). (2)
2. Record the class results (posted on D2L) in the following table. (3)

Number of Plaques
No sunscreen
No sunscreen
A B C D No UV
(Control 1)
(Control 2)

0 min

5 min

10 min

15 min

20 min

30 min

3. Prepare one figure (Page 50) showing the effect that length of exposure to UV radiation has on the titer of
T4 for each of the sunscreens tested. Label the figure with as much information as you can determine from
your results (i.e. assign an SPF value to each sunscreen). Attach the figure to your submission. (10)
4. Explain why a germicidal tube of the 264 nm is used in this experiment. (2)
5. Explain the results of this experiment. Make sure you refer to the data, controls and the figure in your
discussion. (5)
Lab Exam 2
You will write Lab Exam 2 in your scheduled section on April 3/5. The exam will cover material presented
throughout the term and will be a mix of factual recall, practical and application questions. The exam will be 2
hours in length and consist of short answer/practical questions. You may use a pen or pencil, ruler and a non-
programmable calculator. You may not bring personal electronic devices (PDAs, MP3s, cell phones, etc.)

What should you study?


Terminology - know how to use the proper terms at the proper times. I.e. a bacterial cell is cocci, not
circular
Procedures and theory - know how to do a Gram stain, a streak plate, etc. and how to perform an
experiment to find information about an organism based on test results.
Results and calculations - know how to interpret any of the tests we have done, how to determine phenol
coefficients, frequency of transposition, etc.
Conclusions and study questions - know how to draw conclusions from results. Study the questions in the
lab manual and your submissions.
Experimental design - understand how/why an experimental procedure works and be able to design an
experiment to answer a question
Aseptic technique - you should follow proper safety guidelines and know how to maintain a safe, aseptic
work environment

What should you NOT study?


Quantities and lists - you will not be asked how many grams of agar need to be added to 1000 ml water
or list the ingredients of DOC agar, or to reproduce the specific procedure for transposing E. coli with
Rhizobium, etc.
Specific results - you will not be asked to recall the specific results of the endospore stain for unknown a,
etc.
Unknowns - you will not be asked any specific questions about your unknowns
Pathways - you will not be asked to illustrate the dissimilative nitrate reduction pathway of E. coli, etc.

45
46
Appendices
Appendix 1:
Laboratory Exercises
For the laboratory component of CMMB 343, no formal lab reports will be submitted for grading. Instead, you
will be required to complete:
1. Exercise submission sheets, which are located at the end of each exercise and handed in for grading. On the
submission sheets, the results of the experiments will be recorded and questions answered and/or discussion
included.
2. Online (D2L) quizzes to be completed between labs.
3. A journal (format to be discussed with your TA) detailing the information you collect about an unknown
organism that you will identify.
You may complete your submissions in pen or pencil, but exercises completed in pencil cannot be submitted for
regrading.
A list of the literature you have cited in answering the questions or discussing your results does not need to be
included (except for the journal - see page 32). Generally, the resources required to answer the questions will be
found within your textbook - additional references are available in the library.
When a list of references is required, you must be follow CSE citation rules. The references are alphabetized
according to the last name of the first author of each work, followed by the date of publication. The title of the
paper and the journal, the volume of the journal, and the pages cited, follow in that order. If a book is being
referenced, the edition, publisher and place of publication is included. In-text citations will not be necessary,
unless specifically called for. The use of general interest websites, wikipedia, and dictionaries is not acceptable,
if you start with these sites, ensure you go to the original source that informed these sites.
Example:
Brock, TD and Madigan, MT. 1991. Biology of Microorganisms, 6th edition. Prentice Hall Inc., New
Jersey. Pages 87-99.
Lechevalier, H 1986. Nocardioforms. In Bergey’s Manual of Systematic Bacteriology. Vol. 2. Edited by
P.H. Sneath, N.S. Mair, M.E. Sharpe, and J.G. Holt. Williams and Wilkens Co., Baltimore, MD. p. 1458-
1484.
Morrin, M and Ward OP. 1989. Investigation of cell wall composition of different mycelial forms of
Rhizopus arrhizus. Mycology Research. 93: 524-528.

Feel encouraged to work on the results, questions and discussions with colleagues in the course, but remember
that writing the final version of the lab submission is an individual effort. Using or copying another student’s
lab submission and handing it in as your own is plagiarism. If this situation occurs the students will be
referred to the Department Head for disciplinary action.

It is suggested that you keep a lab note book for notes and preliminary results. Remember, two lab exams will
be written during the term (see the schedule on page v).

For the majority of the exercises, it will be necessary to return to the lab the day after your scheduled lab
to make observations of your results. In these cases the technician will remove the plates and tubes from the
incubator and place them in the lab. Discard, in the appropriate place, all your plates and tubes after you have
made your observations, unless directed otherwise.

49
Figures
Figures must be completed on appropriate paper
• Computer-generated figures are acceptable, as long as they meet the format guidelines
that follow
• Graph paper is always acceptable

Figures must show what experiment was performed and the results obtained
• Figures are self-contained, which means someone looking only at the figure should
understand the experiment without seeing the procedure or any other results
• Figures include all related treatments and controls

Figures must have an appropriate caption


• Captions are placed below the graph and are labelled (“Figure 1”, etc.)
• Captions must indicate the variables involved, the names of all organisms, chemicals or
molecules used, and all appropriate statistical information (sample size, etc.)

Figures must have properly formatted and labelled axes


• Independent variable is plotted on the x-axis; dependent variable is plotted on the y-axis
• Axes start at “0”, and scale must be continuous and appropriate (arithmetic, logarithmic,
etc.), such that there isn’t a lot of empty space. You may use breaks if appropriate (i.e.
they do not affect the trend)
• Axes are labelled appropriately and units are clearly indicated (if the variable has units)

Figures displaying multiple treatments must clearly delineate each treatment


• Each line of best fit must use a distinguishable colour and/or style (solid, dashed, etc.)
• A legend must demarcate each line

Figures requiring labels must be clear and neat


• Labels appear close to the area being identified
• A bar clearly indicates the area being identified

50
Appendix 2:
Microscopy
The care and use of the brightfield microscope:
This procedure applies to the Olympus microscope (model CX31) that will be used in the laboratory:
A. Handling the microscope
1. Microscopes are calibrated instruments - handle with care and avoid sudden or severe impact.
2. Carry the microscope upright with one hand under the base and the other hand holding the recessed
handle on the rear of the arm. Never hold by the stage, nosepiece, or eyepiece.
3. Always lift the microscope to move it. Never slide the microscope across a work surface.
4. Position the microscope so that you can sit comfortably at your stool when observing a specimen.
B. Preparation before use
1. Remove the dust cover and place it in the cupboard (not on the bench) until you are finished.
2. Ensure the main switch is set to “O” (off) and the light intensity control knob is set to “1”.
3. Plug the power cord into the nearest electrical outlet. Keep the excess cord neat and behind the
microscope to prevent pulls and tangles.
4. Ensure:
a. the 4x (red) objective is engaged
b. the stage is in its lowest position (rotate the course adjustment knob toward you)
c. the condenser lens is in the highest position (rotate the condenser height adjustment knob)
d. the aperature iris diaphragm is closed (slide the adjustment lever to the right as far as it will go)
e. the field iris diaphragm is open (turn the adjustment ring clockwise as far as it will go)
C. Placing the specimen
1. Always place the specimen with great care to avoid damaging the microscope or breaking the glass slide
2. Open the curved finger on the specimen holder and place the slide on the stage from the front
3. Gently release the spring-loaded curved finger until it contacts the slide.
4. To adjust the position of the specimen, turn the upper (Y-axis) and lower (X-axis) stage control
knobs. Do not adjust the stage or specimen holder directly. The specimen should be centered over the
condenser lens.
D. Observing the specimen
1. Set the main switch to “|” (on) and adjust the light intensity knob to “4”. You may adjust the intensity
knob to make the illumination brighter or darker as needed.
2. Rotate the revolving nosepiece to engage the 10x (yellow) objective.
3. While looking through the eyepieces, adjust the interpupillar distance until the left and right fields of
view coincide completely. Don’t pinch your fingers!
4. Focus on the specimen by rotating the coarse adjustment knob (rotate away from you). Rack the stage
up slowly until the specimen becomes visible. Watch to ensure the objective lens does not come into
contact with the slide. Improve the focus with the fine adjustment knob (rotate away from you) until the
image is fully resolved.
5. You may move the specimen by gently adjusting the X- and Y-axis knobs.
51
E. Adjusting the diopter
1. Looking through the right eyepiece with your right eye, bring the image into focus.
2. Looking through the left eyepiece with the left eye, turn the diopter adjustment ring to focus the image.
3. This will reduce eye strain as you manipulate many specimens.
F. Adjusting Magnification
1. The objective lenses are parfocal. Once the 10x objective is focused, the 40x or 100x objective can be
engaged and focused with only slight manipulation of the fine adjustment knob.
2. As you adjust magnification, you may need to adjust the light intensity, field iris diaphragm, and aperture
iris diaphragm to maximize focus and resolution.
G. Using the Immersion Objective
1. Focus on the specimen with the 10x objective engaged.
2. Rotate the revolving nosepiece to engage the 4x objective.
3. Carefully add 1 drop of immersion oil where the light from the condenser is shining through the slide.
4. Engage the 100x objective (directly from 4x) and adjust the focus using the fine adjustment knob.
5. When finished, CLEAN THE OIL FROM THE OBJECTIVE WITH LENS PAPER THAT HAS
BEEN MOISTENED WITH CLEANING SOLUTION. WIPE DRY WITH LENS PAPER.
*NEVER LEAVE OIL ON THE OBJECTIVES OR ON THE STAGE OF A MICROSCOPE*
H. Maintaining the microscope
1. Remove the slide and dispose properly!
2. Clean the objectives with cleaning solution, engage the 4x objective, lower the stage completely, set the
light intensity to “1”, turn the power switch to “O” (off), wrap the cord around the microscope, replace
dust cover and place in the correct cupboard.
3. Report any malfunction or accident immediately.

Interpupular distance adjustment

Diopter adjustment ring

Arm

with curved finger

height adjustment knob

lens
Field iris diaphragm adjustment ring

Base

(X- Y-axis knob)


52
Appendix 3:
Media and Methods Used For Isolating and Cultivating Bacteria
A. General preparation of media

Bacteria can rarely be identified only from a study of their cellular morphology and it is necessary
therefore to obtain pure cultures of these organisms growing in the laboratory. Bacteria display many
variations for the major nutritional requirements and so the utilizable sources of energy also vary.
However, media for the cultivation of bacteria must contain a) water, b) a source of energy, usually in
the form of a carbon compound, c) essential elements including N, P, and S, d) metallic elements such
as K, Mg, Ca, Fe, Mn, Cu, and Zn in varying proportions, and e) organic nutrients. These are known
as growth factors, compounds that an organism requires as a precursor of its organic cell material but
which it cannot synthesize from simpler carbon sources. They include: i) amino acids ii) purines and
pyrimidines and iii) vitamins.

In prepared media, chemicals of defined composition are to be preferred to those of undefined


composition. Since the nutritional patterns of many bacteria are not sufficiently well known, it is often
necessary to add substances of complex organic composition e.g. peptone. In addition, media are often
solidified with agar, a complex polysaccharide extracted from red algae. It is essential therefore, to
use the same source of these materials for any comparative experiments. Most chemicals should be
dissolved in distilled water. Tap water may contain toxic elements, particularly copper.

Most bacteria can only grow within a restricted pH range. The reaction of the medium must be adjusted
so that the final pH, after sterilization is between 6.8 and 7.2, unless a different reaction is needed for
some special purpose.

Buffers, which prevent large changes in pH, are often required to facilitate growth. This is particularly
true of media composed of simple compounds or in which acid-producing bacteria are cultivated.
Mixtures of sodium and potassium phosphates are often employed. In complex media, buffering is
provided by the peptides and amino acids.

To determine pH changes during growth, indicators may be included in the medium. Where critical
experiments are being performed it is often advisable to add the indicator to a sample tube only as a
control, and to a small quantity of the actual culture fluid at the end of the experiment. Otherwise, it is
possible that the indicator or the alcohol it is dissolved in might act as a substrate for bacterial growth.

B. Types of media used for cultivating bacteria

The nutritional classification of organisms is based on three parameters: the energy source, the
principal carbon source and the source of reducing power. With respect to energy source, phototrophs
are photosynthetic organisms that use light as their energy source and chemotrophs are organisms
that depend on a chemical energy source. Organisms able to use CO2 as a principal carbon source are
autotrophs. Heterotrophs depend on an organic carbon source. To designate the source of reducing
power, the term lithotroph or organotroph is applied. Lithotrophs use inorganic compounds as their
source of reducing power, and organotrophs use organic compounds as their source of reducing power.

53
To summarize:
source of
energy source carbon source reducing power

photoautotroph light CO2 inorganic oxidizable


(photolithotroph) substrate

photoheterotroph light
organic organic
(photoorganotroph)

chemoautotroph chemical oxidation of CO2 inorganic


(chemolithotroph)* reduced inorganic
compounds
e.g. NH3, NO2- and H2

chemoheterotroph chemical organic organic


(chemoorganotroph)

*All chemoautotrophs are chemolithotrophs, but not all lithotrophs are autotrophic. For example, the
methylotrophic bacteria can use organic carbon as their carbon source*

1. Media for autotrophic bacteria

Autotrophic bacteria are cultivated in solutions of mineral salts without organic additives (except in
those species requiring growth factors). The media will be variable depending on whether the organism
to be grown is a phototroph or a chemotroph.

2. Media for heterotrophic bacteria

Many different types of media have been perfected for the cultivation of heterotrophic microorganisms
depending on their metabolic processes and nutritional requirements. Those organisms with complex
requirements are said to be nutritionally exacting and include several of the pathogenic organisms and
certain soil species. They are usually grown on complex natural media while the less exacting types (and
the autotrophs) are grown on synthetic media of known composition.
Recipes for various types of media can be found in the DifcoTM & BBLTM Manual (http://www.bd.com/
ds/technicalCenter/documents.asp).

C.  The cultivation of anaerobic bacteria

Many species of bacteria are facultative aerobes, i.e. they can grow under aerobic or anaerobic
conditions, the latter ability being dependent upon the presence of some substance that can be utilized
as a hydrogen acceptor by the species concerned. Generally, facultative organisms prefer to grow
aerobically rather than anaerobically. Aerobically, a larger amount of ATP and a larger amount of all
mass is produced. Some bacteria are obligate aerobes, unable to use anything but oxygen as a final
electron acceptor. Aerotolerant anaerobic organisms are fermentative organisms that are able to grow in
the presence of oxygen. Others are obligate anaerobes which cannot use oxygen as an electron acceptor.
A few bacteria are somewhat intermediate, growing best in low oxygen tensions. These are called
microaerophilic bacteria.

54
Anaerobic forms occur in several families of bacteria such as Bacteroidaceae and Peptococcaceae.
Other anaerobic organisms include the Genus Clostridium, an endospore-forming organism, certain
autotrophic bacteria e.g. the purple sulphur bacteria, and some other soil or water bacteria e.g.
Desulfovibrio.

During growth aerobic bacteria tend to utilize all available oxygen and so reduce the medium. Thus, in
mixed cultures the oxidation-reduction potential (Eh) of the medium may become low enough to allow
anaerobes to develop.

1. Methods involving removal of oxygen:

a. Stab and shake cultures

Many anaerobes can grow in deep stab or shake cultures in glucose agar. The method is particularly
useful for microaerophilic species. A seal of liquid paraffin or vaseline is sometimes advocated to help
maintain anaerobic conditions, e.g. Hugh and Leifson fermentation test. This method is not entirely
reliable and some evidence has been collected showing that oxygen diffuses through paraffin quite
quickly.

b. Anaerobe jar culture

If surface cultures are required, the plates or slopes should be placed in an anaerobe jar e.g. the McIntosh
and Fildes jar. This is a heavy metal or glass jar with a lid that can be clamped firmly down to form an
effective seal. Inlet and outlet valves allow the air in the jar to be replaced with hydrogen, after slow
and careful evacuation of most of the oxygen. When this has been done, the valves are closed and a
current is passed through a small heating coil packed with palladinized asbestos and enclosed in a wire
gauze cage, attached to the inside of the lid. The gently heated palladium catalyses the combination of
any remaining traces of oxygen with the hydrogen. A tube of methylene blue agar fitted to the jar acts
as an indicator. Recently, many adaptations of this jar have been introduced. The one used presently is
a commercially prepared envelope by Merck. The envelope called an Anaerocult A is placed in the jar.
The envelope incorporates a system which allows for the chemical binding of oxygen which creates an
oxygen-free milieu and a CO2 atmosphere simply by adding 35 ml of water to the contents. To assure
that anaerobic conditions are provided, a disposable anaerobic indicator, methylene blue, is included in
the jar.

c. Thioglycollate medium

This is prepared by adding 0.1% (up to 0.4%) thioglycollic acid to nutrient broth before adjusting
the pH. 1% glucose must also be added. Although the medium may be solidified with agar, it is more
usual to use a semi-solid medium, i.e. 0.5% agar. The increased viscosity of the medium prevents the
distribution of oxygen (dissolved at the exposed surface), by convection currents. Resazurin is added to
act as an indicator of reducing conditions. It is colourless when reduced (i.e. between Eh + 0.12 and 0.3
volts, which will allow most anaerobes to develop). A pink layer at the surface shows the depth to which
oxygen has diffused into the medium. Inocula should be introduced carefully by means of a fine pipette,
at the bottom of the tube.

55
D. Methods of isolating bacteria:

1. Isolation from contaminated cultures

Progressive dilution is the basis of isolation techniques. This can be applied to suspensions containing two
or more bacterial species as follows:

a. Poured plate method

Melt and cool to 45°C two tubes of an agar medium. Inoculate one tube with one loopful of the
suspension and mix well by rotating it between the hands. Flame the loop thoroughly and transfer one
loopful of the mixture from the first tube to the second. Mix again. Pour the contents of both tubes into
sterile Petri dishes and allow to set. Take care that the agar does not set before it is poured.

The second plate should show well separated colonies after incubation. Distinct colonies can then be
picked off and examined. If the suspension is very heavy initial dilution in sterile saline may have to be
made.

b. The streak plate method

This is a quicker, though less reliable method. Prepare a plate of solid medium. Dry the plates at
50°C for 30 minutes before use. Prepare a streak plate for single colony isolates using the method on
pages 62-63. After incubation, well separated colonies should be found along the streak marks. Before
deciding that a culture is pure by either method, colonies should be picked off, grown and then re-
separated, until all colonies are the same. Morphological variants can upset strict application of this
principle. Staining can be used as a check on the purity of the final isolations.

2. Isolation from natural sources

The objective of many viable counting techniques is to estimate the number of all live organisms in a given
sample of material. To do this, a medium satisfying the nutritional requirements of as many of the bacteria
in the sample as possible, is required. Thus, in soil bacteriology, soil extract media have been developed as
most resembling an ideal non-selective medium. Other types of viable counts and all isolation techniques
embody the reverse principles. Here, it is necessary to pick out and encourage one type of organism and to
prevent or depress the development of other types. In natural habitats, the organisms which one is isolating
may be present only in small numbers. The first step in such cases is thus to obtain an enrichment culture
by one or more of three methods:

a) by using selective media


b) by using selective conditions of incubation
c) by selective pretreatment of the material

Several generations of sub-cultures on liquid, or solid media may be necessary, but the final step will consist
of plating out the organisms by one of the methods described above.

56
a. The use of selective media
A completely selective medium, allowing the growth of only a single species, is not attainable in
practice, but media can be obtained which will discourage all but the required species.

Some selective media are also differential. Certain organisms, when grown on them, exhibit distinctive
biochemical or morphological characters which enable them to be recognized easily. This may be very
important from the medical point of view, where the range of species concerned is small. Where a
large range of species is involved, e.g. in soil, differential media cannot be used to separate taxonomic
groups, but are useful to distinguish groups concerned in some biochemical process, e.g. cellulose
decomposition.

Selectivity may be achieved in three ways:


i) Addition of substances to a medium
A classic example of selective media is MacConkey’s bile-salt lactose broth. Bile salts discourage
the growth of most bacteria except those of intestinal origin. The presence of 1% lactose and a pH
indicator make the medium differential as well. Coliform organisms ferment the lactose to produce
acid and gas from the medium indicating fecal pollution in water and milk, etc. The same medium
solidified with agar is used to isolate the pathogenic Salmonella/Shigella group of bacteria. While the
coliforms produce acid and therefore a color change, the Salmonella and Shigella group are not able
to ferment lactose and no color change occurs.
Other examples of substances added to media to make them selective are:

potassium selenite (0.4%) inhibits coliforms

sodium azide inhibits the cytochrome oxidase enzyme in the


electron transport chain where the transport is
coupled with ATP synthesis
0.025% used to isolate fecal streptococci

potassium tellurite (0.002%) inhibits coliforms and pseudomonads used in blood


agar to  isolate Corynebacterium diphtheriae

crystal violet a triphenylmethane dye that exerts its inhibitory


effect by interfering with cellular oxidase processes
inhibits Gram-positive bacteria at very low
concentrations (0.01%)
Used for isolating Gram-negatives since they are
affected by much higher concentrations.

rose bengal (0.006%) discourages all bacterial growth

actidione inhibits yeasts and fungi, 0.0005% used to


enumerate bacteria in mixed flora

nystatin inhibits yeasts and fungi

antibiotics e.g. penicillin (1000 units/ml)


57
ii) Alteration of the pH of a medium
Organisms tolerating a high pH, e.g. Vibrio and Pseudomonas sp., can be encouraged by adjusting the
reaction to pH 9-10, e.g. Dieudonne’s blood-alkali agar for isolating Vibrio cholerae. Acid tolerant
bacteria can be more readily isolated if the pH is reduced to 5 or lower. Thus, Lactobacilli are isolated
on media containing 0.5% acetic acid. After isolation, the organisms should be grown on normal media.
iii) Omission of substance
Media without organic substances are used to isolate and grow autotrophic microorganisms. Several
transfers are usually needed. Although the first cultures show much growth, some organic matter is
inevitably introduced with the inoculum causing contamination. Mannitol phosphate media used for
nitrogen fixing organisms is an example of this method of selection.
b. The use of selective incubation
Selective incubation of cultures can select quite different parts of the microbial population.
i) Temperature
Microorganisms have more or less definite temperature requirements, with maximum temperatures
above which they fail to grow and minimum temperatures below which they fail to grow. These limits
vary depending on cultural conditions, but are sufficiently precise to be used in isolation procedures.
The temperature range of a given microorganism is relatively easy to establish. One culture of a given
species, uniformly inoculated, should be incubated over a range of temperatures, e.g. 50°C, 45°C,
37°C, 25°C, 18°C, 10°C, 2°C. Tubes incubated at high temperatures must be enclosed in polythene
bags to prevent excessive desiccation. The amount of growth occurring after one day, two days or two
weeks incubation should be noted visually and recorded as a scale from zero to three i.e. 0, 1, 2, or 3.
The method may be made more precise by employing stringent inoculation procedures and using an
spectrophotometer to assess the turbidity of a suspension.
The results obtained from this experiment will vary, thermophiles growing at 45°C-50°C and above,
mesophiles rarely growing above 40°C, body organisms having an optimum at 37°C, soil organisms
growing at about 10°C-25°C. Organisms growing at low temperatures are usually termed psychrophiles.
These temperature distinctions are made for convenience and the precise numbers should not be taken as
absolute.
Thermophiles occur widely in soil and are important in the production of silage and compost, and in
the canning and sugar-refining industries. To isolate such organisms from soil, a moist sample should
be incubated at 45°C-60°C or higher for three days. A suspension of this material should then be made
in sterile Ringer’s solution and heated in a water bath at 100°C for twenty minutes. Add l ml of this
suspension to 15 ml of nutrient agar melted and cooled to 45°C. Pour into a sterile petri dish. Incubate
at 45°C-60°C with the dishes in a polythene bag to prevent drying.
Thermophiles are thermoduric. However, a similar sample should be incubated at 25°C to demonstrate
that some thermoduric organisms are not thermophiles. The distinction between mesophiles and
thermophiles may be imprecise.
Extreme thermophiles have been found in hot springs such temperatures at the boiling point, in steam
vents (fumaroles) where temperatures may reach 150°C - 500°C and in geothermal vents at the bottom
of the ocean which have temperatures of 350°C and higher.
ii) Aeration
Bacteria have a definite relationship to aeration conditions. Some will only grow with a supply of
oxygen, the obligate aerobes. Others will only grow in the absence of oxygen, the obligate anaerobes.
The great bulk of organisms will grow in either state to a certain degree. These are known as facultative.

58
Appendix 4:
Review of microbiological techniques
I. Preparation of bacterial smears for staining

To make a good film for staining, obtain a clean grease-free slide. Draw a circle on it approximately 2 cm in
diameter with a grease pencil. Turn the slide over.
If the inoculum is from solid (agar) media, add a drop of water within the circle. With a sterile loop or
stick, remove a very small quantity of surface growth from the agar, mix this with the water to make a
homogenous suspension of the cells, and allow to air dry.
If the slide is prepared from a broth culture, use a sterile loop or swab to place a drop of the broth culture
onto the slide (do not add water). A loop must be flamed again before replacing it in the holder; a swab can
be discarded in the autoclave garbage.
When dry, the film should be only faintly visible; a thick opaque film where the bacteria are piled on top of
one another is use­less.
During the staining process, cells can be washed away, leaving very few cells for observation. To prevent this
from happening, the cells must be fixed to the slide using one of the following methods:
Heat Fixing: pass the end of the slide with the smear through a Bunsen flame several times. Do not hold the
slide in the flame: if the slide is over-heated, the bacteria will become distorted, making it hard to observe
under the microscope.
Methanol Fixing: add a drop of methanol to the thoroughly dried smear. Allow the methanol to air dry.

II. Staining procedures:


A. Acid-fast stain (Kinyoun method)
1. Prepare a bacterial smear as outlined in I above.
2. Flood the smear with Kinyoun carbolfuchsin for 10 minutes.
3. Rinse the slide with water to remove excess stain.
4. Decolourize with acid-alcohol until the run-off is clear. Rinse with water.
5. Counterstain with Brilliant Green for 1 minute. Rinse with water, blot dry and observe.

ACID FAST ORGANISMS ARE RED; OTHERS ARE BLUE/GREEN

B. Capsule stain (does not require a bacterial smear)


1. Aseptically place a loop of culture in a drop of Congo Red on one end of a glass slide.
2. Using the end of a second slide, draw the Congo Red to the opposite end of the slide. The Congo Red
should form a thin translucent layer on the slide. Dispose of the second slide appropriately.
3. Do not heat fix, but allow to air dry.
4. Flood the slide with Maneval’s Stain for 1 minute. Rinse gently. Air dry and observe.

CELLS ARE RED, CAPSULES ARE CLEAR, AND THE BACKGROUND IS BLUE.
DIFFERENTIATE BETWEEN BACTERIAL CELLS AND ARTIFACTS ON THE SLIDE. WHEN THE
EDGE OF THE CAPSULE IS IN FOCUS, THE RED CELLS SHOULD BE VISIBLE.
59
C. Endospore stain
1. Generally, the organism is grown on a sporulation agar plate for 48 hours before doing an endospore
stain.
2. Prepare a bacterial smear as outlined in I above.
3. Place the slide on a heating rack in the fume hood. Cover the smear with a small piece of paper towel
and flood the smear with Malachite Green. Steam gently for 5 minutes, keeping the paper moist with
stain.
4. Remove the paper towel with forceps and rinse the slide with water to remove excess stain.
5. Counterstain with Safranin for 1 minute. Rinse with water, blot dry and observe.

The endospores will stain green and the rest of the cell pink

D. Gram stain
1. Prepare a bacterial smear as outlined in I above.
2. Flood the smear with Crystal Violet for 1 minute. Rinse with water. Remove excess water by tapping
slide gently on the staining rack.
3. Flood the smear with Lugol’s iodine for 1 minute. Rinse with water. Remove excess water by tapping
slide gently on the staining rack.
4. Decolourize with 95% ethanol. Drip alcohol down the slide for approximately 10 seconds and rinse with
water. Alcohol and water are alternated until the drippings are colourless. Remove the water after the last
rinse.
5. Counterstain with Safranin for 1 minute. Rinse with water, blot dry and observe.

Gram-positive organisms stain blue (purple) and Gram‑negative organisms stain pink (red).

III. Cellular morphology


Cellular morphology is generally obtained from a Gram-stained slide. The cellular morphology includes:

size as determined by calibration of the microscope


diameter in mm (cocci)
length in mm (bacilli)
shape cocci (spherical), bacilli (rod)
arrangement of cells random, pairs, tetrads, chains, clusters
Gram reaction positive, negative

A diagram drawn to scale usually accompanies the cellular morphology

60
A. Diagrams of Cells:
1. Make all diagrams with pencil. Diagrams are to be prepared on blank white paper.
2. Draw what you observe through the microscope rather than your interpretation or what you expect to
see.
3. Guidelines for scientific diagrams:
a) Diagrams should be large. Usually only one representative cell needs to be drawn in detail, other
cells are drawn in rough to represent the surrounding medium.
b) Do not shade.
c) Do not write notes on the diagrams.
d) Label identifiable structures. A ruler is used to draw a straight line horizontally from the structures to
the label (usually the right side).
4. All diagrams must be labelled, below the diagram, with:
a) Title of the diagram.
b) Total magnification of the specimen. Multiply the power of the oculars by the power of the objective
used. For example, under the 10x objective, the total magnification is 100x since the oculars also
magnify 10x.
c) All diagrams must be drawn to scale. To demonstrate the magnification of the drawing, divide the
size of cell as drawn by the size of cell as measured on the slide. Remember scale!
5. The diagram should be drawn in proportion so that one measured dimension is sufficient when drawing
to scale. Use a size bar to indicate the actual size of the cell. See Figure Ap4.1 below:
Your microscope was calibrated so that under the 100x objective, 1 ocular division = 1.0 micrometer,
and the rod shown measures 5 ocular divisions in length, the length of the rod is therefore 1.0
micrometers x 5 = 5 micrometers. The length of the rod that has been drawn is 73 mm, so:

5 µm

Cell Wall

Figure Ap4.1: Bacilli bacterial cell


Total Magnification: 1000x
Magnification of drawing: 73 000 µm / 5 µm = 14 600x

61
IV. Colony morphology
The following characteristics are those most commonly used to describe colony morphology. A single colony is
observed when describing colony morphology.
1. Shape:

2. Surface:
smooth rough mucoid
moist dry powdery
3. Elevation:

4. Size:
in mm, as measured with a ruler
5. Pigment:
cream, white or beige coloured organisms are usually considered to be non-pigmented.
Pigments may be purple, red, pink, yellow, brown, blue, grey, etc. Water soluble
pigments diffuse into the media.
6. Opacity:
Transparent (can see through) or opaque.

V. Streak plate:

If a colony morphology is to be obtained from a plate culture, the agar plate must be streaked in such a manner
so as to obtain single colonies of the bacteria. See Figure 5.12 in your text and Figure Ap4.2 on page 63.
1. A loop/swab of liquid culture, or a small amount (1 colony) of bacterial growth from a plate culture, is
transferred aseptically to a sterile plate and spread across the surface of one quadrant of the plate in a
zigzag pattern.
2. The loop is flamed to re-sterilize the surface and cooled by touching the loop to the surface of the agar
near one edge of the plate. This will cause the agar to sputter, so make sure your plate is close to your
bunsen flame to reduce the spread of aerosols. To avoid aerosols, sterile sticks can be used and discarded
between each set of streaks. This will be demonstrated by your TA.
3. NOTE: The loop/stick is not to be reintroduced into the broth or plate culture used for the first
streak. Make a second set of zigzags on a second quadrant of the plate, being sure to pass through part
of the first set of streaks to pick up inoculum from the first quadrant. This will dilute the number of cells
spread in the second streak compared to the first streak.
4. Flame the loop, cool in the agar, and make a third set of zigzag streaks. Repeat for a 4th set. After
incubation, fewer and fewer bacterial cells should appear in successive streaks resulting in isolated
colonies in the area of the 4th set of streaks.
62
Figure Ap4.2: How to do a steak plate for single colony isolation
VI. Labeling of Cultures:
Individual tube and plate cultures should be labelled legibly with the date, your initials, lab section, and
specimen information. The plates are labelled along the outside edge on the bottom only (agar side) since the
lids may get mixed up. Plates should be incubated agar side up so any condensation will fall on the lid. Tubes
are labelled on the test tube, NOT on the lid.
VII. Incubation of Cultures:
Cultures should be incubated at the temperature most favorable to growth or the specific activity being studied.
Human pathogens and commensal species grow best at body temperature, i.e. 37°C. Soil organisms and plant
pathogens are normally incubated at 20-25°C. The optimum temperature is that temperature at which the growth
rate is maximal for a particular organism. Cultures will usually grow at their optimum temperature in 24-
48 hours. Some specific tests may take 48-96 hours of incubation to reach maximum growth.
VIII. Pipetting using sterile technique:
Whenever pipetting is done in the microbiology laboratory, strict aseptic technique must be used. In CMMB
343, glass (or plastic disposable) pipettes will be used to pipette large volumes and micropipettes will be used to
dispense smaller volumes.
Pipetting with a sterile glass (or disposable plastic) pipette and a propipette
The can of pipettes has been sterilized and all the pipettes must be kept sterile. Take the can of pipettes to the
work bench. Remove the top of the can, flame the opening and remove one pipette, making sure that only
the top of the pipette is touched. Reflame the opening and replace the top. Carefully place the propipette on
the pipette without contaminating the lower 3/4 of the pipette. When the pipetting is completed, remove the
propipette carefully and place the used pipette in the pipette bucket.

63
Pipetting using a micropipette
These micropipettes are very expensive and very delicate. Please handle them carefully and do not exceed
the maximum or minimum volumes. When not in use, the micropipettes are kept in the micropipette stands.
Micropipettes consist of a barrel (that houses a spring-loaded piston) attached to a button that is depressed
with your thumb to draw up and expel liquid. Micropipettes must be used in conjunction with plastic tips
that have been sterilized in special boxes. A tip must be placed securely on the barrel of the pipette so that
there is an air-tight seal. If it is too loose, the tip will leak and not transfer an accurate volume. Since the tip
is the only sterile part of the instrument, only allow the tip to come into contact with the bacterial culture,
not the barrel. These tips are disposable and a clean tip should be used every time a solution is measured.
Discard used tips into the disposal bags on your bench.
1. Eppendorf micropipette

Figure Ap4.3: Eppendorf micropipette

a) The control button on top of the pipette has three positions:

At Rest - The position of the button when it is not being used

Stop 1 - This measures the volume selected in the window. Depressing the button from the
“At Rest” position to “Stop 1” measures the volume selected in the window.

Stop 2 - This blows out the last drops when expelling liquid from the tip.

b) To set the volume required:


i) Turn the setting ring. Note the “-” and “+” directions and turn accordingly. For the blue-coded
micropipettes, never turn below 100 μl (shown as 0100 from top to bottom) or above 1000 μl
(shown as 1000 from top to bottom). The yellow-coded micropipettes have a range between 10 μl
and 100 μl.
64
* Remember to use aseptic technique *
c) To measure a required volume of liquid:
i) Securely attach a micropipette tip from a sterile tip box. Keep the lid of the box closed as much as
possible to maintain sterility
ii) Press the control button down to the “First Stop” and hold.
iii) Hold micropipette vertically and immerse tip approximately 3 mm into the liquid.
iv) Allow control button to glide back SLOWLY to the “At Rest” position. Never allow it to snap back.
v) Slide tip out of the liquid along the inside of the vessel to remove any remaining droplets. The
volume in the tip should now be the volume shown in the window.

d) To transfer this volume of liquid:


i) Place the tip in the receiving vessel, holding the tip at an angle against the inside of the vessel.
ii) Press the control button slowly down to the “First Stop” to dispense the liquid.
iii) Press the control button down to the “Second Stop” (blow-out) to empty the tip completely.
iv) Hold down the control button. Slide tip out along the inside of the vessel.
v) Let the control button glide back slowly to the “At Rest” position.
vi) To eject the tip after the liquid has been dispensed, hold the micropipette over the disposal bag and
depress the eject button. This will shoot the tip off the end of the micropepette into the disposal bag.
Be careful to aim correctly.

Figure Ap4.4: Technique of holding micropipette, test tube and cap

Figure 5.12 in your text shows a technique to aseptically retrieve a loop of broth culture from a tube. The
technique to use a micropipette to aliquot liquid between vessels is similar.

65
IX. Serial ten-fold dilutions
It is sometimes necessary to determine the concentration of the organism with which you are working. Working
concentrations are often very high, which makes counting difficult, so the stock solution must first be diluted
before plating and subsequent counting. A series of 10-fold dilutions is typically made, and a sample from
each dilution is then plated and incubated. It is assumed that each colony on the resulting plates originated
from a single organism from the dilution, so we call each colony a colony forming unit (CFU) and we
measure bacterial concentration as the number of CFUs per ml of original culture. The dilution that produces
a colony count between 30 and 300 CFU/ml is normally chosen to perform the calculation. It is not considered
statistically reliable to count from a plate containing more than 300 colonies and a plate with less than 30
colonies. Thus, only one set of plates will normally be counted if serial ten-fold dilutions are used. Figure Ap4.5
below shows how to perform serial 10-fold dilutions.
Stock
Culture
Solution

100 µl

Dilution
100 µl 100 µl 100 µl 100 µl 100 µl

900 µl
sterile
broth

1/10 1/100 1/1000 1/10 000 1/100 000 1/1 000 000
10-1 10-2 10-3 10-4 10-5 10-6

Figure Ap4.5: Serial dilutions (refer to Figure 5.15 in your text)


X. The dilution-plate method of viable counts
a) Serial ten-fold dilutions are prepared. 1000 μl of each dilution is mixed with 15 or 20 ml of an agar medium,
previously melted and cooled to 45°C and poured into separate sterile petri dishes and even distribution is
ensured. When set, the plates are incubated at the required temperature. The appropriate plate is counted and
the concentration is determined in CFU/ml.
b) Alternatively, 100 μl of each dilution is added to a separate agar plate and the dilution is spread with a
sterile glass spreader. When dry, the plates are incubated at the required temperature. The appropriate plate
is counted and the concentration is determined in CFU/ml.
For example: If you count 185 colonies on the 10-4 dilution plate (theoretically 1850 on the 10-3 and 18.5 on the
10-5 plates), you need to multiply by 10 (you plated 100 μl onto the plate) and multiply by 104 (the inverse of the
dilution) to determine the concentration of the stock culture solution: 1.85 x 107 CFU/ml.

66
XI. Biochemical tests
1. Oxidase test
Cytochrome c is a major enzyme involved in many metabolic electron transport systems and becomes
reduced when it oxidizes the cytochrome bc1 complex. The electrons are then passed to Complex IV, which,
in turn, reduces O2 to H2O. If present, cytochrome c will also oxidize the artificial reagent dimethyl-p-
phenylenediamine, which changes from colourless (reduced) to blue (oxidized).
2. Catalase test
Flavoprotein (FMN) is the first enzyme in many metabolic electron transport systems and becomes reduced
when it oxidizes NADH. FMN normally passes electrons onto other proteins within Complex I, but can reduce
oxygen directly. When this occurs, toxic oxygen radicals like hydrogen peroxide (H202) and superoxide radical
(O2-) are produced. The enzyme catalase functions by converting hydrogen peroxide to water and oxygen gas
(02). When hydrogen peroxide is added directly to cells that produce catalase, oxygen bubbles form instantly.
3. Hugh and Leifson (H&L) test
Utilization of a carbohydrate (glucose, lactose, etc.) results in the production of acidic end products or
intermediates. Excretion of these acidic compounds into the medium results in the pH indicator, bromthymol
blue, changing from green to yellow. This is considered a positive result for carbohydrate utilization.
The location of the colour change, from green to yellow is also important. If the organism is can only ferment
the carbohydrate, or both respire and ferment the carbohydrate, then the colour change will occur in both the
sealed (mineral oil) and unsealed media. If the organism can respire only, then the colour change will occur in
the unsealed media only. Also note that each tube will contain an oxygen gradient, with the aerobic zone near
the top of the tube and the anoxic zone near the bottom of the tube.
If the organism cannot utilize the carbohydrate, but can grow in the medium utilizing peptones, the
organism will produce alkaline end products. Excretion of these basic compounds into the medium results
in the bromthymol blue changing from green to blue at the top of the medium. This is a negative result for
carbohydrate utilization.
3. Phenol red test
Utilization of a carbohydrate (glucose, lactose, etc.) results in the production of acidic end products or
intermediates. Excretion of these acidic compounds into the medium results in the pH indicator, phenol red,
changing from red to yellow. This is considered a positive result for carbohydrate utilization.
When this medium is prepared as a broth, gas production can be detected by placing an inverted Durham vial in
the tube. If the organism produces gas as a result of fermentation, the medium turns yellow and a bubble forms
in the Durham vial. When the medium is prepared as a gel (with agar), gas production can be detected when
cracks appear in the agar.
4. Citrate test
Simmons citrate medium is a mineral medium with 0.3% sodium citrate as the sole carbon source. The indicator
bromthymol blue is incorporated into the medium. It is green at pH 6.8 and blue at pH 7.6. The organism is
streaked onto the surface of the slant and incubated. Organisms able to utilize the citrate grow on the surface of
the medium. Due to oxidative formation of sodium carbonate, the medium becomes alkaline and changes colour
from green to blue.

67
5. Coagulase test
The organism being tested is inoculated into a tube of 0.5 ml blood plasma which is prepared fresh daily. The
tube must be incubated for at least 6 - 8 hours and at longest 24 hours. A solid clot indicates a positive result.
The organism produces the enzyme coagulase which coagulates the blood plasma to form a clot.
6. H2S test
Hydrogen sulfide may be produced from sulphur containing amino acids, e.g. cysteine, present in peptone.
The organism is inoculated into a tube of peptone water and a piece of lead acetate paper is suspended into
the tube (not touching the liquid) by the cap. After overnight incubation, if the organism breaks down cysteine
and produces H2S, the lead acetate paper turns black due to the formation of lead sulfide. A negative result is
indicated by the lead acetate paper remaining white.
7. Indole test
Indole is produced by some bacteria upon degradation of the amino acid tryptophan. The organism is inoculated
into a tube of peptone water and incubated overnight. Several drops of Kovac’s reagent are added after
incubation. If the organism breaks down tryptophan to produce indole, the indole and the amyl alcohol present
in the Kovac’s reagent produce a cherry red colour at the interface of the medium and the Kovac’s reagent. A
negative result is indicated by a brown colour.
8. Litmus milk
Litmus milk is composed of skim milk with sufficient litmus to produce a lilac colour. There is a small amount
of glucose present in the milk and a larger amount of lactose. Casein is the main protein in milk and gives the
milk its opacity. Reactions occur due to the fermentation of the carbohydrates or utilization of the proteins. The
pH indicator, litmus, changes colour as a result of the reactions that occur. The organism is inoculated into the
medium and incubated for 2 - 5 days.
Litmus indicates changes in the pH of the media and also in the oxidation-reduction state of the media:
a) acid production litmus turns pink
indicates lactose (glucose) fermentation with acidic end products
b) alkaline reaction litmus turns purple/blue
partial casein digestion with alkaline end products (ammonia)
c) reduction litmus turns white
litmus reduction by a fermentation reductase enzyme (anaerobic)
d) coagulation the medium solidifies
acid reaction precipitates casein
gas production can create fissures in the clot (coagulated casein)
e) peptonization the medium loses its opacity (sometimes white, pink or brown on top)
Casein is digested leaving whey (straw colour)
9. Mannitol yeast extract congo red agar
The organism is streaked onto a plate of mannitol yeast extract congo red and incubated for 2-5 days. The
organism grows on the mannitol yeast extract agar. If it can absorb the congo red, the colonies are red. A
negative result is indicated by growth of translucent colonies on the agar.

68
10. Methyl Red - Voges Proskauer (MR-VP) test
The Methyl Red (MR) test and the Voges-Proskauer (VP) test determines two possible end products of the
fermentation of glucose. The organism is inoculated into two tubes of MR-VP broth and incubated overnight.
In the MR test, the organism ferments the glucose in the medium to the end products acetic, lactic and succinic
acids, and ethanol, CO2 and H2. The high concentration of organic acids overcomes the phosphate buffer in the
medium and the pH becomes very low. Several drops of methyl red are added: a red colour indicates mixed acid
fermentation (a positive result). A yellow (or orange) colour indicates that little acid has been produced by the
organism (a negative result).
In the VP test, the organism ferments the glucose in the medium to pyruvate, which is then converted to acetoin
and 2, 3-butanediol. 15 drops of a-naphthol and 5 drops of 40% KOH are added and the tube is vortexed gently
to expose the medium to oxygen in order to oxidize the acetoin (if present) to diacetyl. The diacetyl then reacts
with peptones, which results in a red pigment (a positive result). If there is no acetoin present, the reagents will
turn the medium a brown (copper) colour.
11. Motility
A stab is used to inoculate the organism into the TTC motility medium. Stab carefully to obtain only a single
stab line and incubate overnight. The triphenyl tetrazolium chloride is reduced when broken down by the
organism and turns red. Therefore, the medium turns red where it is inoculated. If the organism is a facultative
organism and motile it swims throughout the medium and the whole tube becomes red. If the organism is an
aerobic organism, the stab line turns red and the entire top of the medium turns red where the organism swims
in the presence of oxygen.
12. Ornithine decarboxylase test
Some amino acids can be utilized as a carbon source after decarboxylation. In this process, the carboxyl group
is converted to CO2 and the amino acid is converted to an amine in the presence of the coenzyme pyridoxal
phosphate. The production of the amine results in a rise in pH which changes the colour of the indicator in the
media used.
The amino acid decarboxylases are lysine, arginine and ornithine. This test will use the amino acid ornithine
which is not found as a constituent of protein but does occur in the free form. The medium used is Møller’s
amino acid medium. It incorporates basal salts, glucose, the indicator bromcresol purple, as well as 1% L-
ornithine.
A tube of ornithine decarboxylase TEST medium and a tube of decarboxylase CONTROL medium is inoculated
with the organism, and approximately 1 ml of sterile mineral oil is poured into each tube. The tubes are
incubated overnight.
The indicator, bromocresol purple is yellow at pH 5.2 and purple at pH 6.8. If the organism can utilize the
glucose in the control tube, the medium turns yellow because of acid production. If the organism can utilize
the glucose and the ornithine in the test tube, the medium first becomes yellow because of acid production
from glucose, later if decarboxylation occurs, the medium turns alkaline and back to purple. A positive result is
indicated by a yellow control tube and a purple test tube. A negative result is indicated by a yellow control and a
yellow test tube.
13. Pigment Solubility Tests
The pigment produced by some microorganisms can be either a water soluble pigment or organic solvent
soluble. To determine water solubility, observe the NA or BHI agar on which the organism is grown. Water-
insoluble pigments remain confined within the cells; water-soluble pigments dissolve into the surrounding
medium. To determine organic solvent solubility, add 10 drops of alcohol: acetone (1:1) mixture to a test tube.
Add a loop of the cells grown on a NA or BHI agar plate. If the pigment is soluble, the liquid will be tinted the
colour of the pigment and will be transparent. If the pigment is insoluble, the liquid may appear to be tinted due
to the suspended cells, but the suspension will be opaque.
69
14. Urea test
A solution of urea is added aseptically to a sterile cooled basal medium in which phenol red is added as a pH
indicator. Phenol red is red at pH 8.0 and yellow at pH 6.6. The organism is inoculated into the medium and
incubated overnight. If the organism possesses the enzyme urease, it will break down the urea, ammonia is
produced and a red colour results. A negative result is indicated by the medium remaining yellow.

70
Appendix 5:
Bacterial Culture Media
The following media is prepared from Difco dehydrated culture media:

1. Blood agar base + 5% defibrinated whole blood


2. Brewer thioglycollate gelatin media
3. MacConkey agar
4. Mannitol Salt agar
5. Phenol Red Broth base + millipore filtered lactose, sucrose, maltose, xylose or sorbitol added to a
concentration of 1% after autoclaving.
6. SF agar
7. SIM media

8. Mannitol nitrogen free agar

MnSO4•4H20 trace
dH20 1.0 L
CaCO3 1.0 g
NaCl 0.2 g
MgSO4 0.2 g
K2HPO4 0.5 g
Mannitol (sugar) 10 g
FeCl3•6H2O granule
agar (1.5%) 15 g
molybdenum salt trace

To remove the precipitate which forms on autoclaving, autoclave for 5 minutes at 15 lb. Cool. Filter
through 3 pieces of Whatman #1 in a Buchner or if necessary use a sintered glass funnel with suction.
Distribute in flasks. Re-autoclave - 15 minutes at 15 lb.

9. Carbon free agar

Prepare minimal glucose agar, but omit glucose and do not amend with any amino acids.

10. Nitrate reduction broth

beef extract 1.2 g


peptone 2.0 g
KNO3 0.4 g
dH20 400 ml

Distribute 10 ml in large test tube. Add Durham vials upside down for trapping nitrogenous gases.

71
11. Butlin’s medium

K2HPO4 0.5 g
NH4Cl 1.0 g
Na2SO4 2.0 g
CaCl2•6H20 0.1 g
MgSO4•7H2O 1.0 g
yeast extract 1.0 g
FeSO4 0.002 g
dH20 1000 ml
Na lactate 3.5 ml of 60% solution

Adjust pH to 7.5 before autoclaving. Fill screw cap test tubes approximately 5/6 full. An iron nail (sterile)
may be added to the tube, however FeSO4 in the media provides the iron required for formation of FeS.

12. Rogosa SL agar

Bacto Tryptose 10.0 g


yeast extract 5.0 g
Dextrose 10.0 g
Arabinose 5.0 g
Saccharose (sucrose) 5.0 g
Sodium acetate 15.0 g
Ammonium citrate 2.0 g
KH2PO4 6.0 g
magnesium sulfate 0.57 g
manganous sulfate 0.12 g
ferrous sulfate 0.03 g
Tween 80 (sorbitan
  monooleate liquid) 1.0 g
agar 15.0 g
dH20 1000 ml

Heat to dissolve. Add 1.32 ml glacial acetic acid/liter. Mix and then heat to 90 - 100°C for 3 minutes. Do
not autoclave. Distribute in tubes or Petri plates.

13. Sucrose agar

Tryptone 1.0 g
yeast extract 0.5 g
K2HPO4 0.5 g
ammonium citrate 0.5 g
sucrose 5.0 g
agar 1.5 g
dH20 100 ml

72
14. Minimal glucose agar (MG)

2X salts

K2HPO4 21.0 g
KH2PO4 9.0 g
(NH4)2SO4 2.0 g
MgSO4•7H20 0.2 g
dH20 1000 ml

2X agar

Bacto agar 6.0 g


dH20 200 ml

Prepare salts in 400 ml bottles. Make 200 ml of salts/bottle.

Prepare agar and distribute into 200 ml quantities. Autoclave separately. Add 2 ml of 40% glucose
(volumetric) to the cooled salts. Pour hot agar into salts, mix and pour plates.

15. Amino acids

Add the following amounts of millipore filtered a.a. to 200 ml of cooled 2X salts.

1-leucine 1.2% solution 1 ml


threonine 1.2% solution 1 ml
thiamine 1.0% solution 0.125 ml
tryptophan 1.2% solution 1 ml

16. Nitrate reduction broth

Peptone 1.0 g
NaCl 0.5 g
KNO3 1.0 g
dH20 100 ml

17. Stevenson’s medium


Inorganic Compound Ammonium Medium  Nitrite Medium

(NH4)2SO4 2.0 g -
NaNO2 - 1.0 g
MgSO4•7H20 0.5 g 0.5 g
FeSO4•7H20 0.1 g 0.1 g
NaCl 0.3 g 0.3 g
MgCO3 5.0 g -
Na2CO3 - 1.0 g
K2HPO4 1.0 g 1.0 g
dH20 1000 ml 1000 ml

73
18. Peptone Yeast Extract agar
Ferric ortho Phosphate 0.01 g dH20 1.0 L
Yeast Extract 1.0 g agar 15.0 g
Peptone 5.0 g

19. Starkey’s medium


(NH4)2SO4 0.3 g CaCl2 0.25 g
KH2PO4 3.0 g Ferric sulfate 2.0 ml of a 0.5% solution
MgSO4•7H20 0.5 g dH20 1000 ml

Mix well, check pH. Should be at pH 4.5. Autoclave. Add 1 g of sulfur that has been sterilized by steam for 30
minutes on 3 consecutive days. Sulfur is sieved (20 mesh or less) and blown or gently poured onto surface of
media. Positive growth is indicated by pH changing to 1.0, increased turbidity, and sulfur granule precipitation.

20. Tryptic Soy Broth


tryptone 15.0 g NaCl 5.0 g
polypeptone 5.0 g dH20 1000 ml

21. Tryptose dextrose agar


Tryptose 20.0 g agar 15.0 g
Dextrose 1.0 g dH20 1000 ml
NaCl 5.0 g
Dissolve in steamer. Autoclave.

22. Yeast extract - mannitol agar (YMA)


mannitol 10.0 g CaCO3 4.0 g
K2HPO4 0.5 g yeast extract 0.4 g
MgSO4•7H20 0.2 g agar 15.0 g
NaCl 0.1 g dH20 1000 ml
Adjust pH to 6.8 - 7.0.
Before autoclaving, add 2.5 ml of a 1% congo red solution

23. Winogradsky column


In the 1880s, Sergei Winogradsky devised a medium and procedure to study the development of soil
microorganisms in vitro
Mix together 1 teaspoon of finely chopped grass and 2 tablespoons mud
Add 1 teaspoon of Plaster of Paris and mix until a thick, gummy consistency is achieved
Use a glass rod to tightly pack the mixture into the bottom of a glass tube
When the column is half full of the mixture, add bog water, leaving one inch of space at the top of the tube.
Screw the cap on loosely and incubate under a light rack at room temperature for 2-3 days
Remove any air bubbles trapped within the column, top up with bog water, screw the cap on tightly and
observe the development of the miniature ecosystem

24. Rhizobium enrichment culture


Aseptically add 4-6 clover seeds to the surface of 2 water agar slants
Once the clover has germinated, inoculate one of the tubes with Rhizobium, cap loosely and observe the
development of the endosymbiotic relationship between the two organisms in the inoculated tube
74
Reagents and Stains
1. Arsenomolybdate Reagent

ammonium molybdate 25.0 g


dH20 450 ml

Add 21 ml concentrated H2SO4


Dissolve 3 g sodium arsenate in 25 ml dH20. Add to the above solution.
Put in a brown bottle and incubate 48 hours at 37°C. Should be yellow with no green tint.

2. Benedict’s reagent for Glucose

copper sulfate 8.65 g


sodium citrate 86.5 g
sodium carbonate, anhydrous 50.0 g
  (or 100 g Na2CO3•10H20)

Dissolve citrate and carbonate in approximately 300 ml of water, heat and filter, Dissolve CuSO4 in 50 ml
of water, heat and pour slowly, stirring constantly into first solution. Cool and add water to bring volume to
500 ml.

3. Kovac’s reagent for Indole

Dissolve 5 g p-dimethylaminobenzaldehyde in 7.5 ml amyl alcohol. Add 2.5 ml hydrochloric acid slowly,
in a fume cabinet.

4. Nelson’s reagent for Glucose

Reagent A

Na2CO3 (anhydrous) 12.5 g


NaK tartrate 12.5 g
Na2SO4 (anhydrous) 100.0 g
dH20 350 ml

Pour into a graduated cylinder and dilute to 500 ml.

Reagent B

CuSO4•5H20 7.5 g
dH20 50 ml
concentrated H2SO4 1 drop

To 100 ml of Solution A, add 4 ml of Solution B just before using.

75
5. Nessler’s reagent for Ammonia
Dissolve 10 g sodium hydroxide in 100 ml dH20 (1)
To 15 ml of dH20 add 15 ml of Nessler’s stock reagent. (2)
Add solution (2) to 70 ml of solution (1).
Do not use if precipitation occurs.

6. Nitrate reagents
A. Diphenylamine reagent
diphenylamine 0.7 g
H2SO4 concentrated 60 ml
dH20 28.8 ml
HCl concentrated 11.3 ml

Dissolve diphenylamine in H2SO4 and add dH20. Cool and add HCl. Allow to stand overnight.

B. concentrated H2SO4

N.B. do not use if black precipitation occurs (or if the solution becomes black)

7. Nitrite reagents
A. Grieses reagent A

glacial acetic acid 29.4 ml


dH2O 70.6 ml
dimethyl naphthylamine 0.25 g

Add acid to water slowly to prevent boiling

B. Grieses reagent B
glacial acetic acid 29.4 ml
dH2O 70.6 ml
sulfanilic acid 0.8 g

Add acid to water

Store in separate containers. Just before use mix equal quantities of A and B. Should be a pale pink-purple
colour.

8. Phenolphthalein solution
phenolphthalein 1.0 g
95% ethanol 100 ml
pH ranges 8.3 - 10.0. Colourless in the acidic range; red when alkaline

9. Sudan Black B stain

0.3 g sudan black B in 70% alcohol. Allow to stand overnight before using.

76
Appendix 6:
WHMIS
WHMIS stands for Workplace Hazardous Materials Information System. It is a nationwide system to provide
information on hazardous materials used in the workplace.

Exposure to hazardous materials can cause or contribute to a variety of health effects such as irritation, burns,
sensitization, heart ailments, cancer, kidney and lung damage. Some materials may also be safety hazards that
can contribute to fires, explosions and other accidents if improperly stored or handled.

WHMIS is an information system which provides the following:

Labels on hazardous materials and their container that alert employers and workers to the danger of the
product and basic safety precautions.

Materials Safety Data Sheets (MSDS) - technical bulletins which provide detailed hazard and
precautionary information on the product.

Please see your TA or the Lab Coordinator if you would like further information on any of the controlled
products that are being used in the laboratory.

77
78
Centers for Disease Control and Prevention. Updated March
13, 2015. Biosafety in Microbiological and Biomedical
Laboratories 5th Edition. http://www.cdc.gov/biosafety/
publications/bmbl5/Accessed December 4, 2017
Table 2. Summary of Recommended Biosafety Levels for Infectious Agents

BSL Agents Practices Primary Barriers and Facilities


Safety Equipment (Secondary Barriers)

1 Not known to consistently cause Standard microbiological practices ■ No primary barriers required. Laboratory bench and sink required
diseases in healthy adults ■ PPE: laboratory coats and gloves;
eye, face protection, as needed

2 ■ Agents associated with human BSL-1 practice plus: Primary barriers: BSL-1 plus:
disease ■ Limited access ■ BSCs or other physical containment ■ Autoclave available
■ Routes of transmission include per- ■ Biohazard warning signs devices used for all manipulations
cutaneous injury, ingestion, mucous ■ “Sharps” precautions of agents that cause splashes or
membrane exposure ■ Biosafety manual defining any aerosols of infectious materials
needed waste decontamination ■ PPE: Laboratory coats, gloves, face
or medical surveillance policies and eye protection, as needed

3 Indigenous or exotic agents that may BSL-2 practice plus: Primary barriers: BSL-2 plus:

79
cause serious or potentially lethal ■ Controlled access ■ BSCs or other physical containment ■ Physical separation from access
disease through the inhalation route of ■ Decontamination of all waste devices used for all open manipula- corridors
exposure ■ Decontamination of laboratory tions of agents ■ Self-closing, double-door access
clothing before laundering ■ PPE: Protective laboratory clothing, ■ Exhausted air not recirculated
Appendix 7:

gloves, face, eye and respiratory ■ Negative airflow into laboratory


protection, as needed ■ Entry through airlock or anteroom
■ Hand washing sink near laboratory
exit

4 ■ Dangerous/exotic agents which post BSL-3 practices plus: Primary barriers: BSL-3 plus:
high individual risk of aerosol-trans- ■ Clothing change before entering ■ All procedures conducted in Class III ■ Separate building or isolated zone
mitted laboratory infections that are ■ Shower on exit BSCs or Class I or II BSCs in com- ■ Dedicated supply and exhaust,
frequently fatal, for which there are no ■ All material decontaminated on bination with full-body, air-supplied, vacuum, and decontamination
vaccines or treatments exit from facility positive pressure suit systems
■ Agents with a close or identical anti- ■ Other requirements outlined in
Biohazard Safety Level Designations

genic relationship to an agent requir- the text


ing BSL-4 until data are available to
redesignate the level
■ Related agents with unknown risk of

Laboratory Biosafety Level Criteria


transmission

59
80
Appendix 8:
Image Reference Sheet
CMMB 343 Laboratory Manual 2018

Page 52
Microscope image from Olympus CX31 compound light microscope user manual
www.olympus-lifescience.com
Olympus Scientific Solutions Americas Corp., Waltham MA

Page 61
Image drawn in-house

Page 62
Images drawn in-house

Page 63
Image drawn in-house

Page 64
Image drawn in-house

Page 65
Image drawn in-house

Page 66
Image from Madigan, Bender, Buckley, Sattley and Stahl. 2018. Brock: Biology of Microorganisms, 15th
Edition. Pearson Education Inc., San Francisco. p. 150.

Page 79
Document from Centers for Disease Control and Prevention. Updated March 13, 2015. Biosafety in
Microbiological and Biomedical Laboratories, 5th Edition. http://www.cdc.gov/biosafety/publications/bmbl5/.
Accessed December 4, 2017.

81
82
2018

83
__________________________________________________________________________________________
TA EVALUATION FORM

84
Notes
Notes
Notes
Notes