MICROBIOLOGY

072-1-MI

PROCESSING AND SET-UP OF

ANAEROBIC SPECIMEN S

THE MICHENER INSTITUTE for Applied Health Sciences

44
Acknowledgements

Author
June Macdonald B.Sc., A.R.T.

Contributors and Reviewers

Greg Buchanan, A.R.T.
Glen Carmichael, A.R.T.

© 1987 by THE MICHENER INSTITUTE for Applied Health Sciences

222 St. Patrick Street
Toronto, Ontario, Canada
M5T lV4

© 2002 Revised Edition

This material has been prepared and developed by The Michener Institute for Applied Health
Sciences. Reproduction of any part of this material, written, audio, visual, or electronic, in any
form, without the written consent of The Michener Institute is forbidden.

45
 DIVISION OF CONTINUING EDUCATION

MICROBIOLOGY

072-1-MI

PROCESSING AND SET-UP OF

ANAEROBIC SPECIMEN S

Table of contents

PAGE

OBJECTIVES 1

RESOURCE MATERIAL 2

MACROSCOPIC APPEARANCE OF THE SPECIMEN 3

GRAM STAIN 3

CULTURE MEDIA 4
SOLID MEDIA 4
FLUID OR BROTH MEDIA 5
PRAS MEDIA 5
QUALITY CONTROL 5

INOCULATION 6

INCUBATION OF CULTURES 7
GAS PAK SYSTEM 7
EVACUATION REPLACEMENT SYSTEM 8
BIOBAGS 10
VPI SYSTEM 10
ANAEROBIC GLOVE BOX 12

46
072-1-MI -1-

OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion of
this module. The self assessment questions and answers will enable you to judge your understanding of the
material.

Upon completion of this module, the student should be able to:

1. List the macroscopic features of a specimen for anaerobic culture.

2. Name the gram counterstain which is best for anaerobes.

3. List the three groups or classes of media set up for anaerobic culture.

4. Give the constituents of both thioglycollate and cooked meat broths indicating reducing substances and
indicators if relevant.

5. State the reason for the inclusion of media incubated aerobically and under carbon dioxide.

6. List four supplements that must be added to anaerobic media.

7. Name two selective media used for anaerobic isolation.

8. Give a procedure for the inoculation of specimens submitted as fluids on one swab and on two swabs.

9. List the steps required to set up a specimen for anaerobic isolation (include types of media).

10. Describe the set up of a holding jar and the types of gasses involved.

11. For each of the following anaerobic incubation systems outline the principles of operation and state the
advantages and disadvantages of each:

 Gas Pak
 Evacuation-replacement
 PRAS/VPI system
 Glove box
 Biobag

12. Explain what is meant by PRAS.

47
072-1-MI -2-

13. Name two redox indicators and state their colours under both aerobic and anaerobic conditions.

14. Name four reducing agents used or found in culture media.

RESOURCE MATERIAL

This module includes the information necessary to meet the objectives. For the references listed below:

[P] designates sources of prerequisite information

All others contain additional information.

TEXTS

Baron, E.J., Feingold, S.M., Diagnostic Microbiology, 8th Edition, The C.V. Mosby Co., Toronto, 1990.

Feingold, S.M., Baron, E.J., Wexler, H.M., A Clinical Guide to Anaerobic Infections, Star Publishing Co.,
California, 1992.

Koneman, E.W., Allen, S.D., Janda, W.M., Schrenkenberger, P.C. and Winn, W.C., Color Atlas of
Diagnostic Microbiology, 4th Edition, Lippincott, New York, 1991.

Balows, Albert, Manual of Clinical Microbiology, 5th Edition, American Society for Microbiology, 1991.

Sutter, V.L., Citron, Diane, Edelstein, M.C., Feingold, S.M., Wadsworth Anaerobic Manual, 4th Edition, Star
Publishing Co. California, 1985.

Shulman, Stanford T., Phair, John P., Sommers, H.M., The Biologic and Clinical Basis of Infectious
Diseases, 4th Edition, W.B. Saunders Co., Toronto, 1992.

INSTRUCTIONAL MODULES DEVELOPED BY THE MICHENER INSTITUTE

[P] 070-1-MI Anaerobes

[P] 071-1-MI Collection and Transport of Wounds, Lesion
and Other Specimens for Anaerobic Culture
[P] 020-1-MI Incubation of Inoculated Media

48
072-1-MI -3-

MICROBIOLOGY
072-1-MI
PROCESSING AND SET-UP OF WOUNDS
AND ANAEROBIC SPECIMENS

Specimens should not be set up for anaerobic culture if improperly taken; however, before rejecting a
specimen, the physician submitting the specimen should be informed. If the specimen is acceptable, yet has
not been submitted in an anaerobic transport medium or system, it should be held under anaerobic
conditions until it can be set up at the earliest opportunity.

MACROSCOPIC APPEARANCE OF THE SPECIMEN

Certain macroscopic features of the specimen will give clues to the presumptive identity of the causative
organisms and perhaps aid in early treatment. Characteristics of note will include:

 Foul odour
 Gas
 Colour of the discharge
 Presence of pus or blood
 Necrotic tissue

GRAM STAIN

A direct gram stain preparation made from the clinical specimen is essential for interpretation of anaerobic
specimens, especially wounds. It is suggested that carbol fuchsin (diluted 1:5) is a better counterstain than
safranin for detection of anaerobic gram negatives. Some authorities also recommend fixing by methanol
rather than by heat.

CULTURE MEDIA

Solid as well as fluid media are required for anaerobic culture. The solid agar separates the various mixed
organisms, whereas, liquid media is included to increase the numbers of scarce or few bacteria.

Media must be fresh and must have the supplements Vitamm K and hemin which encourage the growth of
most anaerobes.

Selective media are useful to prevent facultative anaerobes from overgrowing the anaerobes or to select for
certain anaerobe species or groups.

49
072-1-MI -4-

SOLID MEDIA

Anaerobic Nonselective Media

Anaerobic media should be freshly prepared or "prereduced" in an anaerobic jar for at least 24 hours prior to
use in order to provide media as free of oxygen as possible. Media absorbs oxygen during storage.
Prereduced media is free of oxygen.

Routine blood agar basal media, e.g. BHI or trypticase soy do not supply all anaerobic organisms'
nutritional needs. Supplements such as Vitamin K, hemin, laked blood and yeast extract are added. Often l-
cystine is also included as a nutrient for some anaerobes. The exact formula of these nonselective media will
vary from laboratory to laboratory; however, common examples include supplemented brucella medium and
supplemented trypticase soy.

Anaerobic Selective Media

Because many specimens contain mixtures of aerobes and/or facultative bacteria as well as anaerobes, it is
important to inhibit the growth of these more rapid growers so that they do not overgrow the slower
growing anaerobes.

Phenylethyl alcohol added to a supplemented blood agar base will inhibit facultative gram negatives, yet
will permit most anaerobes to grow.

Kanamycin and vancomycin (in Kanamycin-Vancomycin medium KVL) when added to anaerobic basal
media will select for Bacteroides species.

Ordinary Media

All specimens for anaerobic isolation should be inoculated onto both MacConkey agar aerobically and
sheep blood agar under carbon dioxide to detect facultative or aerobic bacteria in the specimen. Depending
on the specimen site, a chocolate plate may be substituted for the blood.

FLUID OR BROTH MEDIA

The classic example of a fluid anaerobic medium is enriched thioglycollate broth. Its constituents include:
 Sodium thioglycollate, a reducing agent which maintains reduced oxygen conditions

 A small amount of agar to retard convection currents which may introduce air into the lower reaches
of the tube

 Resazurin, a redox indicator indicates the presence or absence of oxygen. The indicator is pink in air
and colourless anaerobically. If the level of resazurin extends more than 1/3 down the tube, the

50
072-1-MI -5-

medium should not be used or it should be boiled once only to drive off oxygen. Some anaerobes are
inhibited by thioglycollate; the supplements vitamin K and hemin are added to overcome this
inhibitory effect to a limited degree. For very fastidious anaerobes, it is recommended that the
resazurin be omitted.

Another important, commonly used broth, is chopped (cooked) meat glucose. The reducing agents in
cooked meat are glutathione and glucose. This medium also is supplemented with Vitamin K and hemin.

In spite of the fact that both these media have reducing agents and theoretically should need to be incubated
only aerobically, many authorities suggest incubation anaerobically.

PRAS MEDIA

Prereduced anaerobically sterilized (PRAS) media can be prepared for culturing of anaerobes. After
preparation, these media are sterilized and stored under anaerobic conditions at room temperature until use.
Care is taken not to expose PRAS media to oxygen during all procedures.

QUALITY CONTROL

Each batch of primary isolation media should be inoculated with Peptostreptococcus anaerobius and
Fusobacterium spp. to ensure it will support the growth of fastidious anaerobes.

INOCULATION

If a fluid has been submitted for culture, use a sterile Pasteur pipette to add drops of the specimen
(preferably a spun-down sediment, if possible) to plate media. To inoculate broth media, insert the pipette to
the bottom of the tube and gently squeeze out the inoculum. Streak plates out in a standard fashion to obtain
isolated colonies.

If a two-swab specimen has been submitted, use one swab to inoculate the main inoculum area of each solid
medium and then the broth. Use the second swab for smear preparation (Figure 1). When inoculating plate
media rotate the swab firmly over the intended main inoculum area and always inoculate nonselective
media before selective. When inoculating a broth, insert the swab to the bottom of the tube and rotate it
gently against the side of the tube. Wring the swab out at the broth surface.

If only one swab is submitted, wring the swab out in a small amount of broth and use the fluid to inoculate
the solid media and to make the smear.

51
072-1-MI -6-

FIGURE 1: Specimen set up when two swabs are supplied

52
072-1-MI -7-

INCUBATION OF CULTURES

Specimens are incubated at 35ºC for 48 hours before examining. There are several systems available,
examples include:

 Gas Pak or anaerobic jars

 Evacuation-replacement system
 PRAS system
 Anaerobic glove box
 Biobag

GAS PAK SYSTEM

The Gas Pak system (Figure 2) consists of an autoclavable plastic jar, a lid containing a catalyst and a
disposable gas generating envelope. (Most jars hold up to 11 plates, although larger jars may be obtained.)

FIGURE 2: Gas pak system

53
072-1-MI -8-

To create an anaerobic environment, water is added to the gas generating envelope, and the gases hydrogen
and carbon dioxide are evolved. The hydrogen combines with the oxygen in the jar to produce condensation
in the jar interior.

A fresh methylene blue indicator is added to the jar just before clamping the jar shut. This mdicator is blue
when oxygenated and colourless under anaerobic conditions.

Use fresh catalysts since they become inactivated due to the moisture and metabolic products of microbial
growth. Catalysts may be rejuvenated by heating at 160ºC for two hours. These catalysts are stored with a
desiccant until used.

Ensure that the jar is free of cracks, the lid has a tight seal and that all rubber hose parts are tightly clamped.

Although these jars are simple to use and relatively inexpensive, the process may take several hours to
achieve anaerobiasis.

EVACUATION-REPLACEMENT SYSTEM

The jar system used for this incubation technique is similar to that used for the Gas Pak System. However,
the lid has a clamped vent which is hooked up to a vacuum pump. The air inside the jar is evacuated by the
pump and is replaced by nitrogen gas. This evacuation-replacement is repeated three times. After a third
evacuation, an anaerobic gas mixture containing 85% nitrogen, 10% hydrogen and 5% carbon dioxide is
added. A palladium catalyst is also included.

Although this method is not as convenient, it is less expensive than the Gas Pak system and it achieves
anaerobiasis faster.

For those laboratories having only a Gas Pak System, specimens can be held in anaerobic transport media
for several hours until there are sufficient specimens for batch processing of all the specimens at once.

54
072-1-MI -9-

Both the Gas Pak system and evacuation-replacement system allow exposure of inoculated plates to oxygen
while enough plates accumulate to fill the jar. To avoid this difficulty, holding jars are used. These jars are
similar to the jars described above but have a piece of tubing feeding a continuous flow of nitrogen into the
bottom of the jar. Plates, as they are inoculated, are placed in the jar and are thus protected from oxygen
until the jar is set up. A plate containing Fusobacterium spp. should be maintained in the holding jar for
quality control.

FIGURE 4: Holding jars

55
072-1-MI - 10 -

BIOBAGS

Biobags consist of small gas-impermeable bags, large enough to hold one plate. In each bag is an anaerobic
indicator and a small gas generating system similar to that found in the Gas Pak generating envelope. This
system permits plate examination at any time through the bag. Laboratories doing only occasional anaerobic
work find this system very practical for the few plates they set up.

FIGURE 5: Biobags

VPI SYSTEM (using PRAS media)

The VPI (Virginia Polytechnic Institute) system uses a very sophisticated holding-media inoculation system.
Agar-based media are not put into petri dishes. Instead, a small amount of agar media is coated onto the
interior of large test tubes. PRAS media are used in these tubes. These tubes are rotated during inoculation.

The inoculum is placed at the bottom of this media-coated tube and the loop is slowly drawn upwards
against the inside of the tube as it rotates. Because the tube is rotating while the line of inoculum is being
drawn up, a spiral inoculum line is produced with a gradual decrease in organism numbers as the inoculum
spirals up the interior of the tube.

072-1-MI - 11 -
56
FIGURE 6: VPI system

While the tube cap is off, a cannula is hooked over the top of the tube to deliver oxygen free, carbon dioxide
during all manipulations. This is called the roll-tube streak method.

This system allows examination and subculture of colonies at any time with total protection of the anaerobe.
However, it is an expensive system requiring skillful handling.

072-1-MI - 12 -
57
ANAEROBIC GLOVE BOX

Glove boxes are enclosed plastic chambers with glove ports attached. Newly set-up plates are passed
through an entry port into the chamber proper or into an incubator within the glove box. Anaerobic
conditions inside the chamber are created by an evacuation-replacement process. A number of catalysts and
desiccants are kept within the chamber.

This system is expensive and, although convenient, it can be awkward to work in the hood using the glove
ports.

FIGURE 7: Glove box

58
 MICROBIOLOGY

073-1-MI

DIAGNOSTICS AND REPORTING OF

ANAEROBIC SPECIMENS

THE MICHENER INSTITUTE for Applied Health Sciences

59
Acknowledgements

Author
June Macdonald B.Sc., A.R.T.

Contributors and Reviewers

Greg Buchanan, A.R.T.
Glen Carmichael, A.R.T.

© 1987 by THE MICHENER INSTITUTE for Applied Health Sciences

222 St. Patrick Street
Toronto, Ontario, Canada
M5T 1V4

© 2002 Revised Edition

This material has been prepared and developed by The Michener Institute for Applied Health Sciences.
Reproduction of any part of this material, written, audio, visual or electronic, in any form, without the
written consent of The Michener institute is forbidden.

60
 DIVISION OF CONTINUING EDUCATION

MICROBIOLOGY

073-1-MI

DIAGNOSTICS AND REPORTING OF ANAEROBIC SPECIMENS

Table of Contents

PAGE
OBJECTIVES 1

RESOURCE MATERIAL 4

OVERVIEW 5

THE DIRECT GRAM STAIN 6

PRIMARY PLATE EXAMINATION 7

TIMING IMPORTANCE 7
EXAMINATION PROCEDURE 7
GROWTH QUANTITATION 7
EXAMINATION OF GROWTH ON ENRICHED
NONSELECTIVE MEDIA 8
DETERMINING ATMOSPHERIC REQUIREMENTS 8
EXAMINATION OF GROWTH ON SELECTIVE MEDIA 11

EXAMINATION OF THE FLUID MEDIA 12

PURPOSE OF SUBCULTURES OR PURITY PLATES 12

LEVEL I IDENTIFICATION 13

LEVEL II IDENTIFICATION 14
IDENTIFICATION OF ANAEROBIC GRAM-NEGATIVE
RODS 14
IDENTIFICATION OF ANAEROBIC COCCI 14
IDENTIFICATION OF SPOREFORMING GRAM-POSITIVE
RODS 17
IDENTIFICATION OF NONSPOREFORMING GRAM-POSITIVE
RODS 19

61
LEVEL III IDENTIFICATION 20
VPI SYSTEM USING PRAS MEDIA 20
GAS-LIQUID CHROMATOGRAPHY 21
COMMERCIAL SYSTEMS 22
ANTIBIOTIC TESTING OF ANAEROBES 22

APPENDIX: OXYGEN REQUIREMENTS 23

073-1-MI -1-
62
OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion of
this module. The self-assessment questions and answers will enable you to judge your understanding of the
material.

Upon completion of this module, the student should be able to:

1. Give the typical Gram-stained appearance of each of the following anaerobic groups or species as seen
on direct smear:

a) Bacteroides fragilis
b) Pigmented Bacteroides
c) Fusobacterium species
d) Peptostreptococcus species
e) Veillonella species
f) Clostridium perfringens
g) Actinomyces israelii

2. State how long anaerobic cultures should be incubated before being observed for growth.

3. State three systems which allow visual examination of colonies earlier than 48 hours while still
maintaining anaerobic conditions.

4. Describe a system that prevents undue exposure to oxygen while handling and observing anaerobic
growth on an open bench.

5. State why the growth on primary isolation plates must be examined closely in relationship to the
organism morphology seen on direct Gram smear.

6. Describe one means of semi-quantitation of anaerobic growth on primary isolation plates and give a
reason for this quantitation.

7. Describe six characteristic colonial morphologies seen on anaerobic blood agar that may be suggestive
of specific species.

8. Describe how the atmospheric requirements for each isolate is determined.

9. Give the purpose for the set up of blood agar (under anaerobic conditions), blood agar (under CO2) and
MacConkey media under aerobic conditions in the bacteriological diagnosis of wound infections.

10. State why the following selective media/atmospheric incubation combinations are used for the
laboratory diagnosis of wound infections:

073-1-MI -2-
63
 a) Aerobic MacConkey agar
b) Anaerobic supplemented phenylethyl alcohol agar
c) Kanamycin-vancomycin laked blood agar

11. Give two reasons for the inclusion of supplemented broth media in the laboratory examination of
wound infections.

12. Give two reasons for the subculture of anaerobic isolates from supplemented enriched agar.

13. Outline an elementary identification scheme for anaerobic gram-negative rods using:
 20% bile
 Susceptibility to high concentration antibiotics
 Fluorescence with long wavelength UV light

14. Name four anaerobic gram-negative rod groups that can be separated by elementary identification
methods.

15. Name three anaerobic cocci that may be separated using simple tests.

16. Describe the typical Gram stain appearance of anaerobic gram-positive cocci.

17. Outline an elementary identification scheme for anaerobic cocci using the Gram stain reaction, the spot
indole and the SPS disk.

18. Describe the appearance of lethicinase and lipase production by bacteria on egg yolk agar.

19. Describe the theory of the reverse C.A.M.P. test.

20. Describe the theory of the Nagler inhibition test.

21. Describe the laboratory diagnosis of Clostridium perfringens including the following characteristics:

 Gram stain and colonial morphology

 Action on egg yolk medium
 Reaction in the reverse CAMP test
 Reaction in the Nagler inhibition test

22. Briefly outline three characteristics of Clostridium difficile.

23. Give two laboratory characteristics of Actinomyces israelii.

24. Give two laboratory characteristics of Propionibacterium.

073-1-MI -3-
64
25. State when level III anaerobic identification should be done.

26. Briefly outline the use of:

a) The VPI/PRAS system

b) Gas chromatography for anaerobic identification

27. Briefly describe a commercial system (microtube or rapid method) for anaerobe identification.

28. State the clinical conditions justifying anaerobic antibiotic testing.

29. State why Kirby Bauer testing is not valid for anaerobes.

30. List the organisms which should be tested for beta lactamase production.

31. Define:
 Capnophile
 Aerotolerant
 Microaerophile

073-1-MI -4-

65
RESOURCE MATERIAL

This module includes the information necessary to meet the objectives. For the references listed below:

[P] designates sources of prerequisite information.

All others contain additional information.

TEXTS

Balows, A., editor, Manual of Clinical Microbiology, Fifth Edition, American Society for Microbiology,
1991.

Baron, E.J., Feingold, S.M., Diagnostic Microbiology, Eighth Edition, The C.V. Mosby Co., Toronto, 1990.

Feingold, S.M., Baron, E.J., Wexler, H.M., A Clinical Guide to Anaerobic Infections, Star Publishing Co.,
California, 1992.

Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenberger, P.E., Winn, W.C., Color Atlas of Diagnostic
Microbiology, Fourth Edition, Lippincott, New York, 1991.

Shulman, Stanford, T., Phair, John P., Sommers, G.M., The Biologic and Clinical Basis of Infectious
Diseases, Fourth Edition, W.B. Saunders Co., Toronto, 1992.

Sutter, V.L., Citron, Diane, Edelstein, M.C., Feingold, S.M., Wadsworth Anaerobic Manual, Fourth Edition,
Star Publishing Co., California, 1985

INSTRUCTIONAL MODULES DEVELOPED BY THE MICHENER INSTITUTE

[P] 070-1-MI Anaerobes

[P] 071-1-MI Collection and Transport of Wound, Lesion and Other Specimens for
Anaerobic Culture
[P] 072-1-MI Processing and Set Up of Anaerobic Specimens

073-1-MI -5-
66
 MICROBIOLOGY
073-1-MI
DIAGNOSTICS AND REPORTING OF
ANAEROBIC SPECIMENS

OVERVIEW

The level of anaerobic diagnostic work will vary with a given laboratory, its size and technological
expertise. There are three levels of service that may be chosen.

LEVEL I provides:

 Presumptive identification from:

- Gram's stain
- Colonial recognition and
- Atmospheric requirements

 Preparation of pure cultures for submission to a reference lab

LEVEL II provides more information than Level I as it includes:

 Simple tests used to place anaerobes in groups and for some speciation

LEVEL III provides:

Complete identification using:

 Biochemicals and/or gas-liquid chromatography (GLC)

 Rapid and/or miniaturized commercial systems

In this module, only Level I identification and a selection of Level II tests will be examined. A brief
overview of the principles of the other systems will also be discussed, but the module is designed as a guide
in how to approach anaerobic diagnosis at a basic level.

073-1-MI -6-
67
THE DIRECT GRAM STAIN

A Gram-stained smear made directly from the specimen should be examined on the day of specimen
receipt in the laboratory. Because anaerobes take longer than aerobes to produce colonies and to be
identified, direct Gram stain reports are valuable for prompt treatment.

Careful listing of all Gram-stained organism types is useful for quality control for it provides a check on
transport of specimens, media and personnel capabilities in ensuring that all organisms seen are actually
isolated either aerobically or anaerobically.

As well, valuable presumptive identification information may be obtained from a direct GramStain.

Gram stain: Presumptive Identification

(Anaerobe Expected)

- Pleomorphic, pale staining Bacteroides fragilis

gram-negative rods - often
bipolar
- Gram-negative coccobacilli Pigmented Bacteroides
- Gram-negative bacillus with Fusobacterium species
pointed ends
- Gram-positive cocci in Peptostreptococcus species
clusters and chains
- Tiny gram-negative cocci Veillonella species
- Large broad gram-positive Clostridium perfringens
"boxcar" shaped rods
- Filamentous gram-positive Actinomyces israelii
rods with sulphur granules
and clubs

Some laboratories refuse to carry out culture work on wounds, respiratory and urogenital tract specimens
that demonstrate only squamous epithelial cells. The presence of epithelial cells and a lack of inflammatory
cells indicate poor collection techniques.

073-1-MI -7-
68
PRIMARY PLATE EXAMINATION

TIMING IMPORTANCE

Primary plates should, theoretically, not be examined before 48 hours' incubation. Many anaerobes are not
visible before this time and exposure to oxygen before 48 hours may be lethal for slow growers. However,
cultures incubated in glove boxes, biobags or in the VPI roll tubes may be examined earlier and, in the case
of glovebox and VPI systems, faster growing anaerobes may be actually subcultured. These systems permit
the technologist to examine the colony growth without exposing the organisms to oxygen.

EXAMINATION PROCEDURE

Keeping in mind the number and morphological types of bacteria seen on direct smear, examine all plates to
ensure all types have been isolated. Plates must not be exposed to air for an excessive time period.
Technologists using a Gas Pak System must work swiftly so that the anaerobes are not killed. Plates should
be kept in holding jars when not actually being examined and a steady flow of oxygen-free carbon dioxide
maintained in both the jars or the opened PRAS tubes.

GROWTH QUANTITATION

Examine each plate initially to semi-quantitate the relative amount of growth. Growth quantitation gives a
rough indication of the concentration of the organism(s) in the actual specimen. The following scheme is
frequently used.

FIGURE 1: Quantitation of Growth

Some workers prefer to use the terminology:

 Light
 Moderate or
 Heavy growth
073-1-MI -8-

69
EXAMINATION OF GROWTH ON ENRICHED NONSELECTIVE MEDIA

The anaerobic enriched blood agar is carefully examined using a hand lens or stereoscopic microscope and
the colonies described. The following criteria should be noted for each colony: colour, pigment, hemolysis,
diameter, elevation, surface and optical properties.

Certain distinctive features are suggestive of certain anaerobic groups or species:

Characteristics Possible Anaerobe

- Pitting colonies Bacteroides ureolyticus

- Large colonies with a double
zone of hemolysis
- "Bread crumb"- like with
speckling of the colony
- Molar tooth colony
- Black colonies
- Medusa head colony
Clostridium perfringens
Fusobacterium nucleatum
Actinomyces israelii
Pigmented Bacteroides
Clostridium septicum

DETERMINING ATMOSPHERIC REQUIREMENTS

Compare the growth on the anaerobic blood agar with the growth under capnophilic and aerobic conditions
(Figure 2). Attempt to determine the atmospheric requirements of each type of colony

FIGURE 2: Comparison of Growth on Blood Agar Under Various Atmospheric Conditions

073-1-MI -9-
70
Since it may be difficult to determine the atmospheric requirements on primary isolation media, each colony
type on the original anaerobic nonselective blood agar is Gram-stained, subcultured and incubated under
different atmospheric conditions to determine its precise atmospheric requirements (Figure 3):
 Anaerobic blood agar incubated anaerobic
 Ordinary blood (or chocolate) agar incubated with increased carbon dioxide
 Ordinary blood (or chocolate) agar incubated in presence of oxygen

FIGURE 3: Examples of a Subculture from Anaeorobic Plates

The patient's specimen illustrated in Figures 2 and 3 contains:

 2 anaerobes (one strict anaerobe; one aerotolerant)
 1 facultative anaerobe (aerobe)
 1 capnophilic organism

Aerotolerant bacteria, (e.g. some Clostridium species) grow best anaerobically and do not use molecular
oxygen yet may grow slightly without anaerobic conditions.

073-1-MI - 10 -
71
EXAMINATION OF GROWTH ON SELECTIVE MEDIA

Aerobic MacConkey

This plate is included to detect aerobe, and particularly facultatively anaerobic gram-negative rods which
may be involved synergistically in the pathogenic process. MacConkey plates are not incubated
anaerobically.

Anaerobic Phenylethyl Alcohol

Because anaerobic infections are often polymicrobic, it is important that faster growing bacteria not
overtake the slower growers. Supplemented phenylethyl alcohol supports the grow of both anaerobic gram-
positives and negatives but inhibits facultative gram-negatives.

Kanamycin-Vancomycin Laked Blood Medium (KVL)

The vancomycin in this medium inhibits most gram-positive bacteria whereas the kanamycin inhibits
facultative gram-negative rods. The laked blood permits earlier production of colour by pigmented
bacteroides. KVL medium selects for Bacteroides and Fusobacterium species.

EXAMINATION OF THE FLUID MEDIA

The broth is included as a "back-up" should no growth occur on solid media. As well, the broth is routinely
subcultured onto supplemented anaerobic blood agar for anaerobic incubation and an ordinary blood agar
to, hopefully, isolate any bacteria seen on primary Gram's stain yet not isolated on solid media.

073-1-MI - 11 -
72
PURPOSE OF SUBCULTURES OR PURITY PLATES

All isolates suspected to be anaerobes must be subcultured to two blood agar or chocolate agar plates. One
is incubated in air or CO2 and the other anaerobically. Only by comparing these two plates after incubation
can you be sure that the suspected colony is an anaerobe. This is sometimes called an aerotolerance test.

These plates are used, not only to determine atmospheric requirements but to provide an abundant pure
growth of the anaerobe for:

 Further tests to give a specific identification

 Submission of the culture to a reference laboratory for identification
 Susceptibility testing

073-1-MI - 12 -

73
LEVEL I IDENTIFICATION

The use of just the Gram stain, atmospheric requirements and growth on primary plates allow the
presumptive identification outline in Table 2.

073-1-MI - 13 -

74
LEVEL II IDENTIFICATION

Level II identification provides presumptive identification using simple tests easily preformed in small
laboratories. Before attempting these tests, ensure that the organism is in pure culture and that It is truly
anaerobic by subculturing under aerobic and anaerobic conditions.

Once isolated in pure culture and checked for anaerobiasis, do another Gram smear to ensure you have the
correct gram reaction.

IDENTIFICATION OF ANAEROBIC GRAM-NEGATIVE RODS

There are four species or groups that may be relatively easily separated using simple tests:
 Bacteroides fragilis group
 Pigmented Bacteroides
 Bacteroides ureolyticus group
 Fusobacterium

There are five commonly used tests which used in conjunction with the Gram stain and cultural
characteristics are useful to separate these gram-negatives.

These tests are:

 Growth in 20% bile
 Susceptibility or resistance as demonstrated using the following high potency antibiotic disks:
- Kanamycin (1 mg)
- Vancomycin (5 mg)
- Colistin (10 mg)
Coloured fluorescence due to long wavelength UV light

20% Bile test

Growth in the presence of bile may be tested using a broth form or on bile esculin medium. A positive result
on bile esculin medium is demonstrated by growth with or without esculin hydrolysis.

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Susceptibility to Antibiotics

The disks are placed on an area of the plate streaked for confluent growth. After incubation anaerobically
zones of inhibition around each disk are noted.

FIGURE 4: Antibiotic Disks Used for Identification

Fluorescence Using Long Wavelength UV Light

A hand held UV lamp is held over suspected colonies to detect the characteristic brick-red fluorescence of
Prevotella melaninogenicus.

With the above tests, the following patterns may be achieved (Table 3).

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IDENTIFICATION OF ANAEROBIC COCCI

Anaerobic cocci may be roughly separated using Gram stain reactions, the spot indole test and the zone of
inhibition surrounding a SPS (sodium polyanethal sulfonate) disk.

Gram Stain

Anaerobic gram-negative cocci are not major human pathogens. For presumptive purposes, anaerobic gram-
negative cocci may be reported as Veillonella. However, technologists should be aware that anaerobic gram-
positive cocci may overdecolourize easily and may, therefore, be misidentified.

Spot Indole

Organisms to be tested must be picked from a tryphophane containing medium and are rubbed onto a disk
containing paradimethylaminocinnamaldehyde. A positive indole is detected by a blue colour on the disk.
Only Peptostreptococcus asaccharolyticus is positive.

SPS Disk

A zone of inhibition between 12 to 18 mm is indicative of Peptostreptococcus anaerobius.

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IDENTIFICATION OF SPOREFORMING GRAM-POSITIVE RODS

here is one anaerobic sporeforming genus, Clostridium, which contains many species. In this module only
Clostridium perfringens will be discussed. Other Clostridia isolated from specimens sent for anaerobic
culture can be reported as "Clostridium species not Clostridium perfringens."

Reverse CAMP Test

One inoculum line of a group B streptococcus is streaked down the centre of a blood agar plate. Clostridium
perfringens, when streaked at a right angle to (but not touching) this streptococcus, produces, after
overnight anaerobic incubation, a typical arrow-head hemolysis. This arrow configuration is produced by
the synergistic combination of the two organisms' hemolysis.

FIGURE 6: The Reverse CAMP Test

Lecithinase and Lipase Production on Egg Yolk Agar

Egg yolk agar is used to detect the production of the lecithinase and lipase enzymes produced by some
anaerobes.

Lecithinase is detected by an opaque white zone of precipitate in the medium surrounding the colonies
growing on the egg yolk medium. Lipase is detected by an irridescent sheen ("pearly layer" or and "oil
slick") on the surface of the colony.

Clostridium perfringens is lecithinase positive and lipase negative.

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Nagler Inhibition Reaction

If an antitoxin to Clostridium perfringens is spread onto a half egg yolk plate and an isolate of Clostridium
perfringens is streaked across the plate from the side free of antitoxin to the antitoxin-containing side, the
plate will demonstrate, after incubation anaerobically, an inhibition of lecithinase activity. Clostridial
species other than Clostridium perfringens which are lecithinase producers, show no inhibition of the
opacity.

(The specific clostridial antitoxin inhibits the specific lecithinase which produces the opacity)

FIGURE 7: Nagler Inhibition Reaction

Although Clostridium difficile is isolated from stools and not wounds, it is an important anaerobic pathogen.
On a special selective medium, cycloserine cefoxitin fructose agar, (CCFA) Clostridium difficile produces a
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yellow "ground glass" colony with a distinctive "horse-stable" odour. Under long wavelength UV the colony
appears greenish-yellow.

IDENTIFICATION OF ANAEROBIC NONSPOREFORMING GRAM-POSITIVE RODS

Only a few of these organisms can be specifically identified without advanced tests. The genera included in
this group are:

 Actinomyces
 Propionibacterium
 Bifidobacterium
 Lactobacillus
 Eubacterium

Actinomyces may be characterized by its branching filaments and "molar-tooth" colonies which take about
one week to grow.

Propionibacterium are long narrow gram-positive rods which are catalase positive.

Report other organisms as gram-positive anaerobic asporogenous rods.

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LEVEL III IDENTIFICATION

Complete identification to species level should be attempted (or the culture sent to a reference laboratory) if
the anaerobes were isolated from surgical, peritoneal or gangrene specimens. Unidentified isolates can be
sent to a referral laboratory in cooked meat broth or Cary-Blair medium.

VPI SYSTEM USING PRAS MEDIA

The VPI system was originally developed at the Virginia Polytechnic Institute and may be referred to as the
VPI method. This system is the standard method by which all others are compared.

As mentioned in previous modules, PRAS media are prereduced and anaerobically sterilized. (They are
prepared and sterilized under oxygen-free gas.) To help maintain the low redox potential, a reducing agent is
included in the medium.

All inoculation of PRAS biochemicals may be done in two ways:

 By needle inoculation through a sealed rubber closure

 Open tube method where the cap is removed and a cannula containing sterile oxygen-free CO2 is
allowed to continuously flow into the open tube. The open tube with flowing gas also allows
inoculation of roll-streak tubes for isolation of individual colonies.

The inoculated tubes are incubated until good turbidity is visible and the growth can be graded. Acid
production from carbohydrates is detected not by using pH indicators but by manually measuring the pH in
each tube. The VPI/PRAS system is expensive and labour intensive.

GAS-LIQUID CHROMATOGRAPHY

Many anaerobes produce large amounts of certain organic acid metabolic byproducts that are characteristic
of a given genus or species.

Propionibacterium = propionic acid

Actinomyces = succinic acid
Lactobacillus = lactic acid
Fusobacterium = butyric acid

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To detect these byproducts, organisms for testing are inoculated into a strictly prescribed basal medium
containing peptone and glucose. Volatile fatty acids are extracted with ether and nonvolatile acids with
chloroform. The ether and chloroform extracts are injected into the chromatograph so that the fatty acids
may be separated by their molecular weight or polarity.

FIGURE 8: Gas Chromatograh

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The presence of the various acids are recorded on a chart which can be interpreted by comparison with
controls.

FIGURE 9: Chromatographic Charts

COMMERCIAL SYSTEMS

Rapid Enzymatic Tests

These test systems are based upon detection of anaerobic preformed enzymes. They permit rapid
identification within four hours incubation.

Organisms are suspended in a diluent to a fairly heavy density and are inoculated into the substrates used by
the system. Coloured end results are read visually, a numerical biocode obtained and the species determined
from a computerized data base.

Other Systems

There are two other microtube systems:

 Api20A (Analytab Products Inc.)
 Minitek BBL which rely on bacterial generation of enzymes during a 24 hour incubation

All of these commercial systems may be unable to identify all anaerobic species without additional PRAS
tests or gas-liquid chromatography.

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SUSCEPTIBILITY TESTING OF ANAEROBES

Susceptibility testing of all anaerobic isolates remains controversial; however, anaerobes isolated from
blood, spinal fluid, cases of osteomyelitis plus those from brain abscesses and pelvic infections that fail to
respond to treatment may be tested. Certain resistant anaerobes such as the Bacteroides fragilis and
Prevotella-Porphyromonas groups should be tested.

At present most approved tests for anaerobic susceptibility testing are difficult to carry out in routine
microbiology laboratories. Testing is frequently done by reference laboratories.

Bacteroides fragilis, Prevotella and other Bacteroides species are frequently beta lactamase producers and
should be tested using nitrocefin disks (Cefinase).

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 APPENDIX

OXYGEN REQUIREMENTS

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