MICROBIOLOGY

068-1-MI

CAMPYLOBACTER , VIBRIO, AEROMONAS

AND SIMILAR ORGANISMS

THE MICHENER INSTITUTE for Applied Health Sciences

175
Acknowledgements

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

Contributors and Reviewers

R. Anstey, A.R.T.
G. Carmichael, A.R.T.
G. Chipperfield, R.T.
C.J. Semple, R.T.
R. Smith, 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.

Materials copyrighted to The Michener Institute may be purchased from the bookstore at the address above.

176
 DIVISION OF CONTINUING EDUCATION

MICROBIOLOGY

068-1-MI

CAMPYLOBACTER , VIBRIO, AEROMONAS

AND SIMILAR ORGANISMS

Table of Contents

PAGE
OBJECTIVES 2

RESOURCE MATERIAL 4

CAMPYLOBACTER S 5
CAMPYLOBACTER CLINICAL FEATURES 5
CAMPYLOBACTER LABORATORY DIAGNOSIS 6

HELIOBACTER SPECIES 10

VIBRIO 12
VIBRIO SPECIES 12
CLINICAL FEATURES 13
LABORATORY DIAGNOSIS 13

AEROMONAS 13

PLESIOMONAS 13

CHROMOBACTERIUM 14

SUPPLEMENT 068-1-MI-1 16

APPENDIX A: CLASSIFICATION 23

APPENDIX B: CLINICAL SIGNIFICANCE AND ORIGIN OF

CAMPYLOBACTER S 24

APPENDIX C: CLINICAL SIGNIFICANCE OF VIBRIOS, AEROMONAS

AND PLESIOMONAS 25

APPENDIX D: BIOCHEMICAL COMPARISON OF AEROMONAS,

PLESIOMONAS, VIBRIO AND CAMPYLOBACTER 26

068-1-MI -1-

177
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. Name two disease syndromes caused by the genera discussed in this module.

2. Compare and contrast these bacteria with the Enterobacteriaceae and Pseudomonas based upon the
oxidase and O/F tests, their flagella location and cellular appearance.

3. State the incidence of Campylobacter -caused gastro-enteritis relative to gastroenteritis caused by

Enterobacteriaceae.

4. Name the one species most frequently responsible for Campylobacter gastroenteritis symptoms and
indicate the usual characteristics of these symptoms.

5. State the source of Campylobacter jejuni infections.

6. Name a medium useful for transport of specimens containing Campylobacter .

7. State why selective agents must be incorporated in any medium designed for Campylobacter
isolation.

8. Explain why Campylobacter cannot grow in ordinary ambient air or candle jars.

9. State the recommended gas composition and concentration needed for Campylobacter growth..

10. Describe three ways to obtain the atmosphere suitable for Campylobacter growth.

11. State why 42°C is used for Campylobacter isolation when the organism grows well at 37°C.

12. Describe the typical colonial appearance of Campylobacter on isolation media such as Campy
blood agar.

13. Give the typical Campylobacter microscopic appearance.

14. Explain why saffranin is not used as a gram counterstain for Campylobacter .

15. Name three Campylobacter sp. screening tests and describe the organism's reactions or appearance
in these tests.

068-1-MI -2-

178
 16. Name five tests used to identify pathogenic Campylobacter species.

17. Using identification charts, differentiate between C. jejuni, C. coli and C. lari.

18. For Helicobacter pylori:

a) state the most common disease syndrome
b) differentiate from Campylobacter s based upon:
 isolation medium constituents
 humidity requirements
 urease production

19. Briefly describe the transmission of Vibrio cholera and the distinctive clinical features of V. cholera
gastroenteritis.

20. State what is meant by "rice water" stool.

21. Briefly describe the clinical features and transmission of V. parahemolyticus and V. vulnificus.

22. Give two clinical instances where a search for Vibrio should be considered.

23. State the purpose of thiosulfate citrate bile salts sucrose agar and describe the appearance of
pathogenic Vibrio growth upon it.

24. Indicate that Vibrio species tolerate increased salt concentration and that some species are halophiles.

25. Give the natural habitat and pathogenicity of Aeromonas and Plesiomonas.

26. Give the oxidase reaction, O/F reaction and the growth on MacConkey of Aeromonas and
Plesiomonas.

27. State the pigment produced by Chromobacterium violaceum.

28. Given a set of biochemical reactions, speciate a member of the Vibrio, Aeromonas or Plesiomonas
genera using an identification chart or a commercial system biocode.

068-1-MI -3-

RESOURCE MATERIAL

179
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.

Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenberger, P., Winn, W.C., Color Atlas of Diagnostic
Microbiology, 4th Edition, Lippincott, New York, 1992.

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

INSTRUCTIONAL MODULES DEVELOPED BY THE MICHENER INSTITUTE

[P] 065-1-MI Laboratory Identification of the Enterobacteriaceae

[P] 067-1-MI Pseudomonas and Related Organisms

068-1-MI -4-

MICROBIOLOGY
068-1-MI
CAMPYLOBACTER S, VIBRIO, AEROMONAS
180
 AND SIMILAR ORGANISMS

These Gram-negative rod genera are similar in that they all include some species that can cause
gastroenteritis or gastritis.
Also, these genera are oxidase-positive and are fermenters (except Campylobacter s). As well, they have
polar flagella and most are comma or spiral-shaped.

CAMPYLOBACTER S

Until relatively recently, Salmonella and Shigella were the primary bacteria sought as the cause of
gastroenteritis. Due to changing social conditions and the introduction of new selective media,
Campylobacter species have gained prominence as causative agents of gastroenteritis. In some geographic
areas, Campylobacter species are now isolated more frequently than Salmonella. It is estimated that 200,000
Canadians are afflicted yearly by Campylobacter-caused enteritis. Closely related to Campylobacter genera
are the Helicobacter spp. One of these, H. pylori, is the causative agent of chronic antral gastritis and
gastroenteritis.

Campylobacter Species

 C. jejuni
 C. coli
 C. lari

Helicobacter Species

 H. pylori

and many others.

CAMPYLOBACTER CLINICAL FEATURES

The most important Campylobacter species is C. jejuni which causes a watery, often bloody, diarrhea.
Usually accompanying the diarrhea are fever, abdominal pain, nausea and vomiting. Two other species, C.
coli and C. lari, may produce similar symptoms but less frequently.

Campylobacter infections often follow consumption of water, raw milk or food. One study indicated that
over 80% of chicken excrete the bacterium. Unlike food infections caused by Salmonella and
Staphylococcus. Campylobacter does not multiply in food.

068-1-MI -5-

It is thought that both an enterotoxin and a cytotoxin are responsible for the diarrhea since the presence of
red and white blood cells in the specimen indicates intestinal wall invasion.

Erythromycin is most frequently used for treatment.

181
CAMPYLOBACTER LABORATORY DIAGNOSIS

Presumptive Identification From Stool

It may be possible to make presumptive diagnosis of Campylobacter enteriditis by observing characteristic

gram-negative curved, S-shaped, gull wing, spiral forms in gram stain preparation of diarreheral stools.

Transport

Fecal specimens for Campylobacter isolation should be cultured within two hours of collection or, if a delay
is anticipated, the specimens should be refrigerated. Campylobacter species in stool survive best if they are
collected in Cary Blair transport medium.

Isolation Requirements

These organisms cannot be isolated on blood agar or MacConkey under ordinary atmospheric conditions.
Three conditions are required for isolation:

 A special growth medium

 Suitable atmospheric conditions (15% oxygen, 10% CO2, 85% Nitrogen)
 An elevated temperature for isolation (42°C)

Selective Isolation Media

The purpose of any Campylobacter isolation medium is not only to provide suitable nutrients for bacterial
growth, but also to inhibit the normal fecal flora by various antimicrobial agents.

Many media have been formulated, but the one most commonly used is Campy-BAP (formulation by
Blaser's medium).

068-1-MI -6-

The following are the constituents of CAMPY-BAP:

 Brucella blood agar base

 0.10 (10%) sheep blood
 Vancomycin (inhibits Gram-positives)
 Polymyxin (inhibits some Gram-negatives) 182
 Trimethoprin (broad spectrum -inhibits both Gram-positives and some Gram-negatives)
 Cephalothin (inhibits Gram-positives and some Gram-negatives)
 Amphotericin B (inhibits fungi).
Another media used routinely is by formulation of Skirrous media which has:

a) Lysed horse blood 7%

b) Vancomycin
c) Polymyxin B
d) Trimetophrim

Atmospheric Conditions

Campylobacters are true microaerophiles: (they require reduced oxygen conditions) and capnophila requires
increased CO2. The 0.21 (21 %) oxygen content of ordinary air must be reduced to approximately 0.05-0.06
(5%-6%) to grow them. Candle jars do not produce suitable growth conditions since the oxygen content,
after the candle has been extinguished, is 0.17-0.19 (17-19%). The easiest way to produce the needed
microaerophilic conditions is to use a Campy Pak envelope, a disposable commercial product. Some
workers simply evacuate and add the recommended gas composition of 85% nitrogen, 10% carbon dioxide
and 5% oxygen to a jar with a catalyst. Others merely evacuate 75% of the air from a jar and add a mixture
of 10% carbon dioxide and 90% nitrogen.

FIGURE 1: Campylobacter Gas Generating System

068-1-MI -7-

Temperature

Although the pathogenic Campylobacters grow well at 35°C to 37°C, they produce larger colonies at 42°C
to 43°C. There is an initial isolation advantage because some of the other bacteria in the specimen may not
grow as well or are inhibited at this higher temperature.

183
Length of Incubation

Plates may be examined at 24, 48 and 72 hours incubation. Forty-eight hours is the usual incubation period.
Plates examined after overnight incubation should be reincubated as soon as possible.

Colonial Appearance on CAMPY-BAP

Campylobacter colonies are nonhemolytic, slightly "wet" or mucoid and tend to spread along the streak
lines. The colonies themselves are pinkish to yellowish grey.

FIGURE 2: Campylobacter Colonies

Screening Tests

The three primary screening tests for Campylobacter identification include:

 Oxidase
 Wet-preparation motility
 The characteristic stained appearance

All Campylobacters are oxidase-positive.

They produce a characteristic darting motility in wet preparations viewed ideally with darkfield microscopy.
However, ordinary light microscopy may also be used. If a flagellar stain is used, single polar flagellae are
seen.

068-1-MI -8-

Gram Reaction

The Gram-stained cells appear as small Gram-negative rods which have a classic "sea-gull" or "gull-
winged" appearance. The cells may also be curved, "S"-shaped or demonstrate long spiral forms. Since
Campylobacter may not counterstain well with safranin, carbol fuschin is recommended.

FIGURE 3: Campylobacter Gram Stain

184
Species Identification

Pathogenic strains are differentiated from nonpathogenic strains by:

 A catalase positive reaction and
 The ability to grow at 35ºC and 42ºC but not at 25ºC
 C. jejuni is hippurate-positive whereas the other pathogenic Campylobacters causing gastroenteritis
are negative.
 Nalidixic acid and cephalothin disk tests are also routinely performed in many laboratories.

N.B: Some labs omit the hippurate test, preferring a report of C. jejuni/coli.

The only biochemical test that differentiates C. jejuni and C. coli Is hippurate hydrolysis. C. jejuni positive
(although 5% -10% can be hippurate negative), and C. coli is negative. There are molecular methods
available (such as PCR) for the differentiation of Campylobacter spp.
068-1-MI -9-

Although enteric infections with Campylobacter spp. appears to be identical, it is important to identify
Campylobacter isolates to species level for epidemiological purposes.

HELICOBACTER:

H. pylori: This species was initially called Campylobacter pyloridis and then Campylobacter pylori,
however recent evidence suggests that it does not belong to Campylobacter group.
185
Helicobacter pylori is found only on the mucus secreting epithelial cells of the stomach. In addition to
causing antral gastritis, Helicobacter pylori is suspected to be a factor in peptic/duodenal ulcer disease.
Specimen submitted for diagnosis are usually gastric biopsies, aspirates or brushings.

Culture and Isolation of H. pylori:

Specimens for recovery of H. pylori

For the disease of H. pylori -associated gastritis, histologic staining and culturing of biopsy specimens has
been considered "gold standard." Gastric and duodenal biopsies are the suitable specimens. Specimens
should be processed without delay and should be transported within 3 hours after collection, however,
specimens may be kept for at least 5 hours if kept @ 4°C. Tissues should be kept moist by the addition of 2
ml, or less of sterile saline.

Isolation Procedure

Grinding of specimens in a ground glass grinder yields a heavier growth than micing or rubbing the
specimens into an agar surface.

Media used for isolation of H. pylori include:

a) Nonselective blood agar medium or some institutions have included medium using Brucella brain
heart infusion (BHI) and tryptic Soy agar plates with 5% sheep or horse blood added.
b) Skirrow's formulation for Campylobacter isolation is also a suitable medium, since H. pylori will
not grow on Blaser's Campy-BAP or any selective medium containing cephalosporins.
c) Many laboratories have had good results using modified Thayer Martin agar as selective medium for
the isolation of H. pylori in mixed cultures.

068-1-MI - 10 -

Plates are incubated under microaerophilic conditions in a humid environment. The optimum temperature
for isolation is 35°C to 37°C, although some strains will grow at 42°C. Growth is usually observed in 3-5
days.

Identification of H. pylori

Colonies of H. pylori are small gray translucent and weakly beta haemolytic. Gram stain reveals pale
staining, curved gram negative bacteria with characteristic gull-wing and "U" shapes. Presumptive

186
identification can be made with positive reactions for oxidase and catalase and extremely rapid (within
minutes) urease reaction.

To diagnose H. pylori will include invasive procedures that require endoscopy (culture, stain PCR CLO
tests).

Non-invasive assays are available recently such as Urease and Serologic methods:

1) Urease Method: Urea breath test -patient will ingest 14C -labelled urea dissolved in water followed
by collection of breath samples that are analyzed for the presence of carbon dioxide after 1 hour.
2) A second method utilized radiolabelled urea containing 15N134, after oral ingestion, radiolabelled
urea is broken down into ammonia and carbon dioxide by H. pylori urease in the stomach. The
ammonia is absorbed into the blood and excreted in the urine. The sensitivity of the 15NH4
excretion test is reported to be 96% with 100% specificity when compared to patients who were H.
pylori positive by culture and Gram stain.

Serologic Method

Serologic test for detecting antibodies to H. pylori have been used mainly for epidemiological studies but
can be used also to monitor for efficacy of treatment. The principal format is the ELISA test for the
detection of IgG. Latex agglutination tests are available. IgA and IgM can also be detected but are less
useful diagnostically. The inherent problem is establishing a baseline for the positivity, since elevated
antibody titers is relatively high in certain populations.

Other Medically Important Helicobacter species:

Several species of Helicobacter other than H. pylori have been isolated from humans and are associated
with human disease. H. cinaedi, have been isolated from rectal swabs taken from symptomatic or
asymptomatic homosexual men, in patients with AIDS and another who are HIV positive. H. cinaedi has
also been isolated from women without any record of sexual contact from chilldren who manifest symptoms
of fever, bacteremia, cellulitis, skin infections and arthritis. The only known natural reservoir of H. cinaedi
is hamsters which may serve as reservoir for human ifections.

Reference: Diagnostic Microbiology, Koneman, et al: Fifth Edition

068-1-MI - 11 -

VIBRIO

The Vibrios are unique bacteria characterized by their curved or comma-shaped, polar-flagellated cells.

Vibrio species cause cholera and, as well, other seafood-related poisonings. Vibrios tolerate increased salt
levels and some are halophiles, requiring elevated sodium chloride concentrations for growth.

VIBRIO SPECIES
187
  Vibrio cholerae
 Vibrio parahemolyticus
 Vibrio vulnificus
 Vibrio alginolyticus

CLINICAL FEATURES

Cholera occurs after Vibrio cholera ingestion through fecally-contaminated food or water. In areas of the
world where hygiene is poor, cholera is very common. Cholera is usually thought of as a tropical disease,
endemic in the Far East, but in the nineteenth century up to 0.30 (30%) of immigrants to Canada died of
cholera either on shipboard or after debarking.

Vibrio cholera produces a severe diarrhea. The organism produces a powerful enterotoxin which acts on the
bowel mucosa, producing a profuse discharge of fluid. So rapid is the fluid loss that without replacement
therapy, severe dehydration and death occurs. The stool is frequently referred to as "rice water" stool
because it is flecked with mucus and is grey and watery.

Although cholera is now rare in developed countries, another species, Vibrio parahemolyticus, has caused
many cases of acute enteritis in the United States and is responsible for half the cases of bacterial food
poisoning in Japan. Poorly cooked shrimp, lobster and crab are most often implicated in transmission of the
bacterium.

Vibrio vulnificus produces a diarrhea after ingestion of undercooked or raw seafood such as oysters and
other shellfish. This organism also causes wound infections and a very serious septicemia which is
accompanied by a 50% mortality.

Vibrio alginolyticus is the most halophilic of all the Vibrios. Most strains can grow in the media with salt
concentrations as high as 10% NaCl. Vibrio alginolyticus are usually isolated from wounds on patients who
were in contact with salt water. Although pathogenic Vibrio, species grow well on many routine media, it is
important that the laboratory receive the appropriate clinical information (e.g., travelers with history of
seawater exposure) to ensure optimal set-up and isolation procedures.

068-1-MI - 12 -

LABORATORY DIAGNOSIS

Vibrio infections should be considered if diarrheal disease is related to ingestion of seafood or seawater. As
well, oxidase positive Gram-negative rods isolated from blood and wounds should also be suspected as
possible Vibrios.

Isolation

188
Isolation of Vibrios is improved by using Cary Blair transport medium. Since Vibrio cholera is killed by
acid, it may be selectively isolated in a medium having a very high pH (about 8.5 to 9.5). As well, Vibrios
tolerate increased concentrations of salt; those Vibrios which require increased levels of sodium chloride for
growth are called halophilic Vibrios.

Although Vibrios can be isolated on blood agar and MacConkey, selective media, thiosulphate citrate bile
salts sucrose (TCBS) medium is used to help inhibit normal fecal flora and to isolate the Vibrios. The
appearance of smooth yellow opaque colonies on this medium is highly suggestive of V. cholera. Both V.
parahemolyticus and V. vulnificus produce green-grey translucent colonies on TCBS medium. Vibrios may
be identified using traditional Enterobacteriaceae biochemicals and most commercial identification
systems.

Colonies will appear as nonlactose fermenters on MaConkey agar. Colonies should be spot tested for
oxidase, (using the patient's clinical history as a guide) and positive colonies subcultured for further
identification. TCSBS agar (thiosulfate -citrate -bile salts -sucrose) is the selective medium that may be used
particularly in area where infections are endemic or seasonal. On TCBS media, Vibrio alginolyticus colonies
are sucrose fermenting and appear yellow (as well as Vibrio cholerae) after overnight incubation.

AEROMONAS

Aeromonas is a Gram negative rod which has a similar habitat to Pseudomonas. It is more often found in
water and moist environments such as sink traps and drain pipes, and can be recovered from tap water
faucets and distilled water which are potential sources of organisms involved in nosocomial infections. The
name of the most significant species, Aeromonas hydrophila, is a useful reminder of its environmental
preferences.

Aeromonas hydrophila (water loving) is one of the Aeromonas species which can produce an acute diarrhea
similar to Campylobacter and Shigella. These species can also cause many nosocomial as well as wound
infections which develop after exposure to soil and water.

068-1-MI - 13 -

Types of infections caused by Aeromonas, other than the common gasteroenteritis also include:

1) Cellulitis and wound infections following exposure to contaminated water

2) Septicemia
3) Miscellaneous infections: UTI, hepatobilliary, Ear infections, Endocarditis, and
septicemia secondary to Aeromonas hydrophila.

Although Aeromonas is oxidase positive, it may appear in every other respect to resemble to that of an
Enterobacteriaceae. It produces:

189
 a) A uniform Gram negative rod
b) Its colonies appear similar to those of the Enterobacteriaceae on both blood agar and
MacConkey

In fact, on MacConkey agar it may even be a lactose fermenter. It has a fermentative O/F and TSI of A/A or
NA/A. In short, Aeromonas may be overlooked as ordinary Escherichia coli or other Enterobacteriaceae
species both morphologically and biochemically.

Aeromonas species have been misidentified as Vibrio species often with the use of commercial systems.
Supplemental media and biochemical tests are necessary for definitive identification of pathogenic Vibrio
species.

Selective agars used to culture Aeromonas:

1. Blood agar (with or without Ampicillin): blood agar can be made selective by incorporating 10µg/ml
of ampicillin, to improve recovery of Aeromonas from stool specimens.
2. CIN agar: originally developed for the isolation of Yersinia enterocolitica. CIN agar is also suitable
for recovery of Aeromonas from faeces.
3. Enteric agars such as MacConkey agar, Deoxycholate agar, Xylose, Lysine deoxycholate.

An oxidase test can easily exclude Aeromonas (Oxidase positive) from Enterobacteriaceae (Oxidase
negative).

Most Aeromonas species are indole positive.

PLESIOMONAS

The term Plesiomonas derived from the Greek word meaning "neighbour", indicating close association with
Aeromonas.

068-1-MI - 14 -

The one Plesiomonas species, Plesiomonas shigelloides is ubiquitous in surface waters and in soil and
commonly infects turtles, snakes, lizards. Primarily ingesting contaminated water or unwashed food infects
humans.

Plesiomonas related gastroenteritis in humans usually manifests as a mild water diarrhea in which the stools
are free of blood and mucin. Pathogenicity is probably related to the production of enteropathogenic
enterotoxin. Few isolated cases of extraintestinal infections include septicemia, neonatal meningitis,
cellulitis, septic arthritis and acute cholecystitis.

Laboratory isolation and identification:

190
 - Plesiomonas shigelloides is a straight, rounded short, motile gram negative rod.
- Grows well on sheep blood agar in 24 hours as grey, shiny, smooth and opague.
- Readily isolated on enteric agars such as MacConkey, deoxycholate, Hektoen and xylose lysine
deoxycholate.
- On MacConkey agar in non-lactose fermenter and sometimes can be confused as Shigella species.
- Oxidase and indole: positive

GENUS CHROMOBACTERIUM

Brief mention of the genus Chromobacterium is made because some strains are oxidase positive fermenters
and can be confused with Aeromonas and Vibrio species.

Chromobacterium violaceum is the most commonly encountered in clinical laboratory, however, it is seldom
associated with human disease. C. violaceum grows well on blood agar and most strains produce abundant
violet pigmentation that make recognition easy.

068-1-MI - 15 -

SUPPLEMENT 068-1-MI-l
USE OF THE CAMPY GAS PAK SYSTEM

PURPOSE

To generate gases to grow microaerophilic organisms such as Campylobacter.

REQUIREMENTS

 Gas Pak jars

191
  Fresh palladium catalyst
 10mL syringe
 Campy Pak envelope

METHOD

1. Place fresh activated catalyst in the lid of the jar.

2. Place only six* inoculated plates in the jar, media side up.**

3. Without crushing or depressing the envelope, cut the corner of the envelope as indicated.

4. Using a syringe, add 10 mL of tap water to the envelope. (Keep the envelope vertical).

5. Immediately, carefully place the envelope in the jar and clamp the lid (no indicator is necessary).

6. Visible condensation within an hour indicates active gas generation.

PRINCIPLE

Each envelope contains sodium borohydride, sodium bicarbonate and citric acid.

Upon addition of 10 mL of water, sufficient hydrogen and carbon dioxide are released so that:

 After the hydrogen has combined with available oxygen to form water, a residual (0.06) 6% oxygen
concentration remains in the jar
 A (0.10) 10% carbon dioxide concentration results

068-1-MI - 16 -

* The amount of oxygen remaining after 24-48 hours' incubation is influenced by the number of organisms
growing in the jar. When growth of organisms is heavy, colony size may be reduced.

** Include control plates of Campylobacter, Pseudomonas aeruginosa and Clostridium perfringens. If true
microaerophilic conditions exist, all these organisms will grow.

192