Laboratory Methods For The Diagnosis of Vibrio Cholerae Chapter 4
Laboratory Methods For The Diagnosis of Vibrio Cholerae Chapter 4
Laboratory Methods For The Diagnosis of Vibrio Cholerae Chapter 4
Although V. cholerae O1 will grow on a variety of commonly used agar media, isolation from
fecal specimens is more easily accomplished with specialized media. Alkaline peptone water
(APW) is recommended as an enrichment broth, and thiosulfate citrate bile salts sucrose
(TCBS) agar is the selective agar medium of choice for isolating V. cholerae O1. In certain
instances (for example, when the patient is in very early stages of illness and is passing liquid
stool), it may not be necessary to enrich specimens or use selective plating media. However,
enrichment broth and a selective plating medium should always be used with convalescent
patients, suspected asymptomatic infections, environmental specimens, and whenever high
numbers of competing organisms are likely to be present in the specimen.
Figure IV-1. Overnight colonies of V. cholerae on TCBS agar are large (2-4 mm) and yellow
because of the fermentation of sucrose. They are characteristically round, smooth, glistening, and
slightly flattened.
Figure IV-2. On TTGA medium, colonies of V. cholerae are grey, flattened, and are surrounded by
a cloudy halo formed by the production of gelatinase.
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and translucent peripheries (Figure IV-1). The yellow color is caused by the fermentation of
sucrose in the medium. Sucrose nonfermenting organisms, such as V. parahaemolyticus,
produce green to blue-green colonies. Suspicious colonies for further testing should be
subcultured to a noninhibitory medium, such as gelatin agar, heart infusion agar (HIA), Kligler’s
iron agar (KIA), or triple sugar iron agar (TSI).
2. Taurocholate tellurite gelatin agar (TTGA or Monsur’s agar)
TTGA is a selective and differential agar specifically designed for the isolation of V. cholerae.
TTGA has a relatively long shelf life after preparation, and growth directly from the medium may
be used for oxidase and agglutination tests (Table IV-1). The disadvantages of this medium are
that it is not commercially available, and overnight colonies of V. cholerae on TTGA tend to be
smaller (1 to 2 mm) than those from the TCBS agar. Potassium tellurite, which is added to the
medium to increase selectivity, also varies in its quality, and each lot should be titrated to
determine the optimal concentration to use in TTGA medium (see Chapter XI, “Preparation of
Media and Reagents).
Overnight growth of V. cholerae on TTGA agar appears as small opaque colonies with slightly
dark centers (Figure IV-2). After 24 hours, the centers of the colonies become darker, and
eventually the entire colony becomes “gunmetal” grey in color. In addition to the dark coloration,
which is due to the reduction of tellurite, there is also an opaque zone around colonies which
resembles a halo. The halo effect, which is due to the production of the enzyme gelatinase, can
be intensified by brief (15- to 30-minute) refrigeration of the plate. Because many members of
the genus Vibrio have similar characteristics on TTGA, additional tests (antisera and/or
biochemicals) are necessary to screen isolates from this medium.
Direct testing
Colony Commercially of growth off
Medium Colony size Autoclaved
morphology available a
of plate
Grey, flattened
TTGA opaque zone 1-2 mm No Yes Yes
around colony
b Colorless to
MacConkey 1-3 mm Yes Yes No
light pink
Note: TCBS = thiosulfate citrate bile salts sucrose agar; TTGA = taurocholate tellurite gelatin agar.
a
Direct testing for agglutination in antisera or oxidase reaction.
b
Not all strains of V. cholerae O1 will grow on MacConkey agar.
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Figure IV-3. Overnight growth of V. cholerae on MacConkey agar appears as small (1- to 3
mm), translucent, colorless-to-light pink (lactose-negative) colonies.
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granular and iridescent with a greenish-bronze sheen. Colonies from this medium may be tested
directly for agglutination with antisera as well as oxidase and string test reagents. Salt-free GA
may be used as a screening medium to rule out halophilic (salt-requiring) marine vibrios
resembling V. cholerae, which are frequently isolated from seafood and environmental
specimens. See Chapter XI, “Preparation of Media and Reagents,” for special instructions for
preparation of gelatin agar.
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APW
a
6-8 hr, 35°-37°C
4. Slide agglutination
Fresh growth of suspect V. cholerae on a nonselective agar medium may be tested in V.
cholerae O1 polyvalent antiserum. Usually after 5 to 6 hours of incubation, growth on the
surface of the slant is sufficient to perform slide serology with V. cholera O1 polyvalent antisera;
if not, re-incubate overnight. Isolates that agglutinate in polyvalent antiserum to the O1
serogroup are presumptively identified as V. cholerae O1 (see Chapter VI, “Laboratory
Identification of V. cholerae,” for description of the slide agglutination method). Presumptive V.
cholerae O1 may be confirmed with agglutination in either monovalent Ogawa or Inaba antisera,
but confirmation may not be necessary for all isolates, particularly when the supply of antisera is
limited. The minimum identification of V. cholerae O1 requires only serologic confirmation of the
presence of O1 serotype antigens with suspect isolates. However, a more complete
characterization of the organism is sometimes necessary and may include toxin or hemolysis
testing, as well as determination of antimicrobial sensitivity, biochemical identification, biotype,
or molecular subtype. These types of tests should be performed only on selected isolates, such
as those recovered early in an outbreak or during surveillance in areas threatened by epidemic
cholera. Only isolates that are serologically confirmed to be V. cholerae O1 should be further
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Dark-field procedure
Freshly collected liquid (“rice-water”) stool is the specimen of choice for the dark-field
procedure. If stool is liquid but not rice-water in nature, it should be dilute 1:1 with saline before
direct testing. If freshly collected stool is negative, perform a 6- to 8-hour APW enrichment and
retest the enrichment broth. Antisera may be saved by first determining that the stool or
enriched specimen is motile before preparing the anti-serum-containing specimen for
examination. A known motile strain of V. cholerae O1 should be used as a positive control.
• Microscope with dark-field condenser (if an oil immersion lens is used [100X], the
objective must have an iris diaphragm)
To perform the test, place a drop of polyvalent V. cholerae O1 antiserum near the end of a clean
microscope slide. Next, mix a drop of freshly collected rice-water stool or a drop of a 6- to 8-
hour APW enrichment broth culture with the antiserum, and place another drop of stool or APW
at the opposite end of the slide. Place cover slips over the drops at each end of the slide. Using
dark-field, examine the drop containing the stool or enrichment broth for “shooting star” motility
by using the 40X objective. If this type of motility is detected, examine the antiserum-containing
mixture. If the motile organism is V. cholerae O1, the motility will be completely extinguished. If
the organisms under both cover slips are non-motile or if there is no difference between the
motility of either mixture, the organism is not V. cholerae O1.
2. Immunofluorescence
Immunofluorescence techniques using antisera conjugated to fluorescein isothiocyanate have
been used to visualize V. cholerae O1 cells in a variety of specimen types. Despite the utility of
immunofluorescence methods, they are not widely used as primary diagnostic tools because the
requirements for expensive equipment, high quality immunologic reagents, and trained
technicians.
3. Latex agglutination
A commercially manufactured slide agglutination kit (Denka Seiken, Tokyo, Japan), developed
for serotyping V. cholerae O1 isolates, has been used to detect the organism directly in fecal
specimens. The kit uses latex particles coated with monoclonal antibodies directed against the
A, B, and C antigens of V. cholerae O1. During an investigation of an epidemic of cholera, the
kit was evaluated for its ability to confirm the diagnosis of cholera at bedside using rectal swabs.
The latex agglutination test detected 63% of culture-positive patients from rectal swabs, but
gave false positive results for 12% of culture-negative patients. The sensitivity and specificity of
this test with liquid stool specimens have not been determined.
4. Coagglutination
In the coagglutination method, antibodies against V. cholerae O1 are bound to the surface of
Staphylococcus aureus (Cowan 1) cells while retaining their binding capacity and specificity. In
a positive reaction, staphylococcal cells are bound together in a lattice-like arrangement caused
by the formation of linkages between them created by the binding of the antibody on their
surface to the V. cholerae bacterial cells.
Problems using this technique have been attributed to substances in stool which nonspecifically
inhibit agglutination and lattice formation of staphylococcal cells. Recently, a commercially
prepared monoclonal anti-body-based coagglutination test (CholeraScreen, New Horizons
Diagnostics, Columbia, MD) has been marketed and appears to have overcome these
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obstacles. Reports of evaluations of the product with culture collections in the United States and
with clinical specimens in Guatemala and Bangladesh have been encouraging.
Other techniques for the rapid diagnosis of V. cholerae O1 include methods based on the
multiplication of bacteriophage, addition of antisera to growth medium to precipitate V. cholerae
cells in broth, and the use of antibody-coated magnetic beads to physically aggregate V.
cholerae bacterial cells.
References
1. Benenson AS, Islam MR, Greenough WB. Rapid identification of Vibrio cholerae by darkfield
microscopy. Bull WHO 1964; 30:827-31.
2. Colwell RR, Hasan JAK, Huq A, et al. Development and evaluation of a rapid, simple, sensitive,
monoclonal antibody-based co-agglutination test for direct detection of Vibrio cholerae O1. FEMS
Microbiol Lett 1992; 97:215-20.
3. Gustafsson B, Holme T. Rapid detection of Vibrio cholerae O1 by motility inhibition and
immunofluorescence with monoclonal antibodies. Eur J Clin Microbiol 1985; 4:29-4.
4. Morris GK, Merson MH, Hug I, Kibrya AKMG, Black R. Comparison of four plating media for isolating
Vibrio cholerae. J Clin Microbiol 1979; 9:79-83.
5. Shaffer N, Silva do Santos E, Andreason P, Farmer JJ. Rapid laboratory diagnosis of cholera in the
field. Trans Soc Trop Med Hyg 1989; 83:119-20.
6. World Health Organization. Manual for the Laboratory Investigations of Acute Enteric Infections.
Geneva: World Health Organization, 1987; publication no. WHO/CDD/83.3 rev 1.
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