- Sample: Fresh diarrheal stool or stool collected in Cary Blair transport medium.
- Macroscopic examination: Rice watery stool with mucus flecks
- The laboratory diagnosis of cholera is based on colony morphology, culture characteristics, biochemical reactions, and serological identification by slide agglutination using specific antisera. However, a presumptive diagnosis of cholera can be made by an immobilization test.
I) Immobilization test: A rapid presumptive diagnosis of cholera can be made by observing the wet smear for the distinctive rapid to and fro movement (darting movement) of V. cholerae O1 and O139 due to their single polar flagellum. The movement can be stopped by adding one drop of V. cholerae O1 and O139 antiserum respectively.
Hanging drop method for Vibrio cholerae is one of the easy but most popular tests used for the presumptive diagnosis. Read details about this test here
II) Oxidase test: on performing an oxidase test from a pure sub-culture on nutrient agar, a positive reaction is observed. Oxidase test should not be performed directly from TCBS or MacConkey agar as acidification of these media may result in false-negative oxidase tests.
(Note: However, Aeromonas spp also gives a positive oxidase test result so further confirmation is necessary by culture and serotyping)
1. Cultural characteristics:
- Fresh stool can be directly plated on a non-selective medium like Mac conkey agar and a selective medium such as Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar.
- However, in the case of a rectal swab, the swab stick should be dipped in 10 ml of alkaline peptone water (APW) and incubate for 6-8 hours. After incubation, inoculation on solid media should be done only from the pellicle formed at the upper layer of the broth.
(Note: The broth should not be shaken before plating)
- After 18-24 hrs of incubation at 37°C observe the colony morphology.
- MacConkey agar: Appearance of pale, non-lactose fermenting,1-2 mm in diameter, flat with a serrated margin.
- TCBS: Button shaped yellow colonies of 1-2 mm diameter.
- Biochemical Reactions:
- Serological Reactions:
- Pick up the colonies resembling V. cholerae and perform a slide agglutination test using polyvalent and serotype specific antisera for V. cholerae O1, if negative perform agglutination test with O139 antiserum.
- Place two drops of normal saline on a slide side by side and emulsify colonies resembling V. cholerae from a nonselective medium on both.
- Add a drop of polyvalent O1 antisera to one of the suspension and tilt the slide to and fro. Observe for agglutination within a minute.
- If positive continue the same procedure with monospecific Ogawa and Inaba antisera. If a positive report is seen with Ogawa antisera, report it as Ogawa. Report it is as ‘Inaba’ if a positive result is seen with Inaba antisera but if positive with both antisera, report it as Hikojima.
- If none of the above shows a positive reaction, perform an agglutination test with O139 antiserum.
- V. cholerae O1 can be further differentiated into two biotypes by the following tests.
- Differentiation of the Classical and El Tor biotypes of V. cholerae serogroup O1.
|Chicken cell Agglutination (2.5% RBC)||
|Polymyxin B sensitivity (50 IU/disc)||
Key: S = Sensitive, R = Resistant, + = Positive, – = Negative