How to Identify Vibrio Cholerae Using Biochemical Tests

In order to identify Vibrio cholerae species biochemically, we must first subculture colonies resembling Vibrio cholerae from TCBS agar to a non selective medium such as nutrient agar. This is essential before biochemically testing or serotyping sucrose-fermenting (yellow) colonies. Sufficient growth is usually obtained on a nutrient agar slope after 4 – 6 hours incubation at 35 – 37^oC.

1. cholerae 01 and 0139 reactions

• Oxidase positive. All Vibrio species are strongly oxidase positive.
• Do not ferment L-arabinose. This test is of value in differentiating V. cholerae from V. fluvialis (both produce yellow colonies on TCBS agar). V. fluvialis ferments L-arabinose.

Testing for L-arabinose fermentation can be easily and economically performed using a Rosco L-arabinose identification tablet (code 521 – 21).

Tests for Differentiate Vibrio cholerae 01 Types

In most countries, V. cholerae 01 cholera is caused by the El Tor biotype. In India and Bangladesh where the Classical biotype also occurs, the Voges-Proskaeur (VP) test (e.g. usinga Rosco tablet test) can be used to differentiate the two biotypes. The El Tor biotype is VP positive and Classical biotype is VP negative.

When required other tests (haemagglutination and sensitivity to 50iu polymyxin B) can be performed in reference laboratory. El Tor gives a positive agglutination test and is resistant to 50iu polymyxin. Classical biotypes give a negative agglutination test and sensitive to 50iu polymyxin B.

Serotyping V. cholerae 01 and 0139

Separate antisera are required to identify V. cholerae 01 (Inaba and Ogawa) and V. cholerae 0139 (Bengal). Isolates from TCBS cultures require subculturing to nutrient agar before carrying out serotyping.

Availability of Vibrio cholerae Antisera

Several manufacturers supply V. cholerae (slide agglutinating) antisera. The following range is supplied by S. A. Scientific Inc. in 3ml amounts preserved in glycerine:

• Vibrio cholerae Inaba, code 066911
• Vibrio cholerae Ogawa code 066912
• Vibrio cholerae 0139 Bengal, code 066914
• Vibrio cholerae Poly (Inaba, Ogawa, 0139), code 066913

Note: It is also recommended that in areas where V. cholerae 0139 occurs, microbiology laboratories contact their WHO Regional Office for information on local suppliers of antisera that can be used to identify Vibrio cholerae

Rapid detection of V. cholerae 01 and 0139 antigen in faecal specimens

New Horizon Diagnostics produce the following tests to detect V. cholerae 01 antigen and V. cholerae 0139 antigen directly in faecal specimens.

• Cholera Screen TM which is a latex monoclonal antibody based coagglutination test to detect V. cholerae 01 antigen (code 89-111100). It is priced US$ 750 for 100 tests (1999). Positive and negative controls are included.

• Bengal Screen TM, a latex monoclonal antibody coagglutination test to detect V. cholerae 0139 antigen (code 89-116100). It is priced US$ 750 for 100 tests. Positive and negative controls are included.

• Cholera SMART TM which is an immunochromatographic (IC) sensitive membrane-based antigen. It detects V. cholerae 01 antigen (code 89-113025). It is priced US$ 368.75 for 25 tests (1999). The visually read colour reaction is distinct.

• Bengal SMART TM, an immunochromatographic membrane-based antigen test to detect V. cholerae 0139 antigen (code 89-115025). It is priced US$ 368.75 for 25 tests (1999). Reactions are distinct and easy to read.

Storage: Coagglutination test kits need to be stored refrigerated (2 – 8^oC) whereas the IC membrane test kits can be stored at room temperature (18 – 25^oC).