Isolation and Identification of Vibrio cholerae from faecal sample
- Vibrio is short, curved, Gram-negative rods measuring 2×0.5 µm and that is motile by its single polar flagellum.
- It is a facultative anaerobe that produces acid from carbohydrate fermentation.
- The growth of most species of Vibrio is facilitated by Na + ion but not for Vibrio cholerae and Vibrio mimicus as these both can grow well in absence of NaCl.
- Vibrio produces enzymes like protease, nuclease, lipase, and chitinase.
- Around 12 species of Vibrio are identified to cause gastrointestinal and extra gastrointestinal infections that include V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, and V. alginolyticus.
- Vibrio cholerae (strain O1 and O139) is reported in the number of Cholera outbreak. It is non-halophilic that can grow in media without NaCl and fails to grow in media containing >6% NaCl.
- The optimum temperature for its growth is 370C and pH is 8.2.
- Thiosulfate citrate bile salts sucrose (TCBS) agar: Suspend the agar media in distilled water and heat to dissolve the media completely. Do not autoclave and pour the media in sterile plates.
- Alkaline Peptone Water (APW): Measure 5 gm of peptone and 5 gm of NaCl and dissolve it in distilled water. Adjust the pH 8.6 to 9.0 using 1 mol/L sodium hydroxide. Dispense the medium in a 10 ml screw-cap tube and sterilize by autoclaving at 121°C for 15 minutes.
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Laboratory Diagnosis of Vibrio cholerae
Specimen: The most required specimen for Vibrio spp. is a fecal matter. Rectal swabs are most accepted during the initial phase of the disease. The specimen should be collected in a sterile container and delivered as soon as possible to the microbiology lab. In the case of delay 5 ml, feces can be added in 20 ml Transport medium like alkaline transport medium with boric acid or Cary-Blair medium.
Microscopy: Direct microscopy is not preferred.
- Suspend 2 ml feces in 20 ml Alkaline Peptone Water (APW) with pH-8.6 and then incubate APW at 370C exactly for 5 hrs.
- Take loopful of the surface from APW and inoculate in Thiosulfate citrate bile salts sucrose (TCBS) agar and perform streaking on the agar surface. Incubate the plate at 370C overnight.
- The next day examines the TCBS plate for Vibrio like colonies (Table 1). Pick up the yellow colony of Vibrio from TCBS and inoculate in Nutrient Agar (NA) without NaCl. Incubate the media at 370C for 5 hours.
- After incubation observe the Nutrient Agar (NA) plate for the growth of the culture. Vibrio cholerae can grow with colorless, glistening, translucent colonies in Nutrient agar without NaCl. Vibrio cholerae should be confirmed by its sufficient growth on NA without salt, either on 1% tryptone water without NaCl or in a CLED plate. This conventional test is reliable enough for the isolation and identification of Vibrio cholerae. from other Vibrio sp.
- For further confirmation, perform Gram staining, Oxidase test, and Slide agglutination test. For this test, culture should always be taken from non-selective media like Nutrient agar. For slide agglutination tests take a small volume of culture from NA on a clean slide and make emulsion by adding saline water. Add an equal volume of antiserum O1 and tilt the slide back and forth for 30 sec-1 min. Vibrio cholerae produces strong clumping which is a positive test for slide agglutination. If the result is negative then perform the test again with antisera 0139.
- If Gram’s staining report is a negative rod, the Oxidase test is positive and the Slide agglutination test is also positive then presents the Report to the physician as Cholera.
Table: 1 Key Biochemical and physiological properties of Vibrio spp.
|Species||Colonies in TCBS||Growth in 0.0% NaCl||Gram’s test||Oxidase||Indole|
Agglutination, Vibriocidal, and antitoxin test, and ELISA. However, these tests are not suitable for the diagnosis of infections in Hospital settings.
Note: In the laboratory, if Nutrient agar without NaCl is not available then Vibrio cholerae culture can be identified even from its successful growth in cysteine-, lactose-, and electrolyte-deficient (CLED) media as this media lacks NaCl. For Biochemical and other confirmatory tests the subculture from CLED should be maintained again in ordinary media like Nutrient agar with or without salt. However, this is tedious and time-consuming work.
- Laboratory Methods for the Diagnosis of Vibrio cholerae; Centers for Disease Control and Prevention.
- Tille, P. (2015). Bailey & Scott’s diagnostic microbiology-E-Book. Elsevier Health Sciences.
- McCartney, J. E., Collee, J. G., & Mackie, T. J. (1989). Practical medical microbiology. Charchil Livingstone.