TCBS Agar

Principle And Interpretation

TCBS Agar was first formulated by Nakanishi (1) and further modified by Kobayashi et al (2). It promotes rapid growth of pathogenic Vibrio's after 24 hours incubation at 37°C. The contaminating non-vibrio's are suppressed. Present formulation is recommended by IS:5887(Part V) (3), APHA (4), FDA-BAM (5), ISO21872-1:2017(E) (6), ISO 11133:2014 (7), for isolation of Vibrio cholerae and Vibrio parahaemolyticus. Inoculate the sample in Alkaline Peptone Water (M618S), incubate overnight at 35°C. Subculture the growth on TCBS Agar (M870S). Peptone and yeast extract provide nitrogenous, carbonaceous compounds, long chain amino acids, vitamin B complex and other essential growth nutrients. Bile and the sodium citrate inhibit gram-positive bacteria (8). Sodium thiosulphate serves as a good source of sulphur, which in combination with ferric citrate detects the production of hydrogen sulphide. For the metabolism of Vibrio's, sucrose is added as a fermentable carbohydrate. Bromo thymol blue and thymol blue are the pH indicators. The alkaline pH of the medium improves the recovery of Vibrio cholerae. Strains of Vibrio cholerae produce yellow colonies on TCBS Agar because of fermentation of sucrose. Vibrio alginolyticus also produce yellow colonies. Vibrio parahaemolyticus is a sucrose non-fermenting organism and produces blue-green colonies, as of Vibrio vulnificus. As mentioned previously, occasional isolates of Pseudomonas and Aeromonas species also produce blue-green colonies, but overall TCBS Agar is highly selective and any H₂S-negative colony is possibly Vibrio species. In case of doubt, confirm Pseudomonas using oxidase test. The medium should be inoculated heavily with faecal specimens because some Vibrio species readily die off on the medium, owing to fermentation of sucrose and accumulation of acids.