Thiosulfate Citrate bile salts sucrose (TCBS) agar: Composition, uses and colony characteristics

Thiosulfate Citrate bile salts sucrose (TCBS) agar is a selective as well as differential culture medium used for selective isolation of  Vibrio spp from a variety of clinical and nonclinical specimens.  Vibrio cholerae is the causative agent of cholera. Other Vibrio species have been associated with gastroenteritis and extraintestinal infections, especially of the ear, soft tissue, and blood.

TCBS has a very high pH (8.5-9.5) which suppresses growth of intestinal flora other than Vibrio spp. The bile salts inhibit growth of gram-positive microorganisms. Vibrio spp produce either yellow or green colonies on TCBS, depending on whether they are able to ferment sucrose. If the sucrose is fermented, yellow colonies are produced. Vibrio cholerae produces yellow colonies in TCBS whereas Vibrio parahaemolyticus produces green colonies.

Composition of Thiosulfate Citrate bile salts sucrose (TCBS) agar and their function:

1. Yeast extract, bacteriological peptone: provides the nitrogen, vitamins, and amino acids in TCBS Agar
2. Sodium thiosulphate, Sodium citrate:  selective agents, providing an alkaline pH to inhibit Gram-positive organisms and suppress coliforms
3. Ox Bile: The bile salts inhibit growth of gram-positive microorganisms
4. Sucrose: fermentable carbohydrate
5. Sodium chloride: provide optimum growth and metabolic activity of halophilic Vibrio spp
6. Ferric citrate: Sodium Thiosulfate is also a sulfur source, and acts with Ferric  Citrate as an indicator to detect hydrogen sulfide production.
7. Bromothymol blue/ thymol blue: pH indicator.
8. Agar: Solidifying agent.

pH 8.6 ± 0.2 @ 25°C (Note the alkaline pH): An increased pH is used to enhance growth of Vibrio cholerae, because this organism is sensitive to acid environments.

Procedure for the preparation of TCBS Agar:

TCBS agar is commercially available and easy to prepare, requires no autoclaving.

1. Suspend 88 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. DO NOT AUTOCLAVE.

TCBS agar is green when prepared.

Interpretation of Results

Results of culture are read after overnight culture (18 to 24 hours).

• V. cholerae will produce large (2 to 4 mm in diameter), slightly flattened, yellow colonies with opaque centers and translucent peripheries. V. cholerae and its biotype Eltor ferment sucrose, this results in a decrease in pH and production of yellow colonies.
• V. parahaemolyticus, produce green to blue-green colonies as it does not ferment sucrose.

Suspicious colonies for further testing should be subcultured to a noninhibitory medium, such as triple sugar iron agar (TSI).

Limitation of TCBS Agar

1. TCBS Agar may not support the good growth of some Vibrio spp. (e.g., V. hollisae and V. metschnikovii). The identification of the various Vibrio spp. on TCBS Agar is presumptive and further tests are required for confirmation.
2. It is recommended that a non-selective media be used in conjunction with selective media for optimum recovery of pathogenic organisms.
3. Cultures grown on TCBS should be examined immediately after removal from the incubator as yellow colonies of Vibrio spp. (e.g., V. cholerae) may revert to a green color when left at room temperature.

Quality control

A medium of good quality gives the following mentioned results when cultured at 35°C in aerobic condition for 18-24 hours

1. Vibrio parahaemolyticus ATCC® 17802: Growth; blue-green centered colonies
2. Escherichia coli ATCC® 25922: Partial to complete inhibition; small, clear colonies