TCBS Agar: Introduction, Composition, Uses of Ingredients, Test Procedure, Result Interpretation and Limitation

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5 years ago

TCBS Agar: Introduction, Composition, Uses of Ingredients, Test Procedure, Result Interpretation and Limitation

Introduction of TCBS Agar

Vibrio cholerae is the causative agent of cholera and TCBS agar (Thiosulfate Citrate bile salts sucrose agar) a selective as well as differential culture medium used for selective isolation of this bacterium including its species from a variety of clinical and nonclinical specimens. TCBS agar has a very high pH (8.5-9.5) which suppresses the growth of intestinal flora other than Vibrio spp. The bile salts inhibit the growth of gram-positive microorganisms. Vibrio spp. produce either yellow or green colonies on TCBS agar, 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 agar whereas Vibrio parahaemolyticus produces green colonies.

Composition of Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar

Ingredients Gms / Litre

Proteose peptone: 10.0
Yeast extract: 5.0
Sodium thiosulphate: 10.0
Sodium citrate: 10.0
Bile: 8.0
Sucrose: 20.0
Sodium chloride: 10.0
Ferric citrate: 1.0
Bromo thymol blue: 0.04
Thymol blue: 0.04
Agar: 15.0
Distilled water (D/W): 1000 ml

Final pH ( at 25°C) 8.6±0.2

The function of Ingredients of TCBS Agar

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

Note: The need for alkaline pH is due to enhance the growth of Vibrio cholerae, because this organism is sensitive to acid environments.

Procedure of Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar preparation

Suspend 89.08 grams in 1000 ml distilled water.
Heat to boiling to dissolve the medium completely.
Do not autoclave.
Cool to 45-50°C.
Mix well before dispensing.
Pour Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar into each plate and leave plates on the sterile surface until the agar has solidified.
Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of TCBS Agar

Store at 2-8ºC and away from direct light.
Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Type of specimen

As specimen feces, food samples and water may be used.

Interpretation of result for Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar

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 result in a decrease in pH and production of yellow colonies.
V. parahaemolyticus produces green to blue-green colonies as it does not ferment sucrose.

Quality control of Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar

A medium of good quality gives the following mentioned results when cultured at 35°C in aerobic conditions for 18-24 hours.
Vibrio cholerae ATCC 15748:good-luxuriant growth, yellow colonies
Vibrio parahaemolyticus ATCC 17802: good-luxuriant growth; bluish-green colonies
Escherichia coli ATCC 25922: Inhibited

Limitations of Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar

The medium should be inoculated heavily with fecal specimens because the growth of few species may be inhibited on the medium due to the fermentation of sucrose and accumulation of acids.
However, occasional isolates of Pseudomonas and Aeromonas may also form blue-green colonies on TCBS Agar.
Proteus species that are sucrose-fermenters may form yellow colonies.
TCBS Agar is not a suitable medium for oxidase testing of Vibrio species.
A few strains of V. cholerae may appear green or colorless on TCBS Agar due to delayed sucrose fermentation.
TCBS Agar is highly selective for Vibrio species. Any H2S negative colony of TCBS Agar can be considered presumptive positive for Vibrio.
Further biochemical and serological tests must be carried out for complete identification.

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