TCBS Agar : Introduction, Composition,Principle, Test Requirement,Procedure, Result -Interpretation, Limitation, and Keynotes

Introduction of TCBS Agar

Vibrio cholerae is the causative agent of cholera and Thiosulfate Citrate bile salts sucrose (TCBS) agar is 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. 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

Principle of TCBS Medium

Yeast extract and proteose peptone provide the nitrogen, vitamins, and amino acids in TCBS Agar while Sodium thiosulphate and Sodium citrate act as selective agents, providing an alkaline pH to inhibit Gram-positive organisms and suppress coliforms. Bile salts inhibit the growth of gram-positive microorganisms. Sucrose is a source of carbohydrates. Sodium chloride maintains optimum growth and metabolic activity of halophilic Vibrio spp. 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 are the pH indicators. Agar is the solidifying agent.

Procedure of Thiosulfate Citrate Bile Salts Sucrose 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 Thiosulfate Citrate Bile Salts Sucrose Agar

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

Test Requirements

• Type of specimen  -As specimen feces, food samples and water may be used.
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Vibrio cholerae ATCC 15748 and Escherichia coli ATCC 25922)

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
6. Examine colonial characteristics.

Interpretation of result for 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 the production of yellow colonies.
• V. parahaemolyticus produces green to blue-green colonies as it does not ferment sucrose.

Proteus growth on TCBS agar

Vibrio cholerae on prolonged incubation causing pH shift

Quality control of Thiosulfate Citrate Bile Salts Sucrose 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 parahaemolyticusATCC  17802: good-luxuriant growth; bluish-green colonies
• Escherichia coli ATCC 25922: Inhibited

Limitations of Thiosulfate Citrate Bile Salts Sucrose Agar

1. The medium should be inoculated heavily with fecal specimens because the growth of a few species may be inhibited on the medium due to the fermentation of sucrose and the accumulation of acids.
2. However, occasional isolates of Pseudomonas and Aeromonas may also form blue-green colonies on TCBS Agar.
3. Proteus species that are sucrose fermenters may form yellow colonies.
4. TCBS Agar is not a suitable medium for oxidase testing of Vibrio species.
5. A few strains of V. cholerae may appear green or colorless on TCBS Agar due to delayed sucrose fermentation.
6. TCBS Agar is highly selective for Vibrio species. Any H₂Snegative colony of TCBS Agar can be considered presumptive positive for Vibrio.
7. Further biochemical and serological tests must be carried out for complete identification.

Keynotes

• The need for alkaline pH in TCBS medium is due to enhance the growth of Vibrio cholerae, because this organism is sensitive to acidic environments.
• TCBS medium is a selective and differential agar for the isolation of Vibrio cholerae.

Bibliography

1. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/TCBSAgar.htm
2. http://himedialabs.com/TD/M189.pdf
3. https://www.sigmaaldrich.com/catalog/product/sial/86348
4. Merck Microbiology Manual 12th Edition
5. https://legacy.bd.com/europe/regulatory/Assets/IFU/HB/CE/PA/PA-254432.pdf
6. https://www.thermofisher.com/order/catalog/product/R01865#/R01865
7. https://www.humeau.com/media/blfa_files/265020.pdf
8. https://www.neogen.com/solutions/microbiology/thiosulfate-citrate-bile-salts-sucrose-agar/