Thiosulfate-citrate-bile salts-sucrose (TCBS) Agar, is a type of selective agar that is used in microbiology laboratories to isolate Vibrio species. TCBS Agar can be used to cultivate Vibrio cholerae from clinical specimens or other materials. Kobayashi et. al. developed TCBS Agar, which modified the Nakanishi selective medium. This medium was originally intended to isolate V. cholerae from V. parahaemolyticus. However, Vibrios can grow healthy colonies with many different colonial morphologies.
Composition of TCBS Agar
Final pH ( at 25°C) 8.6±0.2
Principle of TCBS Agar
TCBS Agar can be used to isolate Vibrio cholerae from other enteropathogenic vibrios. Proteose cholerae and yeast extract are essential growth nutrients. They provide nitrogenous compounds, vitamin complex, and other vital growth nutrients. Bile, a mixture of sodium citrate and bile salts, inhibits gram positive bacteria and coliforms. Sodium thiosulphate is a good source for sulphur. This, in conjunction with ferric citrate, detects the formation of hydrogen sulphide. Sucrose is used as a fermentable carbohydrate in Vibrios’ metabolism.
Bromthymol Blue turns yellow due to acidification in the medium from fermentation of sucrose. pH indicators are Thymol blue and Bromthymol. Sodium chloride is essential for the optimal growth and metabolism of halophilic Vibrio species. Agar can be used as a solidifying agent. An alkaline pH in the medium increases the recovery of V.cholerae. Therefore, an increase in pH can be used to boost Vibrio cholera’s growth.
This is recommended for cultivating Vibrio Cholerae and other enteropathogenic Vibrios that cause food poisoning in clinical and food specimens.
Preparation TCBS Agar
- In 1000ml of distilled water or deionized, suspend 89.08 grams of dehydrated medium.
- To dissolve the medium completely, heat to boiling
- Autoclave not
- Keep it at 45-50 degrees Celsius
- Mix everything together and pour onto sterilized Petri dishes.
Result Interpretation on TCBS Agar
|Vibrio cholera||Flat yellow colonies, 2-3 mm in diameter|
|Vibrio alginolyticus||Large yellow colonies|
|Vibrio fluvialis, Vibrio vulnificus||Yellow or translucent colonies|
|Vibrio parahaemolyticus||Colorless colonies with a green center|
|Pseudomonas, Aeromonas||Blue colonies|
|Enterobacteria or others||Tiny transparent colonies|
Quality Control on TCBS Agar
- Positive Control = Vibrio parahaemolyticus ATCC 17802 (Growth, blue green)
- Negative Control = Enterococcus faecalis ATCC 29212 (No Growth)
Uses of TCBS Agar
- TCBS Agar can be used to isolate Vibrio cholerae from other enteropathologic Vibrio, including Vibrio parahaemolyticus in fish, seafood, and biological samples of animals.
- They were also used to combat the spread of crown-of-thorns seastars (Acanthaster Planci).
Limitations of TCBS Agar
- Some strains might not grow well or grow properly on this medium due to nutritional variation. Vibrio species confirmation requires further testing.
- Because of the fermentation of sucrose and accumulations of acids, it is important to inoculate the medium with plenty of faecal specimens.
- However, occasionally isolated Aeromonas or Pseudomonas might form blue-green colonies on TCBSAgar.
- V. parahaemolyticus can be mistaken for Aeromonas hydrophila or Plesiomonas.
- Sucrose-fermenting Proteus spp. Produce yellow colonies that may look like Vibrio.
- TCBS is not a good medium to test Vibrio spp.’s oxidase.
- Due to delayed sucrose fermentation, some strains of V. cholerae might appear green or colorless when grown on TCBS Agar.
- For the best recovery of pathogenic organisms, it is recommended to use a non-selective medium in conjunction with selective media.
- They are very selective for Vibrio species. Any H2S-negative colony on TCBS Agar can also be considered to be presumptive positive. Further biochemical and serological tests are required to confirm the identification.