Abbreviated TCBS, Thiosulfate-Citrate-Bile-Sucrose Agar was original developed by Nakanishi but was subsequently modified by Kobayashy et al.
TCBS is frequently used for the selective isolation of Vibrio cholerae and other enteropathogenic like Vibrios parahaemolyticus from suspected specimen.
TCBS agar is both selective and differential culture media. It is highly selective for Vibrio species and differential due to the presence of sucrose and the dyes. The Sucrose fermentation produces acid, which converts the colour of bromothymol blue or thymol blue. The presence of two dyes rather than one make the medium produce an array of yellow, green, or blue so that differentiating among various Vibrio species is possible.
Vibrio species are Gram-negative motile bacteria, possessing a curved-rod shape (comma shape), several species of which can cause food borne infection, usually associated with eating under cooked seafood. They are typically found in salt water and are facultative anaerobes that do not form spores. They are most widely recognized for their role in human intestinal infections causing causing severe diarrhea and dehydration (Cholera).
Principle of TCBS Agar
It contains high concentrations of sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae. The Inhibition of Gram-positive bacteria is achieved by the incorporation of ox gall, which is a naturally occurring substance containing a mixture of bile salts and sodium cholate, a pure bile salt. Sodium thiosulfate also serves as a sulfur source and its presence, in combination with ferric citrate, allows for the easy detection of hydrogen sulfide production. Saccharose (sucrose) is included as a fermentable carbohydrate for metabolism by Vibrio species. The alkaline pH of the medium enhances the recovery of V. cholerae and inhibits the growth of others. Thymol blue and bromothymol blue are included as indicators of pH changes
Composition of TCBS Agar
The follow are the approximate amounts per liter of ingredients required for the preparation of TCBS Agar in the laboratory
|Yeast extract||5.0 g|
|Proteose Peptone||10.0 g|
|Sodium thiosulfate||10.0 g|
|Sodium citrate||10.0 g|
|Ox gall||5.0 g|
|Sodium cholate||3.0 g|
|Sodium cholate||3.0 g|
|Sodium chloride||10.0 g|
|Ferric citrate||1.0 g|
|Bromothymol blue||0.04 g|
|Thymol blue Agar||0.04 g|
Preparation of TCBS Agar
Preparation of TCBS agar will depend on number of petri dishes to pour.Well note that a normal petri dish has a capacity of 20Ml
- Suspend 88.1 gm of dehydrated medium in 1 liter of distilled or deionized water.
- Slowly bring to boiling, stirring with constant agitation until complete dissolution.
- Do not autoclave.
- Cool to 50°C and pour into sterile Petri plates.
Incubate a petri dish for 24hour to detect if the preparation is contaminated.
Plate reading of Colony morphology on TCBS Agar
|V. cholerae||Large yellow colonies|
|V. parahaemolyticus||Colonies with blue to green centers|
|V. alginolyticus||Large yellow mucoidal colonies|
|V. harveyi/V. fischeri||Greyish-green to bluish-green colonies which show luminescence in dark. Older colonies fail to show bioluminescence.|
|Proteus sp/Enterococci||Partial inhibition. If growth, colonies are small and yellow to translucent.|
|Pseudomonas /Aeromonas||Partial inhibition. If growth, colonies are blue|
|None Vibrio that produces H2S||small black colonies|
- Thompson FL, Gevers D, Thompson CC, Dawyndt P, Naser S, Hoste B, Munn CB, Swings J (2005). “Phylogeny and Molecular Identification of Vibrios on the Basis of Multilocus Sequence Analysis”. Applied and Environmental Microbiology. 71 (9): 5107–5115.
- Madigan, Michael; Martinko, John, eds. (2005). Brock Biology of Microorganisms(11th ed.). Prentice Hall. ISBN 0-13-144329-1.
- Pfeffer, C.; Oliver, J.D. (2003). “A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments”. Letters in Applied Microbiology. 36(3): 150–151. doi:1046/j.1472-765X.2003.01280.x. PMID 12581373
- Kobayahsi, T.; Enomoto, S.; Sakazaki, R.; Kuwahara, S. (November 1963). “A New Selective Isolation Medium for the Vibrio Group; On a Modified Nakanishi’s Medium (TCBS Agar Medium)”. Nippon Saikingaku Zasshi (Japanese Journal of Bacteriology)(in Japanese). 18: 387–392. PMID 14115910 – via http://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TCBSAgar.htm