Thiosulfate Citrate Bile Sucrose Agar ideal for vibrio species

Abbreviated TCBS, source url 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  click here 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

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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 extract5.0 g
Proteose Peptone10.0 g
Sodium thiosulfate10.0 g
Sodium citrate10.0 g
Ox gall5.0 g
Sodium cholate3.0 g
Sodium cholate3.0 g
Saccharose20.0 g
Sodium chloride10.0 g
Ferric citrate1.0 g
Bromothymol blue0.04 g
Thymol blue Agar0.04 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

  1. Suspend 88.1 gm of dehydrated medium in 1 liter of distilled or deionized water.
  2. Slowly bring to boiling, stirring with constant agitation until complete dissolution.
  3. Do not autoclave.
  4. 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. choleraeLarge yellow colonies
V. parahaemolyticusColonies with blue to green centers
V. alginolyticusLarge yellow mucoidal colonies
V. harveyi/V. fischeriGreyish-green to bluish-green colonies which show luminescence in dark. Older colonies fail to show bioluminescence.
Proteus sp/EnterococciPartial inhibition. If growth, colonies are small and yellow to translucent.
Pseudomonas /AeromonasPartial inhibition. If growth, colonies are blue
None Vibrio that produces H2Ssmall black colonies
Vibrio parahaemolyticus
Vibrio parahaemolyticus on TCBS agar
Yellow colonies of Vibrio cholerae  on TCBS
Yellow colonies of Vibrio cholerae on TCBS

References:

  • 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 Microbiology36(3): 150–151. doi:1046/j.1472-765X.2003.01280.xPMID 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
  • https://en.wikipedia.org/wiki/Thiosulfate-citrate-bile_salts-sucrose_agar
  • https://en.wikipedia.org/wiki/Vibrio
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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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