Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar- Composition, Principle, Uses, Preparation and Colony Morphology

TCBS Agar is used for the selective isolation of cholera vibrios and Vibrio parahaemolyticus from a variety of clinical and nonclinical specimens. Vibrio species are most widely recognized for their role in human intestinal infections, and cholera and V. parahaemolyticus diarrhea are important worldwide. All pathogenic Vibrio spp., except Vibrio hollisae, will grow on TCBS Agar.

The original formula developed by Nakanishi was subsequently modified by Kobayashy et al. for the selective isolation of pathogenic Vibrio species.

Composition of TCBS Agar

Ingredients per liter of deionized water:*

Sucrose 20.0 gm
Dipeptone 10.0 gm
Sodium Citrate 10.0 gm
Sodium Thiosulfate 10.0 gm
Sodium Chloride 10.0 gm
Yeast Extract 5.0 gm
Oxbile (Oxgall) 5.0 gm
Sodium Cholate 3.0 gm
Ferric Citrate 1.0 gm
Bromothymol Blue 0.04 gm
Thymol Blue 0.04 gm
Agar 15.0 gm

Final pH 8.6 +/- 0.2 at 25 degrees C. 

Principle of TCBS Agar

TCBS Agar is used for the selective isolation of Vibrio cholerae and other enteropathogenic vibrios. Thiosulfate and sodium citrate, as well as the alkalinity of the medium, considerably inhibit the growth of Enterobacteria. Ox bile and sodium cholate slow the growth of enterococci and inhibit the development of Gram-positive bacteria. The acidification of the medium resulting from the fermentation of sucrose by Vibrio makes bromthymol blue turn yellow. Bromthymol Blue and Thymol Blue are pH indicators. Using thiosulfate as a sulfur source, the production of hydrogen sulfide is visualized in the presence of ferric citrateYeast extract and peptone provides the nitrogen, vitamins, and amino acids in TCBS Agar. Sodium chloride provide optimum growth and metabolic activity of halophilic Vibrio spp. Agar is a Solidifying agent.

An increased pH is used to enhance growth of Vibrio cholerae, because this organism is sensitive to acid environments.

Uses of TCBS Agar

  1. TCBS Agar is used for the isolation of Vibrio cholerae and other enteropathologic Vibrio (in particular Vibrio parahaemolyticus) in fish, seafood and biological samples of animal origin.
  2. TCBS agar has also been used to control outbreaks of the crown-of-thorns seastar (Acanthaster planci).

Preparation of TCBS Agar

  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.

Result Interpretation on TCBS Agar

Typical colonial morphology on TCBS Agar are as follows:

Microorganisms

Characteristics

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

Colony Morphology on TCBS Agar

Colony Morphology on TCBS Agar

Limitations of TCBS Agar

  1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium. Further tests are necessary for confirmation of Vibrio spp.
  2. On initial isolation, V. parahaemolyticus may be confused with Aeromonas hydrophila, Plesiomonas shigelloides, and Pseudomonas spp.
  3. Sucrose-fermenting Proteus spp. produce yellow colonies which may resemble those of Vibrio.
  4. TCBS is an unsatisfactory medium for oxidase testing of Vibrio spp.
  5. A few strains of V. cholerae may appear green or colorless on TCBS Agar due to delayed sucrose fermentation.
  6. It is recommended that a non-selective media be used in conjunction with selective media for optimum recovery of pathogenic organisms.

Quality Control on TCBS Agar

Positive Control =  Vibrio parahaemolyticus ATCC 17802 (Growth, blue green) 
Negative Control = Enterococcus faecalis ATCC 29212 (No Growth)

References

  1. Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France.
  2. Dalynn Biologicals- TCBS Agar, Catalogue No. PT25
  3. Acumedia Manufacturers- TCBS AGAR (7210)
  4. HiMedia Laboratories Pvt. Ltd- TCBS Agar (M189)
  5. Becton, Dickinson and Company- BBL™ TCBS Agar (221872)
  6. Sigma-Aldrich, Inc.- 86348 TCBS Agar
  7. Remel- TCBS AGAR
  8. Conda Laboratories- TCBS AGAR (1074)
  9. Merck Microbiology Manual 12th Edition- TCBS Agar (1.10263)
  10. U.S. Food and Drug Administration- BAM Media M147: Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar
  11. Thermo Fisher Scientific Inc.- Dehydrated Culture Media, CHOLERA MEDIUM TCBS (CM0333)
  12. Hardy Diagnostics- TCBS AGAR (G55)
  13. Bacteria In Photos
  14. Microbe Online
  15. Wikipedia

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