Thiosulfate citrate bile salts sucrose agar : Principle , Composition & Cultural characteristics

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Kobayashi et al.  created TCBS Agar by modifying Nakanishi’s selective medium. Although this medium was designed to isolate V.cholerae and V. parahaemolyticus, most Vibrio’s grow to healthy large colonies with a variety of colonial morphologies. The APHA also recommends Thiosulfate citrate bile salts sucrose Agar for the selective isolation of Vibrio cholerae and Vibrio parahaemolyticus. For routine isolation of V.cholerae, enrichment in Alkaline Peptone Water followed by isolation on TCBS Agar is used.

Principle of Thiosulfate-citrate-bile salts sucrose agar

  • Nitrogenous compounds, vitamin B complex, and other essential growth nutrients are provided by proteose peptone and yeast extract.
  • Bile, a bile salts and sodium citrate derivative, inhibits gram-positive bacteria and coliforms.
  • Sodium thiosulphate is a good source of sulphur, which detects the production of hydrogen sulphide when combined with ferric citrate.
  • Sucrose is added as a fermentable carbohydrate for Vibrio metabolism. Vibrio that can use sucrose will form yellow colonies.
  • The pH indicators are bromothymol blue and thymol blue.
  • The medium’s alkaline pH promotes V.cholerae recovery.
  • Sucrose fermentation causes V. cholerae strains to form yellow colonies on TCBS Agar.
  • Yellow colonies are also produced by V. alginolyticus. V.parahaemolyticus, like V.vulnificus, is a sucrose-non-fermenting organism that produces blue-green colonies.

Composition of Thiosulfate-citrate-bile-salts sucrose agar

IngredientsGms / Litre
Proteose peptone10.000
Yeast extract5.000
Sodium thiosulphate10.000
Sodium citrate10.000
Bile8.000
Sucrose20.000
Sodium chloride10.000
Ferric citrate1.000
Bromo thymol blue0.040
Thymol blue0.040
Agar15.000
Final pH ( at 25°C)8.6±0.2

Type of specimen

  • Faeces
  • Food samples
  • Water samples

Preparation procedure of Thiosulfate-citrate-bile salts-sucrose agar

  • In 1000 mL of purified/distilled water, dissolve 89.08 grams.
  • To completely dissolve the medium, bring to a boil.
  • DO NOT USE AN AUTOCLAVE.
  • Allow to cool to 45-50°C.
  • Pour into sterile Petri plates after thoroughly mixing.

Quality Control of TCBS agar

Uninoculated  Thiosulfate citrate bile salts sucrose agar
Uninoculated Thiosulfate citrate bile salts sucrose agar
AppearanceLight yellow to light tan homogeneous free flowing powder
GellingFirm, comparable with 1.5% Agar gel
Color and Clarity of prepared mediumBluish green colored clear to slightly opalescent gel forms in Petri plates
ReactionReaction of 8.9% w/v aqueous solution at 25°C. pH : 8.6±0.2
Cultural ResponseCultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
pH8.40-8.80

Cultural Response on Thiosulfate-citrate-bile salts-sucrose agar

OrganismGrowthColor of colony
Shigella flexneri ATCCinhibited 
Vibrio cholerae ATCC 15748good-luxuriantyellow
Vibrio fluvialis ATCC 33809 (00137*)good-luxuriantyellow
Vibrio parahaemolyticus ATCC 17802 (00037*)good-luxuriantbluish green
Vibrio vulnificus ATCC 29307 (00187*)fair-goodgreenish yellow

Limitations of Thiosulfate-citrate-bile salts-sucrose agar

  • The medium should be inoculated heavily with faecal specimens because growth of few species may be inhibited on the medium due to fermentation of sucrose and accumulation of acids.
  • However, occasional isolates of Pseudomonas and Aeromonas may also form blue green colonies on TCBS Agar.
  • Proteus species that are sucrose-fermenters may form yellow colonies
  • TCBS Agar is not a suitable medium for oxidase testing of Vibrio species.
  • A few strains of V. cholerae may appear green or colourless on TCBS Agar due to delayed sucrose fermentation.
  • TCBS Agar is highly selective for Vibrio species. Any H2S negative colony of TCBS Agar can be considered presumptive positive for Vibrio.
  • Further biochemical and serological tests must be carried out for complete identification

Storage and Shelf Life

  • Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label.
  • On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation.
  • Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use.
  • Use before expiry date on the label.
  • Product performance is best if used within stated expiry period.

Reference

  1. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed.,
  2. . Baird R.B., Eaton A.D., and Rice E.W., (Eds.), 2015, Standard Methods for the Examination of Water and Wastewater, 23rd ed., APHA, Washington, D.C.
  3. L., Lee J. V. and Donovan T. J., 1978, The Vibrios, Public Health Laboratory Service Monograph Series No. 11, Maidstone Public Health Laboratory, H.M.S.O., London, England.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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