TCBS Agar

TCBS Agar

Thiosulfate-citrate-bile salts-sucrose agar or TCBS Agar is a selective media used for the isolation and cultivation of Vibrio cholerae and other enteropathogenic Vibrio’s causing food poisoning

Principle

TCBS Agar was developed by Kobayashi and co-workers who modified the selective medium of Nakanishi. TCBS agar was originally designed for the isolation of V.cholerae and V. parahaemolyticus however, most Vibrios produce healthy large colonies with many different colonial morphologies.

  • Strains of cholerae ferment sucrose, which result in pH shift produce yellow colonies on TCBS Agar
  • alginolyticus: a sucrose fermenting organism, therefore, produce yellow colonies on TCBS Agar
  • parahaemolyticus: a sucrose non-fermenting organism and produces blue-green colonies on TCBS Agar
  • vulnificus: a sucrose non-fermenting organism and produces blue-green colonies on TCBS Agar
  • Proteus species: sucrose-fermenters may form yellow colonies.
  • harveyi, V. fischeri: Greyish-green to bluish-green colonies which show luminescence in dark. Older colonies fail to show bioluminescence.
  • Proteus/Enterococci: Partial inhibition. If grow, produce small yellow to transparent colonies.
  • Pseudomonas/Aeromonas: Partial inhibition. If grow, produce blue colonies.

Composition                             Ingredients Gms / Litre

Proteose peptone                             10.000

Yeast extract                                      5.000

Sodium thiosulphate                        10.000

Sodium citrate                                  10.000

Oxgall                                               8.000

Sucrose                                             20.000

Sodium chloride                               10.000

Ferric citrate                                     1.000

Bromothymol blue                           0.040

Thymol blue                                     0.040

Agar                                                 15.000

Final pH ( at 25°C) 8.6±0.2

  • Proteose peptone and yeast extract: supplies nitrogenous compounds, vitamin B complex and other essential growth nutrients.
  • Oxgall: a derivative of bile salts and sodium citrate that inhibit the growth  of gram-positive bacteria and coliforms
  • Sodium thiosulphate: a source of sulphur, which in combination with ferric citrate detects the production of hydrogen sulphide.
  • Sucrose: Fermentable carbohydrate. Vibrio that is able to metabolize sucrose will from yellow colonies.
  • Bromothymol blue and thymol blue: pH indicators.

Procedure

  • Suspend 89.08 grams in 1000 ml distilled water.
  • Heat to boiling to dissolve the medium completely.
  • Do not autoclave.
  • Cool to 50°C and pour into Petri plates

Appearance

TCBS agar appear as Light yellow to light tan free flowing powder

Gelling

Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium

Bluish green coloured clear to slightly opalescent gel forms in Petri plates.

Reaction

Reaction of 8.9% w/v aqueous solution at 25°C. pH: 8.6±0.2

pH

The pH of the medium is alkaline 8.40-8.80 of the medium improves the recovery of V.cholerae.

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours

Quality Control on TCBS Agar

Positive Control: =Vibrio parahaemolyticus

Negative Control = Enterococcus faecalis

Limitations of TCBS Agar

  • Further tests are recommended for confirmation of Vibrio spp.
  • Some strains may show poor growth or fail to grow on TCBS agar
  • On initial isolation, parahaemolyticus may be confused with Aeromonas hydrophila, Plesiomonas shigelloides, and Pseudomonas spp.
  • Some species of Proteus may ferment sucrose, therefore, produce yellow colonies which may resemble those of
  • TCBS agar is not suitable medium for oxidase testing of Vibrio spp.
  • A few strains of cholerae show delay fermentation of sucrose and may appear green or colorless on TCBS .
  • a non-selective media should be used in conjunction with selective media for isolation of pathogenic organisms
  • Although TCBS Agar is highly selective for Vibrio species, occasional isolates of Pseudomonas and Aeromonas may also produce blue green colonies on TCBS
  • Any H2S negative colony of TCBS Agar can be considered presumptive positive for Vibrio.
  • The medium should be inoculated heavily with fecal specimens because growth of few species may be inhibited on the medium due to fermentation of sucrose and accumulation of acids

 

 

By | 2018-04-18T11:44:25+00:00 April 18th, 2018|Food Microbiology|Comments Off on TCBS Agar

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