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TCBS Agar (Selective)

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SKU:
M870
Recommended for selective isolation of Vibrio cholerae and other enteropathogenic Vibrio’s.

Intended Use

Recommended for the selective isolation and cultivation of Vibrio cholerae and other enteropathogenic Vibrios.

Composition**

Ingredients g/L
Peptone, special 10.000
Yeast extract 5.000
Sodium citrate 10.000
Sodium thiosulphate 10.000
Sodium cholate 3.000
Bile# 5.000
Saccharose(Sucrose) 20.000
Sodium chloride 10.000
Ferric citrate 1.000
Bromo thymol blue 0.040
Thymol blue 0.040
Agar 15.000
Final pH (at 25°C) 8.8±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Oxgall

Directions

Suspend 89.08 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

TCBS Agar was developed by Kobayashi et al (1), who modified the selective medium of Nakanishi (2). Although this medium was originally designed for the isolation of V. cholerae and V. parahaemolyticus, most Vibrios grow to healthy large colonies with many different colonial morphologies. TCBS Agar is also recommended by APHA for the selective isolation of V.cholerae and V.parahaemolyticus (1,3). Enrichment in Alkaline Peptone Water (M618), followed by isolation on TCBS Agar is routinely used for isolation of V.cholerae (2,4,5). TCBS Agar, Selective has an additional selective ingredient i.e. sodium cholate for improved selectivity.

Peptone special and yeast extract provide nitrogenous, carbonaceous compounds, long chain amino acids, vitamin B complex and other essential growth nutrients. Bile and sodium citrate inhibit gram-positive bacteria and coliforms (6). Sodium thiosulphate serves as a good source of sulphur, which in combination with ferric citrate detects the production of hydrogen sulphide. For the metabolism of Vibrios, sucrose is added as a fermentable carbohydrate. Vibrio that is able to utilize sucrose will form yellow colonies. Bromothymol blue and thymol blue are the pH indicators. The alkaline pH of the medium improves the recovery of V.cholerae. Strains of V. cholerae produce yellow colonies on TCBS Agar because of fermentation of sucrose. V. alginolyticus also produce yellow colonies. V. parahaemolyticus is a sucrose non-fermenting organism and therefore produces blue-green colonies, as does V. vulnificus. Proteus species that are sucrose-fermenters may form yellow colonies (7). TCBS Agar is not a suitable medium for oxidase testing of Vibrio species (8). A few strains of V. cholerae may appear green or colourless on TCBS Agar due to delayed sucrose fermentation (7).

TCBS Agar is highly selective for Vibrio species. However, occasional isolates of Pseudomonas and Aeromonas may also form blue green colonies on TCBS Agar (8). Any H2S negative colony of TCBS Agar can be considered presumptive positive for Vibrio.

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.

Type of specimen

Clinical: faeces, etc; Food samples; Water samples

Specimen Collection and Handling

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (4). For food samples, follow appropriate techniques for sample collection and processing as per guidelines (6). For clinical samples follow appropriate techniques for handling specimens as per established guidelines (9,10). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

  1. 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.
  2. However, occasional isolates of Pseudomonas and Aeromonas may also form blue green colonies on TCBS Agar.
  3. Proteus species that are sucrose-fermenters may form yellow colonies.
  4. TCBS Agar is not a suitable medium for oxidase testing of Vibrio species.
  5. A few strains of V.cholerae may appear green or colourless on TCBS Agar due to delayed sucrose fermentation.
  6. TCBS Agar is highly selective for Vibrio species. Any H2S negative colony of TCBS Agar can be considered presumptive positive for Vibrio.
  7. Further biochemical and serological tests must be carried out for complete identification.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance

Light yellow to light tan homogeneous 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 : 8.8±0.2

pH

8.60-9.00

Cultural Response

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

Organism Growth Inoculum (CFU) Recovery Colour of colony
Productivity
Vibrio parahaemolyticus NCTC 10885 (00185*) good-luxuriant 50-100 >=50% blue
Vibrio furnissii NCTC 11218 ((00186*) good-luxuriant 50-100 >=50% greenish yellow
Specificity
Escherichia coli ATCC 25922 (00013*) inhibited >=104 0%
Escherichia coli ATCC 8739(00012*) inhibited >=104 0%
Escherichia coli ATCC 11775( 00090*) inhibited >=104 0%

Additional Microbiological Testing

Organism Growth Inoculum (CFU) Recovery Colour of colony
Escherichia coli ATCC 25922 (00013*) inhibited >=104 0%
Vibrio parahaemolyticus ATCC 17802 (00037*) good-luxuriant 50-100 >=50% bluish green
Vibrio vulnificus ATCC 29306 fair-good 50-100 >=30% greenish yellow
Vibrio fluvialis ATCC 33809 (00137*) good-luxuriant 50-100 >=50% yellow
Enterococcus faecalis ATCC 29212 (00087*) inhibited >=104 0%
Vibrio cholerae ATCC 15748 good-luxuriant 50-100 >=50% yellow
Shigella flexneri ATCC 12022 (00126*) inhibited >=104 0%
## Proteus hauseri ATCC 13315 inhibited >=104 0%

Key: (*) Corresponding WDCM numbers

## Formerly known as Proteus vulgaris

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 inorder 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. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (11,12).

Reference

  1. Kobayashi T., Enomoto S., Sakazaki R., and Kuwahara S., 1963, Jap. J. Bacteriol., 18: 387.
  2. Nakanishi Y., 1963, Modern Media 9: 246.
  3. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
  4. Lipps WC, Braun-Howland EB, Baxter TE, eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
  5. Morris G. K., Merson M. H., Huq A. K., Kibrya A. K. and Black R., 1979, J. Clin. Microbiol., 9:79.
  6. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
  7. Howard B., 1994, Clinical and Pathogenic Microbiology, 2nd Ed., The C.V. Mosby.
  8. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams & Wilkins, Baltimore, Md.
  9. Forbes B. A., Sahm A. S. and Weissfeld D. F., 1998, Bailey & Scotts Diagnostic Microbiology, 10th Ed., Mosby, Inc. St. Louis, Mo.
  10. Furniss A. 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.
  11. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
  12. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
More Information
Product Name TCBS Agar (Selective)
SKU M870
Product Type Regular
Physical Form Powder
Origin Animal
Packaging type HDPE
References 1. Kobayashi T., Enomoto S., Sakazaki R., and Kuwahara S., 1963, Jap. J. Bacteriol., 18: 387.2.Nakanishi Y., 1963, Modern Media 9: 246.3.Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods,5th Ed., American Public Health Association, Washington, D.C.4.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.5.Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology,8th Ed., American Society for Microbiology, Washington, D.C.6.Furniss A. L., Lee J. V. and Donovan T. J., 1978, The Vibrios, Public Health Laboratory Service Monograph SeriesNo. 11, Maidstone Public Health Laboratory, H.M.S.O., London, England.7.Forbes B. A., Sahm A. S. and Weissfeld D. F., 1998, Bailey & Scotts Diagnostic Microbiology, 10th Ed., Mosby, Inc. St.Louis, Mo.8.Howard B., 1994, Clinical and Pathogenic Microbiology, 2nd Ed., The C.V. Mosby.9.MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams& Wilkins, Baltimore, Md.10.Morris G. K., Merson M. H., Huq A. K., Kibrya A. K. and Black R., 1979, J. Clin. Microbiol., 9:79.11. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.12.Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015)Manual of Clinical Microbiology, 11th Edition. Vol. 1.
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