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TCBS HiCynth™ Agar (Selective)
Intended Use
Recommended for the selective isolation of Vibrio cholerae and other enteropathogenic Vibrios.
Composition
| Ingredients | g/L |
|---|---|
| HiCynth™ Peptone No.3* | 16.000 |
| HiCynth™ Peptone No.5* | 5.000 |
| Sodium citrate | 10.000 |
| Sodium thiosulphate | 10.000 |
| Synthetic detergent | 2.000 |
| 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
* Chemically defined peptones
Directions
Suspend 89.08 gram 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 (3,4). Enrichment in Alkaline Peptone Water / Alkaline HiCynth™ Peptone Water (M618/ MCD618), followed by isolation on TCBS Agar is routinely used for isolation of V.cholerae (5,6,7) .TCBS HiCynth™ Agar is prepared by replacing animal and vegetable peptones with chemically defined peptones to avoid BSE/TSE risks associated with animal peptones.
HiCynth™ Peptone No.3 and HiCynth™ Peptone No.5 provide nitrogenous compounds, vitamin B complex and other essential growth nutrients. Synthetic detergent and sodium citrate inhibit gram-positive bacteria and coliforms (8). 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 from 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 HiCynth™ 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 (9). TCBS HiCynth™ Agar is a suitable medium for oxidase testing of Vibrio species (10). A few strains of V. cholerae may appear green or colourless on TCBS HiCynth™ Agar due to delayed sucrose fermentation (9). TCBS HiCynth™ Agar is highly selective for Vibrio species. However, occasional isolates of Pseudomonas and Aeromonas may also form blue green colonies on TCBS Agar (9). 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
Food samples; Water samples
Specimen Collection and Handling
For food samples, follow appropriate techniques for sample collection and processing as per guidelines (4).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (3).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
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 specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- 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.
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 | Inoculum (CFU) | Growth | Recovery | Colour of colony |
|---|---|---|---|---|
| Productivity | ||||
| Vibrio parahaemolyticus NCTC 10885 (00185*) | 50-100 | good-luxuriant | >=50% | blue |
| Vibrio furnissii NCTC 11218 ((00186*) | 50-100 | good-luxuriant | >=50% | greenish yellow |
| Specificity | ||||
| Escherichia coli ATCC 25922 (00013*) | >=104 | inhibited | 0% | |
| Escherichia coli ATCC 8739(00012*) | >=104 | inhibited | 0% | |
| Escherichia coli ATCC 11775( 00090*) | >=104 | inhibited | 0% | |
Additional Microbiological Testing
| Escherichia coli ATCC 25922 (00013*) | >=104 | inhibited | 0% | |
| Vibrio parahaemolyticus ATCC 17802 (00037*) | 50-100 | good-luxuriant | >=50% | bluish green |
| Vibrio vulnificus ATCC 29306 | 50-100 | fair-good | >=30% | greenish yellow |
| Vibrio fluvialis ATCC 33809 (00137*) | 50-100 | good-luxuriant | >=50% | yellow |
| Enterococcus faecalis ATCC 29212 (00087*) | >=104 | inhibited | 0% | |
| Vibrio cholerae ATCC 15748 | 50-100 | good-luxuriant | >=50% | yellow |
| Shigella flexneri ATCC 12022 (00126*) | >=104 | inhibited | 0% | |
| Proteus hauseri ATCC 13315 | >=104 | inhibited | 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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (11,12).
| Product Name | TCBS HiCynth™ Agar (Selective) |
|---|---|
| SKU | MCD870 |
| Product Type | HiCynth™ |
| Physical Form | Powder |
| Origin | Chemically defined (HiCynth™) |
| Packaging type | HDPE |
| References | 1. Kobayashi T., Enomoto S., Sakazaki R., and Kuwahara S., 1963, Jap. J. Bacteriol., 18: 387. |
| Customized Product Available | No |








