TCBS Agar – Composition, Principle, Preparation, Results, Uses

What is TCBS Agar?

• TCBS Agar, which stands for thiosulfate-citrate-bile salts-sucrose agar, is a specialized type of agar used in microbiology laboratories for the isolation of Vibrio species. It is a selective differential medium specifically designed to cultivate Vibrio cholerae and other Vibrio species from clinical specimens and various materials.
• The development of TCBS Agar can be attributed to Kobayashi et al, who made modifications to the selective medium originally created by Nakanishi. While initially intended for the isolation of V. cholerae and V. parahaemolyticus, TCBS Agar has proven effective in supporting the growth of most Vibrio species, resulting in the formation of healthy, visible colonies with distinctive morphologies.
• TCBS Agar’s selectivity is achieved through the combination of several components. Thiosulfate is included to inhibit the growth of most bacteria, except for Vibrio species, which are able to metabolize it. Citrate serves as a source of carbon, while bile salts and sucrose play a role in differentiating between different Vibrio species.
• When clinical specimens or other materials are streaked onto TCBS Agar, the selective properties of the medium allow Vibrio species to grow while inhibiting the growth of other bacteria present. This selectivity ensures that the isolated colonies predominantly consist of Vibrio species, making it easier to identify and differentiate them based on their characteristic colony morphologies.
• The colonies grown on TCBS Agar exhibit a wide range of appearances, aiding in the identification of different Vibrio species. For example, Vibrio cholerae typically forms large yellow colonies, while Vibrio parahaemolyticus produces green colonies. Other Vibrio species may display variations in colony size, color, and morphology, further contributing to the differentiation process.
• In summary, TCBS Agar is a selective differential medium used in microbiology laboratories to isolate and cultivate Vibrio species. By incorporating specific components that inhibit the growth of unwanted bacteria while promoting the growth of Vibrio species, TCBS Agar enables the isolation and identification of these organisms based on their distinct colony morphologies.

Composition of TCBS Agar

Ingredients	Grams/liter
Proteose peptone	10.0
Yeast extract	5.0
Sodium thiosulphate	10.0
Sodium citrate	10.0
Bile	8.0
Sucrose	20.0
Sodium chloride	10.0
Ferric citrate	1.0
Bromothymol blue	0.04
Thymol blue	0.04
Agar	15.0

Final pH ( at 25°C) 8.6±0.2

Principle of TCBS Agar

The principle of TCBS Agar revolves around its selective and differential properties that facilitate the isolation and identification of Vibrio cholerae and other enteropathogenic vibrios. The components of TCBS Agar work together to create an environment favorable for the growth of Vibrio species while inhibiting the growth of other bacteria.

Proteose peptone and yeast extract present in the medium provide essential growth nutrients, nitrogenous compounds, and vitamin B complex necessary for the growth of Vibrio species. Bile, derived from bile salts and sodium citrate, acts as an inhibitor for gram-positive bacteria and coliforms, selectively preventing their growth on the agar.

Sodium thiosulphate serves as a source of sulfur, and in combination with ferric citrate, it detects the production of hydrogen sulfide. Vibrio species are capable of metabolizing sodium thiosulphate, leading to the production of hydrogen sulfide, which can be detected as a black precipitate.

Sucrose, a fermentable carbohydrate, is included in TCBS Agar to support the metabolism of Vibrios. When Vibrio species ferment sucrose, they produce acidic byproducts that lower the pH of the medium. This decrease in pH causes bromthymol blue, a pH indicator present in the agar, to change from green to yellow, aiding in the differentiation of Vibrio colonies.

The addition of Thymol Blue, another pH indicator, further enhances the differentiation of Vibrio species based on their metabolic activity and resulting pH changes.

Sodium chloride is incorporated into TCBS Agar to provide an optimal environment for the growth and metabolic activity of halophilic Vibrio species, which thrive in high-salt conditions.

Agar acts as a solidifying agent, providing a solid surface for the growth of bacteria and facilitating the isolation of individual colonies.

The alkaline pH of the medium is adjusted to enhance the recovery of Vibrio cholerae. By increasing the pH, the growth conditions are optimized, promoting the growth of Vibrio cholerae colonies and facilitating their isolation.

In summary, the principle of TCBS Agar lies in its selective and differential components that create an environment conducive to the growth of Vibrio species while inhibiting the growth of other bacteria. The medium utilizes specific nutrients, inhibitors, pH indicators, and salts to selectively support the growth of Vibrio cholerae and distinguish it from other bacteria based on colony morphology and color changes.

Intended Use

This is recommended for cultivating Vibrio Cholerae and other enteropathogenic Vibrios that cause food poisoning in clinical and food specimens.

Preparation TCBS Agar

To prepare TCBS Agar, you can either purchase a commercially available pre-made agar or follow the instructions provided by the manufacturer if using a dehydrated powder form. Here is a general procedure for preparing TCBS Agar:

1. Suspend 88 grams of the TCBS Agar medium in one liter of purified water. The concentration may vary depending on the manufacturer’s instructions.
2. Heat the mixture with frequent agitation, ensuring that all the agar is completely dissolved. Boil the medium for one minute. It is important not to overheat the medium during this process.
3. Note that TCBS Agar should not be autoclaved. Autoclaving can adversely affect the composition and performance of the medium.
4. After the medium is prepared, aseptically dispense it into sterile Petri dishes. It is recommended to label the plates with the preparation date and assign a batch number for proper tracking and identification.
5. To maintain the quality of the medium, store the Petri dishes at temperatures between 2-8°C. It is advisable to store them in sealed plastic bags to prevent moisture loss.

Regarding the shelf-life of TCBS Agar, it is typically green in color when freshly prepared. As long as there is no visible contamination or alteration of the medium’s appearance or pH, it can be used for up to four weeks or longer.

The pH of the TCBS Agar medium should fall within the range of pH 8.4-8.8 at room temperature. This pH range is essential for optimal growth and differentiation of Vibrio species.

Result Interpretation on TCBS Agar

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 characteristics

On TCBS Agar, the colony characteristics of different Vibrio species can be observed and used for their preliminary identification. Here are the colony characteristics typically seen on TCBS Agar:

1. Reading Time: The results of the TCBS Agar culture should be read after an overnight incubation period of 18 to 24 hours.
2. Vibrio cholerae: V. cholerae colonies on TCBS Agar are large, measuring 2 to 4 mm in diameter. They have a slightly flattened appearance with opaque centers and translucent peripheries. V. cholerae and its biotype Eltor are able to ferment sucrose, resulting in a decrease in pH and the production of yellow colonies.
3. Vibrio parahaemolyticus: V. parahaemolyticus colonies on TCBS Agar typically appear green to blue-green in color. Unlike V. cholerae, V. parahaemolyticus does not ferment sucrose, which contributes to the difference in colony color.
4. Further Testing: If any colonies on TCBS Agar appear suspicious and require further confirmation, they should be subcultured onto a noninhibitory medium, such as triple sugar iron agar (TSI), for additional testing.

Quality Control on TCBS Agar

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

Uses of TCBS Agar

TCBS Agar finds several important applications in the field of microbiology. Some of the common uses of TCBS Agar include:

1. Isolation of Vibrio cholerae: TCBS Agar is primarily employed for the isolation and identification of Vibrio cholerae, the bacterium responsible for causing cholera. The selective and differential properties of TCBS Agar allow for the growth of Vibrio cholerae colonies while inhibiting the growth of other bacteria, facilitating their isolation and subsequent identification.
2. Isolation of other enteropathogenic Vibrio species: TCBS Agar is also utilized for the isolation and cultivation of other enteropathogenic Vibrio species, with Vibrio parahaemolyticus being of particular interest. It provides a suitable medium to support the growth of these bacteria, aiding in their isolation from various sources such as fish, seafood, and biological samples of animal origin.
3. Control of crown-of-thorns seastar outbreaks: TCBS Agar has been utilized in efforts to control outbreaks of the crown-of-thorns seastar (Acanthaster planci). This marine invertebrate species can cause significant damage to coral reefs. By using TCBS Agar, researchers and conservationists can study and monitor the presence of Vibrio species associated with the crown-of-thorns seastar, helping to better understand its ecology and potentially develop control strategies.

Limitations of TCBS Agar

While TCBS Agar is a useful medium for the isolation and identification of Vibrio species, it has several limitations that should be considered. These limitations include:

1. Nutritional Variation: Some strains of Vibrio may exhibit poor or no growth on TCBS Agar due to nutritional variations. This means that further tests are often required to confirm the presence of Vibrio species.
2. Inhibition of Growth: TCBS Agar may inhibit the growth of certain species due to the fermentation of sucrose and the accumulation of acids. To overcome this limitation, heavily inoculating the medium with fecal specimens is recommended to enhance the growth of Vibrio species.
3. False Positives: While TCBS Agar is selective for Vibrio species, occasional isolates of other bacteria such as Pseudomonas and Aeromonas may also produce blue-green colonies on the medium, leading to potential confusion during identification.
4. Similar Colony Morphologies: On initial isolation, V. parahaemolyticus colonies on TCBS Agar may resemble colonies of other bacteria such as Aeromonas hydrophila, Plesiomonas shigelloides, and certain Pseudomonas species, making differentiation challenging.
5. Similar Colonies with Other Bacteria: Some sucrose-fermenting Proteus species may produce yellow colonies on TCBS Agar, which can resemble Vibrio colonies, further complicating the identification process.
6. Unsatisfactory for Oxidase Testing: TCBS Agar is not suitable for performing oxidase tests on Vibrio species. Oxidase testing is an important step in the identification of these bacteria.
7. Variations in Colony Appearance: Some strains of Vibrio cholerae may exhibit delayed sucrose fermentation, resulting in green or colorless colonies on TCBS Agar, which can lead to confusion during identification.
8. Use in Conjunction with Non-Selective Media: To optimize the recovery of pathogenic organisms, it is recommended to use TCBS Agar in conjunction with non-selective media. This approach helps to ensure comprehensive identification and detection of relevant pathogens.
9. Further Confirmation Required: While TCBS Agar can be used as a presumptive test for Vibrio species, further biochemical and serological tests are necessary for complete identification and confirmation of the specific Vibrio species.

FAQ

References

1. https://microbeonline.com/tcbs-agar/
2. https://microbiologie-clinique.com/tcbs-agar.html
3. https://legacy.bd.com/europe/regulatory/Assets/IFU/HB/CE/PA/PA-254432.pdf
4. https://www.neogen.com/categories/microbiology/thiosulfate-citrate-bile-salts-sucrose-agar/
5. https://www.sigmaaldrich.com/IN/en/product/mm/103854
6. https://microbiologyinfo.com/thiosulfate-citrate-bile-salts-sucrose-tcbs-agar-composition-principle-uses-preparation-and-colony-morphology/