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

Thiosulfate-citrate-bile salts-sucrose (TCBS) Agar is a selective and differential medium for the isolation and identification of Vibrio species, especially Vibrio cholera, the causative agent of cholera. Cholera is an acute diarrheal disease that can be fatal if left untreated. Vibrio cholerae is a gram-negative, comma-shaped, facultative anaerobic bacterium that produces a potent enterotoxin that affects the intestinal mucosa.

TCBS Agar was developed by Kobayashi et al. in 1963 as an improvement over the previous media used for Vibrio isolation, such as alkaline peptone water and taurocholate-tellurite-gelatin agar. TCBS Agar has several advantages over these media, such as:

• It has a high pH (8.6) that inhibits the growth of most other bacteria, except for some alkalophilic species.

• It contains bile salts and sodium citrate that further suppress the growth of gram-positive bacteria and coliforms.

• It contains sucrose as a fermentable carbohydrate that allows the differentiation of Vibrio species based on their ability to ferment it. Vibrio cholera and Vibrio mimicus produce yellow colonies due to acid production from sucrose fermentation, while other Vibrio species produce blue-green colonies due to alkaline by-products.

• It contains sodium thiosulfate and ferric citrate that enable the detection of hydrogen sulfide production by some Vibrio species, such as Vibrio vulnificus and Vibrio parahaemolyticus. These species produce black-centered colonies due to the formation of ferrous sulfide.

• It contains bromthymol blue and thymol blue as pH indicators that change color according to the acid-base reaction of the medium.

TCBS Agar is widely used in clinical microbiology, food microbiology, environmental microbiology, and veterinary microbiology for the isolation and identification of Vibrio species from various samples, such as stool, water, seafood, fish, and animal tissues. TCBS Agar is also useful for monitoring outbreaks of cholera and other vibriosis infections in humans and animals. However, TCBS Agar has some limitations and considerations for its use, which will be discussed in the later sections of this article.

Vibrio cholerae is the causative agent of cholera, a severe diarrheal disease that can cause dehydration, shock, and death within hours of infection. Cholera is endemic in many parts of the world and can cause outbreaks in areas with poor sanitation and water quality. Therefore, rapid and accurate detection of V. cholerae from clinical and environmental samples is essential for the diagnosis, treatment, and prevention of cholera.

TCBS Agar is a standard medium for the isolation of V. cholerae or other Vibrio species from stool specimens of patients suffering from diarrhea, especially after seafood consumption or if outbreaks of cholera are suspected. It may also be used for detecting Vibrio in extraintestinal specimens, such as blood, urine, wound swabs, and food samples.

TCBS Agar is highly selective and differential for Vibrio spp., as it contains ingredients that inhibit the growth of most gram-positive bacteria and coliforms while enhancing the growth and metabolic activity of halophilic Vibrio spp. The medium also allows the differentiation of V. cholerae from other Vibrio spp. Based on their ability to ferment sucrose, which results in yellow colonies on TCBS Agar. V. cholera also produces hydrogen sulfide, which can be detected by the blackening of sodium thiosulfate in the medium.

TCBS Agar is widely used worldwide for the isolation of V. cholerae and has several advantages over other media, such as:

• It is commercially available and easy to prepare, requiring no autoclaving.

• It has high sensitivity and specificity for V. cholerae, as most strains produce large (2 to 4 mm), slightly flattened, yellow colonies with opaque centers and translucent peripheries on TCBS Agar.

• It has a relatively short incubation time (18 to 24 hours) compared to other media.

• It can be used in conjunction with other media, such as alkaline peptone water (APW), for the enrichment and confirmation of V. cholera.

Therefore, TCBS Agar is an important tool for the isolation of V. cholerae from various samples and provides valuable information for the diagnosis and epidemiology of cholera.

TCBS Agar is a selective and differential medium for the isolation and identification of Vibrio cholera and other enteropathogenic vibrios. The medium contains the following ingredients and their functions:

• Protease peptone and yeast extract: These are the sources of nitrogen, vitamins, minerals, and other essential nutrients for the growth of vibrios.

• Bile salts and sodium citrate: These are the selective agents that inhibit the growth of gram-positive bacteria and coliforms, which are normally present in the intestinal tract and can interfere with the isolation of vibrios.

• Sodium thiosulfate and ferric citrate: These are the indicators for hydrogen sulfide (H2S) production, which is a characteristic feature of some vibrios. Vibrios that produce H2S reduce sodium thiosulfate to sulfide, which reacts with ferric citrate to form a black precipitate in the medium.

• Sucrose: This is the fermentable carbohydrate that distinguishes Vibrio cholerae from other vibrios. Vibrio cholerae ferments sucrose rapidly and produces acid, which lowers the pH of the medium and changes the color of the pH indicators from green to yellow. Other vibrios either do not ferment sucrose or ferment it slowly, resulting in green or blue-green colonies on the medium.

• Bromthymol blue and thymol blue: These are the pH indicators that detect the acid production from sucrose fermentation. Bromthymol blue has a transition range from pH 6.0 (yellow) to pH 7.6 (blue), while thymol blue has a transition range from pH 6.0 (red) to pH 8.0 (yellow). The combination of these two indicators gives a green color to the medium at pH 8.6, which is the initial pH of TCBS Agar.

• Sodium chloride: This is the salt that provides the optimum osmotic pressure and metabolic activity for the halophilic (salt-loving) vibrios. The concentration of sodium chloride in TCBS Agar is 2%, which is higher than that in most other media.

• Agar: This is the solidifying agent that gives a firm consistency to the medium and allows the formation of discrete colonies.

The composition of TCBS Agar per liter of distilled water is as follows:

| Ingredient | Amount |

|------------|--------|

| Proteose peptone | 10 g |

| Yeast extract | 5 g |

| Bile salts | 5 g |

| Sodium citrate | 10 g |

| Sodium thiosulfate | 10 g |

| Ferric citrate | 1 g |

| Sucrose | 20 g |

| Sodium chloride | 20 g |

| Bromthymol blue | 0.04 g |

| Thymol blue | 0.04 g |

| Agar | 15 g |

The final pH of TCBS Agar after sterilization is 8.6 ± 0.2.

TCBS Agar is a highly selective and differential medium for the isolation and identification of Vibrio cholera and other enteropathogenic vibrios. The principle of TCBS Agar is based on the following features:

• The medium has a high pH of 8.6, which inhibits the growth of most gram-positive bacteria and coliforms but favors the growth of Vibrio spp., which are alkalophilic and halophilic.

• The medium contains bile salts and sodium citrate, which act as selective agents to further suppress the growth of non-vibrio bacteria.

• The medium contains sucrose as the sole fermentable carbohydrate, which is utilized by Vibrio spp. to produce acid and gas. The acid production lowers the pH of the medium and causes a color change of the pH indicators bromthymol blue and thymol blue from green to yellow. The gas production causes bubbles or cracks in the agar.

• The medium contains sodium thiosulfate and ferric citrate, which serve as indicators for hydrogen sulfide (H2S) production. Some Vibrio spp., such as V. cholerae and V. parahaemolyticus, produce H2S from the reduction of thiosulfate. The H2S reacts with ferric citrate to form a black precipitate of ferrous sulfide in the center or around the colonies.

• The medium contains protease peptone and yeast extract, which provide nitrogenous compounds, vitamins, and other essential nutrients for the growth of Vibrio spp.

By using these features, TCBS Agar can differentiate Vibrio spp. from other bacteria based on their colony morphology, color, and H2S production. For example:

• V. cholerae produces large (2-4 mm), round, smooth, yellow colonies with or without black centers due to sucrose fermentation and H2S production.

• V. parahaemolyticus produces small (1-2 mm), round, smooth, green, or blue-green colonies with black centers due to sucrose non-fermentation and H2S production.

• V. alginolyticus produces small (1-2 mm), round, smooth, green, or blue-green colonies without black centers due to sucrose non-fermentation and H2S non-production.

• Other Vibrio spp. may produce variable colony characteristics depending on their ability to ferment sucrose and produce H2S.

TCBS Agar is a ready-to-use medium that can be purchased as a dehydrated powder or prepared plates. To prepare the medium from the powder, the following steps are recommended:

• Suspend 89.08 g of dehydrated medium in 1000 ml of distilled or deionized water. Mix well to dissolve the powder completely.

• Heat the solution to boiling with frequent agitation to avoid scorching. Do not autoclave, as this may affect the pH and performance of the medium.

• Cool the medium to 45-50°C and pour it into sterile Petri plates. Alternatively, the medium can be stored in a sterile container at 2-8°C for up to one week before use.

• The prepared plates should have a yellow-green color and a pH of 8.6 ± 0.2. If the color or pH is not as expected, discard the medium and prepare a fresh batch.

To inoculate the medium, the following steps are recommended:

• Use a sterile loop or swab to streak the specimen onto the surface of the medium. For fecal specimens, inoculate heavily and spread evenly over the entire plate.

• Incubate the plates aerobically at 35-37°C for 18-24 hours. Some strains may require longer incubation for optimal growth and differentiation.

• Examine the plates for typical colonies of Vibrio spp. and other microorganisms. Refer to the result interpretation section for more details.

TCBS Agar allows the differentiation of Vibrio species based on their ability to ferment sucrose and produce hydrogen sulfide. The following table summarizes the typical appearance of different microorganisms on TCBS Agar:

| Microorganism | Colony color | Sucrose fermentation | Hydrogen sulphide production |

|---------------|--------------|----------------------|-----------------------------|

| Vibrio cholerae | Yellow | Positive | Negative |

| Vibrio parahaemolyticus | Blue-green | Negative | Negative |

| Vibrio vulnificus | Yellow-green | Positive | Negative |

| Vibrio alginolyticus | Blue-green | Negative | Negative |

| Vibrio fluvialis | Yellow-green | Positive | Negative |

| Vibrio mimicus | Yellow-green or blue-green | Positive or negative | Positive or negative |

| Vibrio holiday | Yellow-green or blue-green | Positive or negative | Negative |

| Aeromonas spp. | Blue-green | Negative | Negative |

| Pseudomonas spp. | Blue-green | Negative | Negative |

| Proteus spp. | Yellow | Positive | Positive |

The colonies of Vibrio cholerae are usually large (2-3 mm in diameter), smooth, opaque, and have a raised center. They may also show a characteristic "fish-eye" appearance due to the presence of a central depression.

The colonies of Vibrio parahaemolyticus are usually small (1-2 mm in diameter), smooth, translucent, and have an entire edge. They may also show a characteristic "turquoise" color due to the presence of a blue pigment.

The colonies of other Vibrio species may vary in size, shape, opacity, and color depending on the strain and the environmental conditions.

The colonies of Aeromonas and Pseudomonas species are usually similar to those of Vibrio parahaemolyticus, but they can be distinguished by their biochemical and serological properties.

The colonies of Proteus species are usually similar to those of Vibrio cholera, but they can be distinguished by their swarming motility and their production of hydrogen sulfide.

To confirm the identity of the isolated microorganisms, further tests such as oxidase test, indole test, motility test, salt tolerance test, and serological agglutination test should be performed.

TCBS Agar is not only useful for the isolation and identification of Vibrio cholera and other enteropathogenic vibrios but also has some applications in other fields. Some of the uses of TCBS Agar are:

• Fisheries and aquaculture: TCBS Agar can be used to detect Vibrio infections in fish, shellfish, and other aquatic animals. Vibrios can cause diseases such as vibriosis, cholera, and gastroenteritis in humans and animals. By using TCBS Agar, the presence and prevalence of Vibrio species can be monitored and controlled in aquaculture facilities and fish markets.

• Environmental microbiology: TCBS Agar can be used to assess the water quality and sanitation of natural and artificial water sources. Vibrios can survive and proliferate in various aquatic environments, such as seawater, brackish water, freshwater, estuaries, and coastal areas. By using TCBS Agar, the level of Vibrio contamination can be measured and compared among different water samples.

• Marine ecology: TCBS Agar can be used to study the diversity and distribution of Vibrio species in marine ecosystems. Vibrios are part of the natural flora of marine habitats and play important roles in nutrient cycling, bioluminescence, symbiosis, and pathogenesis. By using TCBS Agar, the ecological interactions and functions of Vibrio species can be explored and understood.

• Biotechnology: TCBS Agar can be used to isolate and screen Vibrio strains that have potential biotechnological applications. Vibrios produce various enzymes, toxins, antibiotics, pigments, polysaccharides, and other bioactive compounds that can be exploited for industrial, medical, and agricultural purposes. By using TCBS Agar, the desired Vibrio strains can be selected and characterized.

TCBS Agar is a versatile medium that can be used for various purposes related to Vibrio research and detection. However, it also has some limitations and considerations that should be taken into account when using it. These will be discussed in the next point.

TCBS Agar is a highly selective medium for the isolation of Vibrio cholera and other enteropathogenic vibrios, but it also has some limitations and drawbacks that need to be considered before using it. Some of the limitations are:

• Not all strains of Vibrio spp. Grow well or at all on TCBS Agar due to nutritional variation or inhibition by the acidic pH of the medium. Therefore, further tests are necessary to confirm the identity of Vibrio spp. from TCBS Agar.

• Some non-Vibrio bacteria, such as Pseudomonas, Aeromonas, Plesiomonas, and Proteus, may also grow on TCBS Agar and produce colonies that resemble those of Vibrio spp. in color or morphology. These bacteria need to be differentiated from Vibrio spp. by additional biochemical or serological tests.

• TCBS Agar is not suitable for oxidase testing of Vibrio spp., as the alkaline pH of the medium interferes with the reaction. Oxidase testing should be performed on a non-selective medium, such as nutrient agar.

• Some strains of V. cholerae may show delayed sucrose fermentation and appear green or colorless on TCBS Agar, which may lead to false-negative results. These strains need to be detected by other methods, such as culture on blood agar or serological tests.

• TCBS Agar is highly selective for Vibrio spp., but it does not differentiate between the different species or serotypes of Vibrio. For example, it cannot distinguish between V. cholerae O1 and O139, which are both responsible for cholera epidemics. Therefore, further tests are required to identify the specific Vibrio species or serotype from TCBS Agar.

Some of the considerations for using TCBS Agar are:

• TCBS Agar should be used in conjunction with a non-selective medium, such as blood agar or MacConkey agar, for optimal recovery and identification of pathogenic bacteria from clinical or environmental samples.

• TCBS Agar should be inoculated heavily with fecal specimens, as the growth of some Vibrio spp. may be inhibited by the fermentation of sucrose and accumulation of acids on the medium.

• TCBS Agar should not be autoclaved, as this may affect the pH and composition of the medium. It should be prepared by dissolving the dehydrated medium in boiling water and cooling it to 45-50°C before pouring it into sterile Petri plates.

• TCBS Agar should be stored in a dark place at 2-8°C and used within one month of preparation. It should not be exposed to light or moisture, as this may alter the color or performance of the medium.

TCBS Agar is a useful tool for the selective isolation of Vibrio cholera and other enteropathogenic vibrios, but it also has some limitations and considerations that need to be taken into account before using it. By following these guidelines, one can ensure the quality and accuracy of the results obtained from TCBS Agar.

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