The genus Vibrio belongs to gram-negative bacteria. They possess a comma shape they are halophilic, meaning highly salt tolerant, and facultative anaerobes, oxidase positive and non-spore-producing. They are motile and have polar or lateral flagella. Its several species can cause foodborne infections, and that’s why the correct and timely identification of Vibrio species is extremely important for public health. In this blog, we intend to discuss the theory behind and the practical usage of Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar in isolating Vibrio species.

An Overview of Vibrio Species:

Vibrio species, such as V. cholerae, V. parahaemolyticus, and V. vulnificus, are important human pathogens that can cause foodborne diseases ranging from mild gastroenteritis to lethal septicemia. Prompt and specific detection of these species is important for early intervention and outbreak control. And in the clinical laboratory where precision matters, easy differentiation of these species is important for the proper treatment. In environmental monitoring, Vibrio identifications aid in evaluating water quality and preventing outbreaks.

Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar

TCBS Agar is a selective medium used specifically for the isolation of Vibrio cholerae and other enteropathogenic Vibrio species responsible for foodborne disease. It is derived from the research of Kobayashi et al., who developed Nakanishi’s selective medium. It favours the growth of numerous Vibrio strains, which have varied colony morphologies. Official procedures, including those from the APHA, recommend TCBS Agar for selective isolation of V. cholerae and V. parahaemolyticus. Enrichment of samples in Alkaline Peptone Water prior to plating on tcbs agar is a common practice to improve V. cholerae recovery. A modified type, Selective TCBS Agar, contains sodium cholate to further improve its selectivity.

In composition, proteose peptone and yeast extract provide fundamental nitrogenous factors, B vitamins, and general growth factors required. Sodium citrate and bile salts are inhibitory and play a selective action in that gram-positive bacteria as well as coliforms are prevented from growth. Sodium thiosulfate acts as a source of sulphur to allow detection of hydrogen sulphide production in combination with ferric citrate. Sucrose, a fermentable carbohydrate source, supports Vibrio metabolism. Acid production resulting from sucrose fermentation causes the pH indicators bromothymol blue and thymol blue to turn yellow. Sodium chloride maximizes growth and metabolism of halophilic species of Vibrio. Agar is used as the solidifying agent. The high pH level of the medium is important to increase the recovery of V. cholerae since the basic environment favours its growth.

Basic Principle of TCBS Agar:

It is a selective and differential medium specifically designed for the isolation of Vibrio species. Its unique formulation contains a number of key components:

1. Thiosulfate and Citrate: These components raise the pH level to approximately 8.6, thus suppressing the growth of most Enterobacteriaceae.
2. Bile Salts: They suppress gram-positive bacteria, further increasing media selectivity.
3. Sucrose: It is the differential component, allowing sucrose-fermenting Vibrio species to be distinguished.
4. Sodium Thiosulfate and Ferric Citrate: These reagents enable the detection of hydrogen sulphide (H₂S) formation.
5. Bromothymol Blue and Thymol Blue: They facilitate monitoring of sucrose fermentation and production of H₂S.

Bile salts and raised pH form a selective medium, while the fermentation of sucrose is responsible for causing yellow colonies due to acid formation. Non-sucrose fermenting species such as V. parahaemolyticus and V. vulnificus form blue-green or green colonies.

Implementation Strategies and Methodologies:

Sample Collection and Preparation

The appropriate sample types are seafood, water, and stool. Sample type must be handled and shipped appropriately in order to preserve integrity.

Inoculation and Incubation

The streak plate method is favoured for inoculation. Optimum incubating conditions are 35-37°C for 18-24 hours.

Colony Morphology and Interpretation

V. cholerae strains, based on their sucrose fermentation, usually form yellow colonies on this agar. V. alginolyticus also has this property. V. parahaemolyticus and V. vulnificus, being sucrose non-fermenters, form blue-green colonies. Although Proteus species that ferment sucrose can also form yellow colonies, certain V. cholerae strains can demonstrate retarded sucrose fermentation, producing green or colourless colonies. Despite strong selectivity towards Vibrio, it will support occasional growth and production of blue-green colonies in Pseudomonas and Aeromonas, albeit atypically. An entirely hydrogen sulphide-negative colony on this agar can be considered as a presumptive positive for Vibrio.

Advantages of TCBS Agar:

It provides excellent selectivity, differential capacity, and comparatively quick results.

Quality Control & Best Practices with TCBS Agar from TM Media:

At TM Media, we understand the importance of reliable culture media. That’s why our TCBS Agar undergoes rigorous quality control processes, adhering to ISO standards, to ensure optimal performance. For best performance, properly store it at 2-8°C.

Comparative Study: TCBS Agar and Other Media:

Alkaline Peptone Water can be used for Vibrio enrichment, but TCBS Agar is more selective and differentiating Culture Media. The selectivity and sensitivity of TCBS Agar towards Vibrio species render it an indispensable tool in environmental and clinical microbiology. However, it has to be known that TCBS is selective, not definitive, and requires additional analysis.

Conclusion:

TCBS Agar continues to be an essential medium for the isolation and preliminary identification of Vibrio species. At TM Media, we’re dedicated to providing microbiologists with quality-assured Agar.