The TCBS agar is a means of highly selective and differential solid culture, used for isolation and cultivation of bacteria of the genus Vibrio, especially Vibrio cholerae, V. parahaemolyticus and V. vulnificus as major pathogens of this kind.
The acronym TCBS stands for Thiosulfate Citrate Bile Sucrose. This agar is also known as a selective medium for Vibrios. The original formula was created by Nakanishi and later modified by Kobayashi.
Commercial TCBS medium and TCBS agar plate seeded with Vibrio cholerae. Source: photo 1: taken by the author MSc. Marielsa Gil / Photo 2: Microrao, JJMMC, Davangere, Karnataka, India
It is composed of yeast extract, meat peptone, triptein, sodium citrate, sodium thiosulfate, ox bile, sucrose, sodium chloride, ferric citrate, bromothymol blue, thymol blue and agar.
This composition allows an adequate development of Vibrio species from water, food and feces samples; with the exception of Vibrio hollisae, which does not grow on this medium. Furthermore, TCBS medium is capable of inhibiting the growth of other accompanying bacteria, especially coliforms.
Due to the serious gastrointestinal and extra-intestinal problems produced by some species of the Vibrio genus, its diagnosis is very important. Humans are mainly infected by eating raw or undercooked foods from the sea or polluted waters, but also through wound infection.
Because of this, clinical laboratories must include TCBS agar in the stool culture study of liquid stool samples, especially with the appearance of rice water. Especially if the patient reports having been in contact with sea water or having consumed shellfish or fish.
Basis
Yeast extract, meat peptones, and triptein are the nutritional source of this medium. However, TCBS agar is an inhospitable medium for most bacteria.
Its high selectivity is given by the addition of sodium citrate and ox bile; both are inhibitory agents that also provide an alkaline pH to the medium, restricting the growth of the accompanying flora and favoring the growth of V. cholerae, among other species. It should be noted that Vibrio cholerae is very sensitive to acidity.
For its part, sodium chloride osmotically balances the medium. Furthermore, as its concentration is high, it also acts as an inhibitory agent, favoring the growth of halophilic bacteria.
Sucrose is the fermentable sugar that, together with the bromothymol blue and thymol blue pH indicators, give the medium its differential character. For this reason, with this medium it is possible to differentiate sucrose fermenting strains from non-fermenting ones.
Colonies of sucrose fermenting strains develop yellow in color and will turn the medium from green to yellow due to acid production. The non-fermenters grow translucent and the medium remains the original color (green).
Likewise, this medium contains sodium thiosulfate as a source of sulfur and ferric citrate as a developing agent. Both show bacteria capable of producing hydrogen sulfide (colorless gas). H 2 S is formed from thiosulfate and subsequently upon reacting with ferric citrate a visible black precipitate forms.
Finally, the agar is what provides the solid consistency to the medium.
Preparation
Weigh 89 g of the dehydrated medium and dissolve in one liter of distilled water. Help dissolution by heating and frequent stirring. The mixture can be boiled for up to 2 minutes.
This medium is not autoclaved. After dissolving, it is served directly on sterile plates. When they solidify, they are arranged in an inverted way on platelets and stored in a refrigerator (2-8 ° C) until use.
The medium after preparation must remain at pH 8.6 ± 0.2.
The color of the dehydrated medium is light beige or greenish-beige, and the color of the medium is forest green or bluish green.
It is important to allow the plates to warm up before seeding the samples.
Use
The most common specimen for Vibrios isolation is diarrheal stool.
Stool samples, if they cannot be immediately seeded on the selective medium, should be transported on the Cary Blair medium.
To increase the sensitivity of the culture, the faeces can be passed through peptone water at pH 8.4 as an enrichment medium for a maximum of 8 hours, from there it is subcultured to the TCBS medium.
It should also be taken into account that some strains of Vibrios can cause septicemia in immunosuppressed patients, therefore they can be isolated from blood cultures. Likewise, samples of water and food from the sea can be analyzed when there are epidemic outbreaks of cholera disease.
Sown
The inoculum of the study sample must be prominent, the sowing is carried out by the striation method by exhaustion. The plates are incubated at 37 ° C for 24 hours in aerobiosis.
The presumptive colonies of Vibrio cholerae are medium in size, smooth, opaque, with thin borders and yellow in color due to the fermentation of sucrose.
The species of V. alginolyticus, V. fluvialis, V. hareyi, V. cincinnatiensis, V. furnissii, V. metschnikovii and some V. vulnificus grow in a similar way. Other clinically important Vibrios species such as V. parahaemolyticus do not ferment sucrose, developing as olive green colonies.
TCBS agar plate seeded with Vibrio parahaemolyticus. Source: isis325 Flickr
On the other hand, it must be borne in mind that some strains of Aeromonas and Plesiomonas that are oxidase (+) can grow in this medium, developing yellow colonies that can confuse the clinician. While some strains of Pseudomonas also oxidase (+) grow as green colonies like V. parahaemolyticus.
Limitation
The oxidase test that is positive for the Vibrio genus should never be performed from colonies obtained from TCBS agar, as the result obtained will be a false negative. Compounds in the middle strongly interfere with this test. Therefore, it must be made from subcultures on blood agar.
QA
To prove that the medium is in good condition, it is advisable to sow known or certified control strains, and observe if the growth meets the expected characteristics.
For this you can use strains of:
-Vibrio cholerae -– satisfactory growth (yellow colonies, translucent border).
-Vibrio parahaemolyticus --– satisfactory growth (colony with green center and translucent border).
-Vibrio alginolyticus ATCC 17749 - satisfactory growth (yellow colonies with halos of the same color around the colony).
-Enterococcus faecalis ATCC 29212 - total or partial inhibition (small yellow or translucent colonies).
-Pseudomonas aeruginosa ATCC 27853-– partial or total inhibition (blue colonies).
-Escherichia coli ATCC 25922 - Completely inhibited.
-Proteus mirabilis ATCC 43071 -– Total or partial inhibition. (Small colonies green center translucent border).
Incubation of uninoculated medium should be unchanged.
References
- Difco Francisco Soria Melguizo Laboratories. TCBS agar. 2009.Available at: f-soria.es
- BD Laboratory. BD. TCBS Agar 2003.Available at: bd.com
- Britannia Laboratories. TCBS Medium. 2015.Available at: britanialab.com
- Acumedia Laboratories. TCBS Agar. 2016.Available at: foodsafety.neogen.com
- Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
- Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.