The TCBS agar is a highly selective and differential solid culture medium, used for the isolation and cultivation of bacteria of the genus Vibrio, especially Vibrio cholerae, V. vulnificus Y V. parahaemolyticus as the main pathogens of this genus.
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.
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; except Vibrio hollisae, that does not grow in this environment. In addition, TCBS medium is capable of inhibiting the growth of other accompanying bacteria, especially coliforms..
Due to the serious gastrointestinal and extra-intestinal problems that some species of the Vibrio genus produce, its diagnosis is very important. Humans are mainly infected by eating raw or undercooked food from the sea or polluted waters, but also through wound infection.
Because of this, clinical laboratories should 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.
Article index
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. Notably Vibrio cholerae is very sensitive to acidity.
For its part, sodium chloride osmotically balances the medium. In addition, 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. 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). The HtwoS is formed from thiosulfate and subsequently, upon reacting with ferric citrate, a visible black precipitate forms..
Finally, the agar is the one that provides the solid consistency to the medium.
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 in plaquers and stored in a refrigerator (2-8 ° C) until use..
After preparation, the medium 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..
The most common specimen for Vibrios isolation is diarrheal stool..
Stool samples if they cannot be sown immediately on the selective medium, they must 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..
The inoculum of the sample under study 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 they are medium in size, smooth, opaque, thin-edged and yellow in color due to fermenting sucrose.
Similarly, the species of V. alginolyticus, V. fluvialis, V. hareyi, V. cincinnatiensis, V. furnissii, V. metschnikovii and some V. vulnificus. Other clinically important Vibrios species such as V. parahaemolyticus do not ferment sucrose, developing as olive green colonies.
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 just like V. parahaemolyticus.
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 should be performed from subcultures on blood agar..
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.
Yet No Comments