The tetrathionate broth or TT broth is a selective liquid culture medium for the enrichment and recovery of strains of the genus Salmonella. It was created by Müeller and later modified by Kauffmann, which is why there are those who call it Müeller-Kauffmann broth.
The original medium contained proteose peptones, calcium carbonate, and sodium thiosulfate. Kauffmann added bile salts to it and created another modality with bright green. These substances inhibit the growth of coliforms, leaving the medium free for the development of pathogenic bacteria, in this case Salmonella..
The modification was very successful because it significantly increased the sensitivity of the medium. For this reason, it is currently useful for the search for Salmonellas in any type of sample, but especially for solid or liquid stools and food..
Its preparation consists of two phases; the commercial medium is a base to prepare the tetrathionate broth, and subsequently so that the tetrathionate can be formed an iodinated iodine solution is added to complete the medium.
The American Public Health Association (APHA) recommends the use of Tetrathionate Broth supplemented with bright green for enrichment of samples for Salmonella, as it is more selective than Tetrathionate Broth and Selenite Broth..
In general, tetrathionate broth is ideal when the presence of bacteria of the genus Salmonella is suspected in small amounts or when they are abused by exposure to inhibitory substances or by industrial processes that minimize their viability..
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The peptones present correspond to pancreatic digest of casein and peptic digest of animal tissue. These provide the source of carbon, nitrogen, and general nutrients for bacterial growth..
For its part, sodium thiosulfate reacts with the iodinated solution to form tetrathionate. This inhibits the growth of coliforms and favors the development of bacteria that contain the enzyme tetrathionate reductase, among them is the genus Salmonella, but also Proteus.
Bile salts also act as an inhibitory substance for most Gram positive and some Gram negative bacteria (coliforms)..
Calcium carbonate absorbs the toxic substances generated by the decomposition of tetrathionate, which forms sulfuric acid. In this sense, calcium carbonate neutralizes acidity, keeping the pH of the medium stable..
In the case of the bright green modality, this substance increases the selective power of tetrathionate broth by inhibiting microorganisms other than the Salmonella genus..
Weigh:
The potassium iodide is dissolved in approximately 5 ml of sterile distilled water, then the iodine is added little by little while the mixture is heated. After it is completely dissolved, make up to the mark with sterile distilled water until reaching a final volume of 20 ml..
Weigh 46 grams of the dehydrated medium and suspend in 1 liter of sterile distilled water. Mix and heat until completely dissolved, it can bring to a boil only for a few minutes. Do not autoclave. The base of the medium is allowed to cool to approximately 45 ° C and at that time 20 ml of the iodinated solution is added..
After adding the iodide solution to the medium it should be used immediately. If you do not want to use the entire mixture, proceed as follows:
10 ml of the base medium is distributed in tubes, and only to those to be inoculated with samples is added 0.2 ml of the iodinated solution..
Those that are not going to be used can still be stored in the refrigerator, however, as the medium is not sterilized, the ideal is to prepare the exact amount that is needed..
The color of the medium before adding the iodine iodine solution is milky white with a white precipitate and after addition it is brown with a dense precipitate. The observed precipitate is normal and corresponds to calcium carbonate that does not dissolve. The final pH of the medium is 8.4 ± 0.2.
To prepare the bright green tetrathionate broth, all the steps described above are carried out, but additionally, 10 ml of the bright green solution prepared at 0.1% will be added to the mixture..
This solution is prepared as follows:
Weigh out 0.1 g of bright green and suspend in 100 ml of distilled water. Heat to a boil to achieve total dissolution. Store in amber bottle.
For stool samples (stool culture) the protocol is as follows:
Inoculate 1 g of solid stool or 1 ml of liquid stool in a tube with 10 ml of ready-to-use tetrathionate broth. Shake vigorously and incubate aerobically at 43 ° C for 6-24 hours.
Subsequently, take a 10-20 µl aliquot of the broth and subculture in a selective medium for Salmonella, such as SS agar, XLD agar, bright green agar, Hektoen enteric agar, among others..
In parallel, selective media for Salmonella should be inoculated with the direct sample (feces) without enrichment. For rectal swab specimens, discharge the collected material into the tube and proceed as described above..
For food samples, weigh 10 g of solid food or 10 ml of liquid food and inoculate a bottle with 100 ml of ready-to-use tetrathionate broth. Proceed in the same way as described above but incubate at 37 ° C.
As can be seen, the relationship between the sample and the broth will always be 1:10.
Known control strains can be used to test the culture medium. The most used are ATCC certified strains.
The strains to use are Salmonella typhimurium ATCC 14028, Salmonella abony DSM 4224, Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 25923.
Excellent development is expected for Salmonella strains, while Escherichia coli may have weak or regular development, and Gram positive strains (Enterococcus and Staphylococcus) are partially or totally inhibited.
-As this medium does not inhibit the growth of Proteus, some laboratories use to place 40 mg / L of novobiocin to avoid the development of this microbial strain. The antibiotic must be added before the iodine iodine solution..
-After preparing the medium including the iodine iodine solution, it should not take more than 2 hours to be inoculated..
-When distributing the medium in the tubes, the mixture must be continuously homogenized to resuspend the precipitate formed..
-In less contaminated samples, the tetrathionate broth is incubated at 35-37 ° C, and in highly contaminated samples incubation at 43 ° C is recommended..
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