Tetrathionate MK Broth Base ISO

Description

During preparation, when iodine is added, tetrathionate is produced from the sulfate, and this salt together with the bile salts in the medium results in a strong inhibition of most of the normal intestinal bacteria, except for those which are capable of reducing tetrathionate, e.g. salmonellae. Reduction reactions liberate sulphuric acid, which is neutralized by the carbonate, avoiding a decrease in the pH, which is harmful even for salmonellae. However, many Proteus species resist the bile salt concentration and they may reduce tetrathionate. So, many authors recommend the addition of other inhibitors simultaneously, such as 0.1% Brilliant Green Solution (10 mL/L) and/or novobiocin at 40 mg/L.

Technique

Note: White precipitate is due to calcium carbonate and does not affect the broth's performance.

Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results.

Immediately before inoculation of Müller Kauffmann Tetrathionate Novobiocin Broth formulated according to ISO 6579:2017, supplement tubes/bottles to get the following concentrations of iodine/iodide solution:

• 2 mL of iodine-iodide solution to each 100 mL medium base in bottles;
• 0.2 mL of iodine-iodide to each 10 mL medium base in tubes.

Inoculate aseptically the tubes/bottles with the prepared sample or its dilution and incubate the tubes tightly closed aerobically at 37 ± 1 °C for 24 ± 3 h. Discard supplemented tubes if not used in less than 10 hours.

After enrichment incubation, inoculate any confirmatory, secondary medium by streaking methodology or by spiral method, like XLD. Enumerate all the colonies that have appeared onto the surface of the agar. Presumptive isolation or recovery of Salmonella must be confirmed by further microbiological and biochemical tests.

Each laboratory must evaluate the results according to their specifications.

Physical/chemical control

Microbiological control

Add supplement for functionality testing - inoculate: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity).

Add 20 mL/L of iodine-iodide solution.

Aerobiosis. Incubation at 37 ± 1 °C, reading after 24 ± 3 h.

Microbiological control according to ISO 11133:2014.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• DIN Standard 10160 Untersuchung von Fleisch und Fleischerzeugnissen: Nachweis von Salmonellen. Referenzverfahren.
• DIN Standard 10181 Mikrobiologische Milchuntersuchung: Nachweis von Salmonellen. Referenzverfahren.
• DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. 4th ed. APHA. Washington. DC. USA.
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. MD. USA.
• FIL-IDF Standard 93 (2001) Milk and milk products: Research of Salmonella.
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• ISO Standard 6579-1 (2017) Microbiology of food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp.
• ISO Standard 6785 (2001) Milk and Milk Products - Detection of Salmonella spp.
• ISO Standard 3565 (1975) Meat Products: Reference Method for detection of Salmonellae.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
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• MARSHALL, R.T. (1993) Standard methods for the examination of dairy products. 16th ed. APHA Washington. DC. USA.
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• U.S. PHARMACOPOEIA (2002) 25th ed. <61> Microbial Limits Test. US Pharmacopeial Convention Inc. Rockville. MD. USA.