Clostridium Perfringens Supplement (TSC)

Description

Sodium metabisulphite and ferric ammonium citrate are used as an indicator of sulphite reduction made by Clostridium perfringens spp. that produce black colonies in TSC Agar.

Technique

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

Reconstitute the vial in aseptic conditions and add it to 500 mL of melted agar base cooled to 50 °C.

Do not overheat once supplemented.

Pour the complete medium into Petri dishes (or tubes) and, once solidified on a flat surface, spread the plates either by streaking, by spiral method or by dilution banks.

Incubate the plates in anaerobic atmosphere at 35 ± 2 °C for 24-24 h. To obtain a more selective medium, incubate at 44 ± 1 °C.

Incubation times longer than those mentioned above or different incubation temperatures may be required depending on the sample or the specifications.

After incubation, count all the colonies that have appeared on the surface of the agar.

C. perfringens grows in black colonies, due to the iron sulfide precipitation.

Presumptive isolation of Clostridium perfringens must be confirmed by further microbiological and biochemical tests.

Reconstitute the original freeze-dried vial by adding 6 mL of sterile distilled water.

Note: each vial is sufficient to supplement 500 mL of medium (TSC Agar Base).

Physical/chemical control

Microbiological control

Reconstitute 1 vial as indicated in Composition; shake and dissolve completely.

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Anaerobiosis. Incubation at 44 ± 1 °C for 21 ± 3 h.

Sterility control

Add 5 mL of the sample to 100 mL of TSB and to 100 mL of Thioglycollate.

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.

• ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London.
• DIN Standard 10165. Referenz Verfahren fur Bestimmung von Clostridium perfringens. Fleisch und Fleischerzeugnissen.
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed. American Public Health Association. Washington.
• DIRECTIVA 2015/1787/UE de la Comisión por la que se modifica la Directiva 98/83/CE relativa a la calidad de las aguas destinadas al consumo humano (DO L260 de 7.10.2015 pg 6 y ss).
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International Inc. Gaithersburg. MD.
• ISO 7937 (2004) Microbiology of Food and Animal Feeding Stuffs. Horizontal Method for Enumeration of C. perfringens. Colony-count technique.
• ISO 6461-2 (1986) Water Quality - Detection and enumeration of the spores of sulfite-reducing anaerobes (Clostridia) - Part 2: Method by Membrane Filtration.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• ISO 14189 (2013) Water quality. Enumeration of Clostridium perfringens - Method using membrane filtration.
• SMITH, L.D. (1981) Clostridial Anaerobic Infections, in Diagnostic Procedures for Bacterial Mycotic and Parasitic Infections. 6th ed. APHA. Washington.
• UNE-EN ISO 11133 (2014). Microbiología de los alimentos para consumo humano, alimentación animal y agua - Preparación, producción, conservación y ensayos de rendimiento de los medios de cultivo.