TSC AGAR (base) INTENDED USE / HISTORY Tryptone Sulfite Cycloserine Agar was described by Harmon for the selective isolation and enumeration of Clostridium perfringens in water and food samples. The medium was recommended for the enumeration of sulfite-reducing anaerobes from foods of animal origin. PRINCIPLES - Sulfur reducing microorganisms reduce the sodium sulfite to sulfide, which with ferric citrate forms a black iron sulfide precipitate around the colonies. - For enumeration of Clostridium perfringens, it is recommended to incubate the inoculated medium at 37 C and to confirm characteristic colonies. - Contaminating flora are by D-Cycloserine which also reduces the size of the black halos around the colonies. PREPARATION - Suspend 42.0 g of dehydrated medium (BK031) in 1 liter of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Dispense 20 ml in 20 x 200 mm tubes or 100 ml per flask. - Sterilize in an autoclave at 121 C for 15 minutes. NOTE : With the ready-to-melt base media BM077, BM039 (or if the media was prepared in advance from the dehydrated base as above), melt the agar for the minimum amount of time necessary to achieve complete liquefaction. Cool and maintain at 44-47 C. 1/6
INSTRUCTIONS FOR USE Use in tubes (with D-Cycloserine) - Add 0.2 ml of reconstituted Selective Supplement D-Cycloserine 200 mg (BS006) to each tube. Mix well. - Heat the product to analyze in order to destroy vegetative cells and activate spores. - Transfer 1 ml of the inoculum and its serial tenfold dilutions to the tubes, avoiding the incorporation of air in the medium. - Homogenize thoroughly. - Cool in an ice water bath. - Incubate at 46 C or 37 C, for 20-24 hours, depending on the analytical protocol applied. NOTE : Avoid heating the tubes after inoculating. Use in Petri dishes ø 90 (Base medium + D-Cycloserine) - Aseptically add 1.0 ml of reconstituted D-Cycloserine Selective Supplement 200 mg (BS006) to 100 ml of base medium. - Inoculate 1 ml of the inoculum and its serial tenfold dilutions to the empty plates. - Pour 15 to 20 ml of complete medium. Mix well. - Let solidify on a flat, cool surface. - Inoculated plates are incubated in an anaerobiosis jar in the presence of a mixture of hydrogen and carbon dioxide for 24 hours at 37 C. - The plates are read immediately after opening the jar, since the colonies may become pale through the oxidation of iron sulfide as a result of exposure to fresh air. - Carry out the tests for the identification of Clostridium perfringens : Gram-staining, catalase, nitrate reduction test, mobility, gelatin hydrolysis, growth in LS Broth (BK140), etc... Base medium + cycloserine + egg yolk - The medium can be supplemented with a suspension of Egg Yolk (BS066) at 8 ml per 100 ml of melted base medium held at 44-47 C, in order to detect the lecithinase produced by Clostridium perfringens. Base medium without cyclosérine (for water control analyses by membrane filtration method ; Petri dishes ø 55) - Pour a first layer of roughly 2 mm. - Let solidify on a flat, cool surface. - Heat the inoculum to destroy vegetative forms and to reactivate the spores. - Aseptically filter a known volume of the water sample through a nitrocellulose membrane. - Deposit the membrane, filtered side down and making sure that the membrane and agar are in close contact. - Quickly pour a second layer, in order to obtain a thickness of 5mm total of agar. - Let solidify on a flat, cool surface. - Incubate under anaerobic conditions for 20 to 44 hours at 37 C. RESULTS Count colonies surrounded by a black halo. When grown in the presence of egg yolk, colonies of Clostridium perfringens are surrounded by an opaque halo due to the production of lecithinase. 2/6
TYPICAL COMPOSITION of the complete medium (can be adjusted to obtain optimal performance) For 1 liter of medium : - Tryptone...15.0 g - Papaic digest of soybean meal...5.0 g - Yeast extract...5.0 g - Sodium metabisulfite...1.0 g - Ferric ammonium citrate...1.0 g - D-cycloserine...0.4 g - Bacteriological agar...15.0 g ph of the ready-to-use medium at 25 C : 7.6 ± 0.2. QUALITY CONTROL - Dehydrated medium : beige powder, free-flowing and homogeneous. - Prepared medium : amber agar. - Typical culture response (complete medium with D-cycloserine) after 20 hours of anaerobic incubation at 37 C in Petri dishes (XP CEN ISO/TS 11133-2) : Clostridium perfringens NCTC 8238 Escherichia coli ATCC 25922 Growth (Productivity ratio : P R ) P R 50% P R 50% - Typical culture response (complete medium with D-cycloserine) after 20 hours of anaerobic incubation at 46 C in Petri dishes (NF V 08-061) : Growth (Productivity ratio : P R ) Escherichia coli ATCC 25922 P R 70% - Typical culture response (complete medium with D-cycloserine) after 24 hours of anaerobic incubation at 36 C in tubes (NF T 90-415) : Groth (Productivity ratio : R 3 ) Escherichia coli RIVM WR1 66% R 3 150% 3/6
- Typical culture response (TSC without D-cycloserine) after 20 hours of anaerobic incubation at 37 C (NF EN 26461-2 ; NF T 90-461) : Groth (Productivity ratio : R 3 ) Escherichia coli RIVM WR1 66% R 3 150% STORAGE / SHELF LIFE Dehydrated base medium (without D-cycloserine) : 2-30 C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Base media in vials : 6 months at 2-8 C. - Complete medium, with supplement : use immediately after preparation. D-Cycloserine 200 mg Selective Supplement, Egg Yolk Enrichment, - Store between 2-8 C, shielded from light. - The expiration dates are indicated on the labels. Ready-to-melt medium (base) : - Store between 2-25 C, shielded from light. - The expiration dates are indicated on the labels. PACKAGING Code Ready-to-melt medium (base) - 10 x 200 ml BM07708-50 x 20 ml tubes BM03908 Dehydrated base medium (without D-Cycloserine) : - 500 g bottle BK031HA D-Cycloserine 200 mg Selective Supplement : - 10 vial kit BS00608 Sterile Egg Yolk Enrichment : - 10 x 50 ml vials BS06608 4/6
PHOTO SUPPORT : Product reference : [BK031HA + BS00608], BM03908, BM07708 Media used for : Isolation and enumeration of Clostridium perfringens. Clostridium perfringens Typical aspect Black colonies, often surrounded by a black precipitate TSC Agar (use in 90 mm Petri dishes) Ref : BK031HA + BS00608 Incubation 24 hours at 37 C (anaerobic conditions) : Back colonies, often surrounded by a black precipitate (reduction of sodium sulfite) 5/6
BIBLIOGRAPHY Harmon, S.M., Kanter, D.A., and Peeler, J.T. 1971. Comparison of media for enumeration of Clostridium perfringens. Appl. Microb., 21: 922-927. Hauschild, A.H.W., Hilsheimer, R., and Griffith, D.W. 1974. Enumeration of faecal Clostridium perfringens spores in eggyolkfree Tryptose-Sulfite-Cycloserine Agar. Appl. Microb., 27: 527-530. Orth, D.S. 1977. Comparison of sulfite-polymyxin-sulfadiazine medium and tryptose-sulfite-cycloserine medium without egg yolk for recovering Clostridium perfringens. Appl. Envir. Microbiol., 33: 986-988. NF T 90-415. Octobre 1985. Essais des eaux. Recherche et dénombrement des spores de bactéries anaérobies sulfitoréductrices et de Clostridium sulfito-réducteurs. Méthode générale par incorporation en gélose en tubes profonds. NF EN 26461-2 (T 90-417). Juillet 1993. Qualité de l'eau. Recherche et dénombrement des spores de micro-organismes anaérobies sulfito-réducteurs (clostridia). Partie 2 : Méthode par filtration sur membrane. NF ISO 15213 (V 08-029). Septembre 2003. Microbiologie des aliments. Méthode horizontale pour le dénombrement des bactéries sulfito-réductrices se développant en conditions anaérobies. XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. Microbiologie des aliments. Guide pour la préparation et la production des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture. NF EN ISO 7937 (V 08-019). Février 2005. Microbiologie des aliments. Méthode horizontale pour le dénombrement de Clostridium perfringens. Technique par comptage des colonies. NF T 90-461. Juillet 2001 et NF T 90-461/A1. Juin 2005. Qualité de l eau. Microbiologie. Contrôle qualité des milieux de culture. NF V 08-061. Décembre 2009. Microbiologie des aliments. Dénombrement en anaérobiose des bactéries sulfito-réductrices par comptage des colonies à 46 C. *Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer s instructions. It is provided as a guide only and no warranty, implied or otherwise is associated with this information. The information provided on the package take precedence over the formulations or instructions described in this document. The information and specifications contained in this technical data sheet date 2010-11-26. They are susceptible to modification at any time, without warning. Code document : BK031/A/2003-01 : 11. 6/6