T.S.C. Agar Base (Tryptose Sulfite Cycloserine) ISO

The superior nutrient base provides optimal conditions for the development of Clostridia. Tryptose and soy peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is source of vitamins, particularly the B-group, essential for bacterial growth. Ferric ammonium citrate and disodium disulfite are H₂S indicators. Bacteriological agar is the solidifying agent. Cycloserine inhibits the accompanying bacterial flora and may cause the colonies which develop to remain smaller.

Colonies producing hydrogen sulfide are characterized by a blackening due to the reaction with the ferric salt. The degradation of the egg yolk lecithin produces insoluble products which accumulate around the colonies, forming a white precipitate. After 24 hours incubation, all black colonies, lecithinase positive as well as the lecithinase negative ones, have to be considered as positive presumptive C. perfringens and the corresponding confirmation tests have to be made.

Preparation

Suspend 42 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 °C for 15 minutes. Cool the medium according to the applicable normative and aseptically add two vials of Clostridium perfringens Selective Supplement (LC-6020). If desired, 25 mL of Egg Yolk Emulsion Supplement (LC-5152) can be added (not indicated in ISO Normative). Homogenize gently and dispense into Petri dishes.

According to ISO 7937

Enumeration of Clostridium perfringens in food according to ISO 7937:

1. Transfer 1 mL of the initial suspension to the empty Petri dishes.
2. Pour 10 to 15 mL of the TSC Agar (44-47 °C) into the dish and mix with the inoculum by gently rotating each dish. When the medium has solidified, add an overlayer of 10 mL of the TSC agar.
3. Incubate under anaerobic conditions at 37 °C for 20 ± 2 hours.
4. After 24 hours incubation, all black colonies, lecithinase positive as well as the lecithinase negative ones, have to be considered as positive presumptive C. perfringens and the corresponding confirmation tests have to be made.

According to ISO 14189

Enumeration of Clostridium perfringens in water samples according to ISO 14189:

1. Filter a measured volume of water, to yield between 10-80 colonies on membrane.
2. Place the membrane on a TSC Agar plate.
3. Incubate the plates with the filters, under anaerobic conditions at 44 ± 1 °C for 21 ± 3 hours, inverted.
4. After 24 hours incubation, all black colonies, lecithinase positive as well as the lecithinase negative ones, have to be considered as positive presumptive C. perfringens and the corresponding confirmation tests have to be made.

• Sahidi S.A. and Ferguson A.R. (1971) Appl. Microbiol, 21 500-506.
• Harmon S.M., Kauttar D.A. and Peeler J.T. (1971) Appl. Microbiol. 21 922-927.
• Hauschild A.H.W. and Hilsheimar R. (1973) Appl. Microbiol. 27. 78-82.
• International standard ISO 7937 Microbiology of food and animal feeding stuffs - Horizontal method for enumeration of Clostridium perfringens - colony count technique
• International standard ISO 14189 Water quality - Enumeration of Clostridium perfringens - Method using membrane filtration