Sigma-Aldrich Chemie GmbH Industriestrasse 25 Postfach CH-9471 Buchs / Switzerland Tel. +41 / 81 755 25 11 Fax +41 / 81 756 54 49 flukatec@sial.com 27548 Clostridium Agar (Reinforced Clostridial Medium, RCM) For the cultivation and enumeration of clostridia and other anaerobes as well as facultative microorganism in food, clinical specimens and other material. Composition: IngredientsGrams/Litre Yeast extract3.0 Meat extract10.0 Meat peptone5.0 Starch1.0 D(+)-Glucose5.0 L-Cysteine hydrochloride0.5 Sodium chloride5.0 Sodium acetate3.0 Agar12.5 Store prepared media below 8C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 2-25C. Directions : Dissolve 33 g in 1 litre water and sterilize by autoclaving at 121C for 15 minutes. Cool, if needed add 0.02 g Polymyxin B (Fluka 81334) or 0.15 g Neomycin sulphate (Fluka 72133) and 0.2 g Sodium acide (Fluka 71290) per litre in form of an aqueous solution and mix. Mix well and pour plates. After inoculation it is advised to incubate the plates under anaerobic conditions. Principle and Interpretation: Yeast extract, Meat extract and Meat peptone provide nitrogen, vitamins and minerals. The D(+)-Glucose is the carbohydrate source and Sodium chloride is for the osmotic balance. Starch detoxifies metabolic by-products.The medium is free from inhibitors and contains cysteine as a reducing agent. Polymyxin B can be added to inhibit Gram-negative bacteria. Neomycin sulphate and Sodium acide inhibit a number of organisms including Proteus, Pseudomonas and most off the cocci. Cultural characteristics after 24-48 hours at about 35C. Organisms (ATCC)Growth without supplementGrowth with supplement Clostidium perfringensis (10543)++++++ Clostidium tetani (10779)++++++ Clostidium sporogenes (10779)++++++ Escherichia coli (25922)+++- Staphylococcus aureus (25923)+++- References: 1.A. Hirsch, E. Grinsted, Methods for the growth and enumeration of anaerobic sporeformers from cheese, with observations on the effect of nisin, J. Dairy Res., 21, 101 (1954) 2.J. Vera, Presented Pa. Soc. Med. Tech., York, Pa. (1962) 3.E.M. Barnes, M. Ingram, The effect of redox potential on the grown Clostridium welchii strain isolated from horse muscle, J. appl. Bact. 19, 177 (1956)