Cooked M Medium

Principle And Interpretation

Clostridium is a large genus of gram- positive spore- bearing anaerobes. They are normally present in soil, some are responsible for human and animal diseases and others are associated with food spoilage. They may be saccharolytic, decomposing sugars to form butyric and acetic acids and alcohols. The meat in Robertsons Medium is reddened and gas is produced. Other proteolytic species attack the amino acids. The mesophilic spore-forming anaerobes are of primary importance in the spoilage of low acid foods packed in sealed containers, because of their high heat resistance, their ability to grow in the absence of oxygen and a growth range which covers the temperature of normal storage of canned and other processed foods including the refrigerated storage of cured meats. Cooked M- Medium was originally developed by Robertson (1) for the cultivation of certain anaerobes isolated from wounds. The present formulation is a modification, also called as Chopped M Medium (2), which supports the growth of many spore forming and non-spore forming strict anaerobes. It has the ability to initiate growth of bacteria from very small inocula and to maintain the viability of cultures over long period. Mixed cultures of bacteria survive in Cooked M- Medium without displacing the slower-growing organisms. The products of growth do not rapidly destroy the inoculated organisms and therefore it is an excellent medium for the storage of aerobic and anaerobic organisms. It is used for cultivation and maintenance of Clostridia and for determining proteolytic activity of anaerobes (2,3).

FDA has recommended this medium for enumeration and identification of Clostridium perfringens from foods (4). Cooked M-Medium contains HMH peptone B, which provides nitrogeneous and carbonaceous compounds, long chain amino acids and other nutrients. The sulphydryl groups, which impart reducing effect, are more available in denatured protein and hence this medium is used. The addition of dextrose allows rapid and heavy growth of anaerobic bacteria in a short time and leads to more rapid identification of important anaerobes. Growth in this medium is indicated by turbidity or bubble formation by some organisms. Blackening and disintegration of the meat particles indicate proteolysis. Aerobes grow at the top whilst more anaerobic species grow deeper in the medium. For the isolation of Clostridium from food, use a stomacher to prepare 10% suspension of the food in Peptone Water (LQ089) diluent. Make dilutions and plate both suspensions and dilutions on Willis and Hobbs Medium Base (M1375) Tryptose Sulphite Cycloserine (T.C.S.) Agar Base (M837). Place a metronidazole disc on the inoculum. Incubate anaerobically at37°C overnight. To count the clostridia, pour the plates with the dilutions on Perfringens Agar Plate (MP579). Incubate duplicate plates aerobically and anaerobically to distinguish between clostridia and other organisms. Add some of the suspension to two tubes of Cooked M-Medium. Heat one tube for 10 min at 80°C and incubate as above. Growth of clostridia is visualized as turbidity or gas bubbles. This medium can be further tested for presence of Clostridium (5).