A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

Abstract

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.