Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37°C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.

A minimal medium was developed for the cultivation of Clostridium perfringens in an anaerobic chemostat. Cultures of C. perfringens ATCC 3624 and NCTC 10240 were grown at 46 and 43°C, respectively, in a glucose-limited, chemically defined medium at pH 7.2. The concentrations of amino acids, minerals, nucleotides, and vitamins, initially present in excess, were varied independently. The minimum concentration of each nutrient which would support 3 x 108 CFU/ml with a generation time of less than 40 min was determined and used to develop a reformulated defined medium. Atomic absorption spectroscopy and amino acid analyses of the reformulated medium indicated additional adjustments in nutrient content which led to the development of a minimal medium for each strain. The nutritional profile for each strain was similar. A decrease in the concentration of arginine, histidine, and tyrosine for strain 3624 and of arginine, histidine, and isoleucine for strain 10240 resulted in an increase in the optical density of each culture.

A constant source of exponential phase cells of Clostridium perfringem ATCC 3624 could reduce the time required to carry out the protein quality evaluation test of Solberg et aI. (1979) to 4 hr. Clostridium perfringens ATCC 3624 was grown in an anaerobic chemostat using the chemically defined medium, R&S, with glucose as the growth limiting nutrient. The dilution rate was set at 0.06 hr-', the pH at 7.2, and the temperature at 43°C. In the transition from a batch culture to a continuous culture an initial oscillatory ceII density response was observed. In the steady state, which was continued for as long as 50 days, the cells were typical Gram positive rods, occurring singly or in chains as long as 15 rods, which were occasionally without septa. The fermentative and biochemical responses of the cells did not change. No sporulation occurred when the cells were growing in the chemostat, but spores were observed in the gtucose free culture effluent after incubation at 37°C for 24 hr. When cells produced in the chemostat, tiere cultured in complex media they demonstrated a growth response similar to cells which had been grown in a batch culture. In defined medium the generation time for the chemostat cultured cells was decreased approximately 17%. The chemostat cultured cells can be used as inoculum for the C. perftingens protein quality assay.