Organic Nutrients Required for Growth and Sporulation of Bacillus Cereus.

E spores. This concentration is obtained by simple mixing of equal parts of absolute ethanol and the enrichment culture prepared as follows. When the presence of type E toxin was demonstrated in the suspect fish by titrations with mice, portions of the sample were cultured in duplicate tubes of cooked meat medium (CMM) prepared from fresh liver and Trypticase-peptoneglucose (TPG) broth (Schmidt, Nank, and Lechowich, J. Food Sci. 27:77, 1962), and were incubated for 3 days at 30 C. After incubation, 2-ml samples of the enrichment culture were placed in sterile test tubes and were mixed with an equal volume of absolute ethanol. This mixture was allowed to stand at 25 C for 1 hr with occasional mixing. The culture-alcohol mixture was streaked on liver veal agar plates containing 4% sterile egg yolk and was subcultured into TPG broth. Plates were incubated in Case Anaero jars under a nitrogen atmosphere for 48 hr at 35 C. After incubation, the plates were examined for characteristic colonies of C. botulinum type E. The opalescence zones (McClung and Toabe, J. Bacteriol. 53:139, 1947) produced by type E colonies aided colony recognition, especially if other sporeforming organisms were present. When typical colonies predominated, the corresponding TPG broth subcultures were assayed for type E toxin. The assaying of TPG broth subculture, rather than reculturing of the individual colonies from the plates, eliminated the possibility of selecting a nontoxic variant frequently encountered in toxic subcultures. Pure culture isolates were rapidly obtained by this procedure from 40 of 41 individual packages of smoked whitefish chubs containing type E toxin, including those associated with victims of the outbreak in the Tennessee-Alabama-Kentucky area in September-October 1963. In addition, pure cultures were obtained from canned tuna fish (Johnston, Feldman, and Sullivan, Public Health Rept. U.S. 78:561, 1963) and the smoked whole whitefish associated with the outbreak of type E botulism in Kalamazoo, Mich., in September 1963. When the enrichment cultures were streaked on plates without alcohol treatment, the overgrowth of facultative microorganisms prevented the recovery of C. botulinum type E. Direct plating of the macerated fish without treatment was not successful for isolating the species, whereas alcohol treatment of the fish often permitted isolation by direct plating. In a few cases, difficulty was encountered when clostridia or other sporeformers were also present. It is felt, however, that the ability of C. botulinum type E to sporulate more readily than many other sporeformers in TPG enrichment broth will aid in its selection by means of the alcohol treatment. Studies are under way on the applicability of this method for the direct recovery of small numbers of C. botulinum type E spores from foodstuffs, water, mud, soil, and other possible sources, in environmental surveys.