A rapid, reliable, and simple method based on the binding of crystal violet (CV) is described for differentiating virulence-plasmid-bearing strains of Yersinia enterocolitica from their plasmidless derivatives. As with other plasmid-mediated properties of this organism, the binding of CV occurs at 37°C but not at 25°C. The CV-binding technique provides a simple and efficient means of screening Y. enterocolitica for virulence and for identifying individual plasmid-bearing colonies.
Oxidation-reduction potentials (Eh) of canned foods ranged from -18 to -438 mV. Foods packed in glass had higher redox potentials than those packed in cans. Only 4 out of 26 products tested reached positive redox values after exposure to air for 24 hr at 4°C. Inoculated containers of mushrooms, whole corn, cream corn, asparagus, beef gravy, kidney beans, green beans, cream of mushroom soup, cheddar cheese soup, and lima beans supported toxin production by C. botulinurn; potatoes and beets did not.
The germination of spores fromn Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-' in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germninated at rates of 0.16% min-' in CTB and 0.04% min-' in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanineand L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains. Complete inhibition of botulinal toxigenesis requires NaCl levels of 5% for type E strains (2, 31) and up to 8 and 10o for type A and proteolytic B strains (1, 26) under otherwise optimal conditions. Lower salt concentrations are, however, inhibitory when used with another inhibitor (10, 32). Complex interactions among pH, salt, and nitrite are responsible Relatively little work has been done on the influence of NaCl on clostridial spore germination (2, 4, 26). While examining the interaction of pH and salt on botulinal physiology (22, 24), we isolated and characterized a salt-tolerant variant of Clostridium botulinum B-aphis. This variant, strain Ba410, differs from the parent strain in salt sensitivity, * Corresponding author. t Publication D-10112-1-85 of the New Jersey Agricultural Experiment Station.
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