The combined effect of low temperature and low pH on survival of, and growth and toxin formation from, spores of Clostridium botulinum

Abstract

An inoculum of 7 × 10 4 spores consisting of approximately equal numbers of spores of two strains each of Clostridium botulinum type A, proteolytic type B, non-proteolytic type B and type E was added to 20 ml volumes of culture medium at pH 7 or adjusted to pHs 5·1, 4·8 or 4·5 with citric acid, which were then incubated under strictly anaerobic conditions, at redox potentials equivalent to approximately −200 mV at pH 7, at 20°C, 16°C, 12°C or 8°C for 8 weeks. Germination, multiplication and formation of toxin occurred rapidly in medium at pH 7 incubated at all temperatures; the toxin detected was type E. In media at pH 5·1, 4·8 or 4·5 no significant change in viable count occurred at any temperature and no toxin formation was detected; counts of viable micro-organisms in heated samples showed that a high number of spores had failed to germinate. From this experiment the probability of growth from single spores in these conditions was calculated to be <3 × 10 −6 , compared with a probability of one of growth and toxin formation in two days in optimal conditions. When inoculated media at pH 5·1, 4·8 and 4·5 that had been incubated at 8°C for 8 weeks were transfered to 30°C, growth and toxin formation were detected at pH 5·1 and pH 4·8 within 23–52 days and 43–61 days respectively, but not at pH 4·5 within 76 days. The toxins detected in these conditions were types A and B.