The inhibition of germination and growth of Clostridium botulinum 62A by BHA, BHT, TBHQ and 8-Hydroxyquinoline

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Virginia Polytechnic Institute and State University


The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary-butyl hydroquinone (TBHQ) and 8- hydroxyquinoline (8-0HQ) upon cell growth and spore germination of Clostridium botulinum type 62A was studied using a complex liquid medium. The inhibition of spore germination was further investigated using a defined liquid medium containing L-alanine, L-lactic acid and NaHC0₃.

Cell growth was monitored by optical density (600 nm) increase and germination was monitored either by optical density (600 nm) decrease or by increase in percent phase dark spores viewed under phase contrast microscopy. Strict anaerobic conditions were maintained throughout the study.

As the concentration of each chemical was increased the rate of germination or growth decreased. This occurred for each pH tested. GHA, BHT, TBHQ and 8-0HQ were more effective inhibitors of growth and germination as the pH was lowered from 7.2 to 5.7. At pH 7.2 50 µg/ml BHA, 200 µg/ml TBHQ and 200 µg/ml 8-0HQ were the minimum concentrations necessary for 90% inhibition of germination. BHT (400 µg/ml) inhibited germination by 65% at pH 7.2. Minimum concentrations at which growth at. pH 7.2 was inhibited by 95% were 60 µg/ml BHA, 20 µg/ml BHT, 250 µg/ml TBHQ and 20 µg/ml 8-0HQ. Inhibition of growth and germination by each chemical was found to be reversible with the exception of the inhibition of growth by BHT.