Low Temperature Growth of Type E Clostridium botulinum Spores. 1. Effects of Sodium Chloride, Sodium Nitrite and pH

SUMMARY— The effects of pH and the addition of sodium chloride (NaCl) or sodium nitrite (NaNO₂) to trypticase-peptone-glucose (TPGI and trypticase-peptone-sucrose-yeast extract (TPSY) media upon spore outgrowth were investigated using seven Type E Clostridium botulinum strains. An inoculum of 1.0 × 10⁵ spores/ml was used and the pH was adjusted with hydrochloric acid to cover the range of 5.2 to 6.6. To define salt tolerance, NaCl was added to the media at intervals of 0.5% in the range of 2.0 to 5.0% and at 0.1% intervals in the 4.5 to 5.0% range. The effect of NaNO₂ was investigated with the addition of 100 and 200 ppm NaNO₂ to the media. Samples were incubated at 30, 15.6, 10, 7.2, 5.0 and 3.4°C. Outgrowth of all strains tested was inhibited at pH 5.2 at 15.6°C. Inhibition occurred at higher pH at lower temperatures. None of the strains showed outgrowth with 4.87% NaCl in the media at any of the incubation temperatures used. Addition of 100 and 200 ppm NaNO₂ to the media inhibited the outgrowth of the Minneapolis, 517, 26080 and A6247 strains but not the Kalamazoo and Seattle Forks strains.