Simple Technique to Determine Heat Resistance of Bacillus stearothermophilus Spores in Fluid Systems ¹

A simple, rapid, highly reproducible procedure was developed to determine heat resistance of Bacillus stearothermophilus spores in milk and soy protein-based formulas at temperatures > 100 C. Plating efficiencies on different media and heat activation temperatures were also studied. The procedure involved use of a serum bottle to which was added formula. The bottle was closed with a rubber septum and sealed air-tight with a crimped aluminum cap. The formula was agitated during heating in a thermostatically controlled oil bath, using a wrist action shaker. When the formula attained the desired temperature, a spore suspension was injected through the rubber septum, using a high-pressure GLC syringe. At selected time intervals, a portion was withdrawn from the bottle, using a sterile GLC syringe. The number of surviving spores was determined by plating on Trypticase Soy agar, which yielded significantly higher spore recovery count than did Trypticase Soy broth fortified with 1.5% agar with and without starch, or Dextrose Tryptone agar. The serum bottle procedure yielded higher D values than did the capillary tube procedure. The difference was significant where p = 0.05 but not where p = 0.01. With the serum bottle procedure, D values for spores in the milk protein base formula were 18.46, 3.56 and 1.13 min at 115, 121 and 125 C, respectively. In the soy protein base formula, D values were 26.1, 3.64 and 1.26 min, respectively. The z values were 7.7 and 7.6 Centigrade degrees (13.86 and 13.68 Fahrenheit degrees). Maximum heat activation of the spore was at 95 C for 10 min in milk protein base formula and at 100 C for 5 min in the soy product.