Immobilized bacterial spores for use as bioindicators in the validation of thermal sterilization processes

Abstract

Spores of Bacillus subtilis ATCC 6051 and Bacillus stearothermophilus NCTC 10003 were immobilized in monodisperse alginate beads (diameter, 550 μm ± 5%), and the capacity of the immobilized bioindicators to provide accurate and reliable F-values for sterilization processes was studied. The resistance of the beads to abrasion and heat was strong enough to ensure total retention of the bioindicators in the beads in a sterilization cycle. D- and z-values for free spores were identical to those for immobilized spores, which shows that immobilization does not modify the thermal resistance of the bioindicators. A D100°C value of 1.5 min was found for free and immobilized B. subtilis spores heated in demineralized water, skimmed milk, and milk containing 4% fat, suggesting that a lipid concentration as low as 4% does not alter the thermal resistance of B. subtilis spores. Providing that the pH range is kept between 3.4 to 10 and that sufficiently low concentrations of Ca2+ competitors or complexants are present in the medium, immobilized bioindicators may serve as an efficient, accurate, and reliable tool with which to validate the efficiency of any sterilization process. The environmental factors (pH, media composition) affecting the thermoresistance of native contaminants are intrinsically reflected in the F-value, allowing for a sharper adjustment of the sterilization process. Immobilized spores of B. stearothermophilus were successfully used to validate a resonance and interference microwave system that is believed to offer a convenient alternative for the sterilization of temperature-sensitive products and medical wastes.