The Influence of Nutrient Depletion on the Properties of Bacillus Stearothermophilus Spores

Abstract

Sporulation frequency and total spore yield were found to depend on specific nutrient depletion of growth as well as on aeration rate and concentration of manganese ions in the sporulation medium. Control of the manganese concentration in the sporulation medium is therefore specially important. Low concentrations of manganese ions (<10 μM) gave rise to a low sporulation frequency. Too high a concentration of manganese (2100 μM), however, exerted toxic effects on the growth of vegetative cells and total spore yields were reduced. Spores of Bacillus stearothermophilus NCTC 10,003 produced from cultures depleted of carbon or nitrogen or sulphur were analysed for population homogeneity. These analyses included germination studies, microelectrophoretic mobility measurements and electron microscopic examinations. Results suggested that the homogeneity of the spore populations was determined both by the specific nutrient depleting growth and by the manganese concentration in the sporulation medium. Magnesium was found to have little effect on spore homogeneity. In general, carbon depleted spores and nitrogen depleted spores containing high intra-sporal manganese contents were more homogeneous than those containing low intra-sporal manganese. Sulphur depleted spores were relatively non-homogeneous. Heat resistance studies revealed that manganese induced different effects on the heat resistance properties of spores from cultures nutrient depleted in different ways. This observation partly explains the current contradictory reports about the effect of manganese on the heat resistance of spores produced from complex media. Glutaraldehyde treatment of nutrient depleted spores delayed spore germination and was related to the integrity of the coat layers. The studies on the sporicidal effect of 2% alkaline glutaraldehyde, coupled with the information obtained by sonication and electron microscopy demonstrate the importance of the coat layers in protecting the spores from otherwise lethal substances. The degree of protection provided by the coat layers was a function of specific nutrient depletion. Carbon depleted or nitrogen depleted spores may provide the necessary stable and reproducible test systems required for the biological monitoring of sterilisation processes.