Impact of Strain, pH and Ethanol Concentration on Ethanol Activation Method of Bacillus Spores Using a Single Spore Approach

Abstract

Aims: This work aims to determine the ideal conditions for ethanol activation of spores during their enumeration and compare to thermal activation which is the reference method. Place and duration: Department of Microbiology of the University of Yaoundé I between May2016 and June 2018. Methodology: Bacillus cereus and Bacillus subtilis spores were activated according to an experimental central composite design with three-factor. The factors considered were exposure time, ethanol concentration, pH and activation temperature according to the types of activation. Germination yield was carried out by individually monitoring each spore of a population on solid medium in order to determine the population germination kinetic parameters (time and rate of colony appearance within a population during germination) and germination yields. These parameters were compared with those obtained after thermal activation known as a reference method. Results: The factors strain and pH, significantly influenced the rate of spore germination within the population after ethanol activation. In the case of thermal activation, the specie and activation temperature were the most influential factors. The best germination yields were obtained for alcoholic activation of spores at 30% ethanol for 60min exposure at pH7, while for thermal activation the best yields varied from one strain to another depending on the activation conditions. Conclusion: Ethanol activation can be considered as a good substitute of thermal activation during spore enumeration provided activation conditions are well controlled. This is in our opinion the first detailed study comparing ethanol activation to heat activation of Bacillus spores. It will impact future revisions of spore enumeration protocols proposed by norms that take into consideration spore activation and reduce bias in spore enumeration.