THE USE OF CONFOCAL SCANNING LASER MICROSCOPY (CSLM) TO STUDY THE GERMINATION OF INDIVIDUAL SPORES OF BACILLUS-CEREUS

Confocal scanning laser microscopy (CSLM) has been used to study the g ermination of Bacillus cereus spores allowing generation of accurate q uantitative data on loss of refractility of individual, germinating sp ores in a population. The technique also allowed the simultaneous stud y of changes in spore permeability to ethidium bromide by fluorescence microscopy. Statistical analyses were performed on the data obtained and the distributions of various germination parameters within the pop ulation studied. All the parameters studied were characterised by posi tively 'skewed' distributions indicating the high level of biovariabil ity present in populations of bacterial spores. The lag time before a spore initiated germination was independent from the subsequent time t o lose refractility, or germinate. The method described in this work, for the first time, allows changes in permeability during germination to be studied using the influx of a compound that is excluded from dor mant spores (ethidium bromide). It is hoped that this will form the ba sis of a useful system for studying the kinetics and sequence of spore germination when combined with other permeability probes.