Does proximity to neighbours affect germination of spores of non-proteolytic Clostridium botulinum?

It is recognised that inoculum size affects the rate and extent of bacterial spore germination. It has been proposed that this is due to spores interacting: molecules released from germinated spores trigger germination of dormant neighbours. This study investigated whether changes to the total number of spores in a system or proximity to other spores (local spore density) had a more significant effect on interaction between spores of non-proteolytic Clostridium botulinum strain Eklund 17B attached to defined areas of microscope slides. Both the number of spores attached to the slides and local spore density (number of spores per mm(2)) were varied by a factor of nine. Germination was observed microscopically at 15 °C for 8 h and the probability of, and time to, germination calculated from image analysis measurements. Statistical analysis revealed that the effect of total spore number on the probability of germination within 8 h was more significant than that of proximity to neighbours (local spore density); its influence on germination probability was approximately four-times greater. Total spore number had an even more significant affect on time to germination; it had a nine-fold greater influence than proximity to neighbours. The applied models provide a means to characterise, quantitatively, the effect of the total spore number on spore germination relative to the effect of proximity to neighbouring spores.