MOISTURE ACTIVITY ON GERMINATION 47 



for counts at intervals. They ol)serve(l an initial significant drop in spore 

 count, but no further drop over a period of several weeks. However, the 

 highest moisture activity they reached was 75%, which they considered in- 

 sufficient for germination; and they attributed their initial spore loss to 

 local areas in which the moisture activities were sufficiently high. It may 

 also be that some spores lose their heat resistance under these conditions 

 without germinating, since products like sugar and flour containing spores 

 are known to gradually lose spores on standing. Dried spores stored in our 

 laboratory showed a slow decrease in viability with time, althought they ex- 

 hibited complete germination as measured by decrease in optical density 

 of their suspensions. Loss of viability may precede this change however, 

 and transcluency may be induced in a nonviable spore by germinating nu- 

 trients. 



We continued the;e studies by the same general method with the difference 

 that we attempted to create the desired moisture levels internally by mixing 

 liquid water directly with the spore-flour mixtures. Though we tried a num- 

 ber of devices, we were only partially successful. Bullock and Tallentire claim 

 to have done this in their peptone-spore powders, but they do not give much 

 detail as to how they obtained uniform distribution. We were not able to 

 create a desired moisture level with good reproducibility, but by preparing a 

 large number of powders we did obtain samples well covering the range 

 we desired. Unfortunately these powders lost moisture activity on standing, 

 and in no case were we able to demonstrate a significant trend in germination 

 that could be correlated with moisture activity. Most of our samples showed 

 an early drop in spore count and none thereafter, recalling the data of 

 Halvorson and Waldhalm. This occurred regardless of the initial moisture 

 activity. While the explanation of local areas of high moisture activity would 

 be applicable here, due to the method of introducing water into the powders, 

 it may also be that the conditions existing in our powders destroyed the 

 viability of some spores, or at least rendered them heat sensitive without 

 actual germination. Or they may have been so near to germination that this 

 treatment was sufficient to nudge them over the top. Spores in a given popu- 

 lation seem to exist in a variety of conditions, and therefore respond differ- 

 ently to a number of treatments, some proceeding toward germination from 

 the fully dormant spore, and others toward loss of viability without germina- 

 tion, branching off from the germination pathway at an undetermined point. 

 That dead spores may have some of the properties of germinated spores is 

 indicated by the fact that one batch of autoclaved spores showed solid staining. 



We went next to a microscopic method patterned after the one that was 

 used successfully with mold spores. Because of their smaller size the mor- 

 phological changes are difficult to follow microscopically in the early stages of 



