48 RUSSELL J. BEERS 



germination; though it has been done in liquid media with phase microscopy, 

 this is not practical in dry mounts. We suspended the spores in a nutrient 

 solution in the cold, prepared replicate smears of this suspension on slides, 

 and dried them in a desiccator. The slides were then incubated in atmos- 

 pheres in which relative humidity was controlled by sulfuric acid solutions, 

 and withdrawn at intervals for staining. When nutrient broth was used as 

 the suspending medium, vegetative cells were seen only rarely, indicating 

 absence of germination. When the relative humidity of the atmosphere was 

 100%, these cells appeared in about 12 hours; in 24 hours the smear was 

 almost solid with growth, in 48 hours extensive resporulation had occurred, 

 while in a week (possibly sooner; this was never checked) much of the 

 vegetative tissue had autolyzed and many of the new spores appeared rela- 

 tively clean. 



Under relative humidities slightly below 100%, the picture was the same 

 up to the point of resporulation, which was not observed at reduced humidi- 

 ties. When the sulfuric acid used to maintain the atmosphere was more 

 concentrated than 5% (98.4% relative humidity), it took longer for the 

 vegetative cells to appear, some times as long as 8 days over 8% acid (97.3% 

 relative humidity), and the final growth was much less dense. With concen- 

 trations of sulfuric acid greater than 8% no change was observed in the 

 spores in an experimental period of 32 days. 



When the suspending medium contained only L-alanine and adenosine, the 

 solidly staining germinated spores disappeared, though vegetative cells were 

 occasionally found. The same limits of humidity affected the germination 

 in the same way as when nutrient broth was used. 



To check these findings we prepared spore suspensions in the same two 

 media as were used for the slides, but dissolved different amounts of sucrose 

 in them to limit the moisture activity. Germination was followed by measur- 

 ing the drop in optical density in a Klett Summerson colorimeter. In solu- 

 tions containing up to and including 20% sucrose (98.6% moisture activity) 

 the drop was th^ same as in the control tube containing no sucrose. In 

 sucrose concentrations between 20%-30% this drop was less, while in con- 

 centrations above 30% (97.6% moisture activity), it became negligible. 

 These moisture limits are in good agreement with those determined by means 

 of smears (see Table I). 



The storage history of the spores is not without effect on these phenomena. 

 The experiments described so far were carried out with spores which had 

 been stored for some time. A recently prepared batch of spores of B. cereus 

 var. terminalis which had, however, been harvested and stored for a short 

 time under refrigeration was slightly more tolerant to lowered moisture 

 levels; it showed some germination in 32% sucrose (97.3% moisture activi- 



