54 RUSSELL J. BEERS 



In a report on a somewhat parallel investigation by Williams and Purneli 

 (1950 and 1953 ( involving limiting growth of the same organism on another 

 substrate, liver, the corresponding moisture activities and total moisture 

 contents were measured. Dehydrated raw liver, inoculated with spores of 

 CI. botulinum, was made into pastes with varying amounts of water in the 

 range of 32 to 60 percent total moisture content, and the extent of spore 

 germination and growth after incubation at 37° C. was determined. Their 

 results indicated that no increase in the viable count took place during 16 

 days' incubation when the total moisture content was 35 percent or less. 

 However, at a moisture level of 40 percent, the growth was irregular and 

 delayed. Good germination and growth were attained at total moisture 

 values of 50 percent or higher. Thus the limiting concentration appeared to 

 occur between 35 and 40 percent total moisture content. This corresponded 

 to a roughly measured moisture activity of 90 to 95 percent. As may be 

 noted from Table I, the hygrostatic technique employed for determining 

 moisture activity did not distinguish between samples of 35 and 40 percent 

 total moisture. As in the case of canned bread, control of pH in the liver 

 paste was an important factor; thus when the pH of the paste was initially 

 adjusted to the broad range of 6.8 to 7.5, irregularities in the results were 

 smoothed out. 



There are some indications that a slow aging effect which occurs during 

 the laboratory storage of spore crops results in altered characteristics of the 

 spores. Recent studies by Halvorson and Murty (1956) suggest that the 

 moisture limits for germination of Bacillus cereus var. terminalis spores have 

 shifted slightly lower over a period of about 10 months, even though the 

 spores were stored in the frozen state. Dr. Beers has also reported that 

 moisture limits for germination vary somewhat with the history of the spores. 

 If it be true, as discussed earlier this afternoon, that vegetative cells require 

 less moisture to permit growth than spores require for germination, then 

 it might appear that aging spores are becoming less spore-like and more like 

 vegetative cells. Perhaps such a phenomenon could assist in explaining the 

 occasional spoilage which occurs during prolonged storage of canned foods 

 arising from the altered germination requirements of long dormant spores. 



One final comment may be of interest and it relates to the moisture-binding 

 properties of different bacterial proteins. Waldhalm and Halvorson (1954) 

 showed that the dehydrated spores and vegetative cells of B. cereus var. 

 terminalis possess different affinities for water. Their work demonstrated that 

 spores are less hygroscopic than vegetative cells and that the spores can give 

 up their moisture to the cells if both are stored together in a closed system. 

 When the difference in structure and physico-chemical properties of spore 

 proteins and bacterial cell proteins is ultimately worked out, we may hope to 



