34 H. O. HALVORSON 



temperature but will not do so in the same menstruum if kept 

 at 65°. 



The germination requirements are also affected by aging. 

 Freshly produced and thoroughly cleaned spores have the most 

 rigid requirements, but as they are aged the requirements 

 generally become less so. The rate at which these changes take 

 place depends upon the temperature of storage. Spores stored 

 in a frozen state can remain unchanged for a very long period, 

 whereas those stored in a refrigerator will change more rapidly. 

 There is a marked difference in different species as to the rate at 

 which these changes can take place. One of the most noticeable 

 effects of aging is the disappearance of the need for heat sensi- 

 tization. The changes that take place during aging are probably 

 the same type as those which occur during heat shock. The only 

 difference is one of time. In our laboratories, we have detected 

 free L-alanine in the same supernatant liquor in which spores 

 have been stored and aged, but this amino acid cannot be found 

 in the supernatant liquor from freshly prepared clean spores^^. 

 Alterations probably occur within the spore during aging, 

 resuhing in the release of chemicals required for germination. 

 It is for this reason that aged spores will germinate with a lower 

 concentration of L-alanine than fresh spores, and in some cases 

 they will germinate with alanine alone and no adenosine, or in 

 other cases with adenosine alone and no L-alanine. 



Various kinds of metals have a marked effect upon germina- 

 tion. Some metal ions, such as cobalt and nickel, will inhibit the 

 process, whereas calcium and magnesium ions are helpful^^. We 

 encountered this phenomenon in a batch of spores which had 

 been produced for us in a pilot plant where the fermentors were 

 made from metal. The spores we obtained from this pilot plant 

 failed to germinate unless we added to the suspension some 

 chelating agent such as versene or heavy concentrations of 

 phosphate^'. Observations made by Brown^^ are of interest in 

 this connection. He found they could germinate spores of the 

 putrefactive anaerobe 3679 with versene only. In this case, it 

 appeared that these spores could germinate spontaneously. 



