THE FORMATION OF SPORES BY BACTERIA 109 



Most of the investigators have been observing germination at room 

 temperature, and it has been their common observation that it will not 

 take place in a refrigerator or at a lower temperature, nor will it occur 

 at 60° C. (Stewart and Halvorson, 1953). On the other hand, Wynne 

 ( 1957 ) claimed that spores from some species of anaerobes germinated 

 at 75° C. when they were suspended in a solution containing glucose 

 that had been autoclaved in an alkaline medium. Recently Foster 

 (1959) reported that Bacillus megaterium spores will germinate at 

 temperatures between 70° and 100° C. In our own laboratories we find 

 that spores of Bacillus cereus will germinate rapidly in the presence of 

 L-alanine and adenosine at room temperature but will not do so in the 

 same menstruum if kept at 65° C. 



The germination requirements are also affected by aging. Freshly 

 produced and thoroughly cleaned spores have the most rigid require- 

 ments, but as they age, the requirements generally become less rigid. 

 The rate at which these changes take place depends upon the tempera- 

 ture 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 

 sensitization. The changes that take place during aging are probably 

 of the same type as those that occur during heat shock. The only dif- 

 ference is one of time. In our own 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 super- 

 natant liquor from freshly prepared clean spores (Halvorson, 1958). 

 Probably alterations that occur within the spore during aging result in 

 the release of chemicals required for germination. This is the reason 

 that aged spores will germinate with a lower concentration of L-alanine 

 than fresh spores require, and in some cases will germinate with ala- 

 nine alone and no adenosine or with adenosine alone and no L-adanine. 



\^arious metals have a marked effect upon germination. Some metal 

 ions, such as cobalt and nickel, will inhibit the process, whereas cal- 

 cium and magnesium ions are helpful. (Gollakota, unpublished data). 

 We encountered this phenomenon in a batch of spores which had been 

 produced for us in a pilot plant where the fermenters 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 ver- 

 sene or heavy concentrations of phosphate (Murty and Halvorson, 

 1957a). Observations made by Brown (1956) are of interest in this 

 connection. He found that spores of the putrefactive anaerobe 3679 

 could germinate with versene only. It appeared that these spores could 

 germinate spontaneously, except for the presence of certain metal ions 



