THE BACTERIAL ENDOSPORE 39 



when spores are broken up by mechanical means, the complex 

 is again decomposed through the same mechanism that occurs 

 during germination. 



The ultimate objective for most of the investigators on 

 bacterial spores is to explain the means by which these structures 

 gain heat resistance. To us it appears we have reached an 

 impasse in our attempts to unravel this mystery by a study of 

 the germination process. We have, therefore, for the time being 

 at least, suspended our studies on germination and focussed our 

 attention on the process of sporulation, hoping this may be a 

 more fruitful study for us. I will devote the rest of this dis- 

 cussion, therefore, to a report on some recent observations 

 we have made on sporulation. 



Although the bulk of this report is going to be based upon 

 studies made on Bacillus cereus, an aerobic sporeformer, the 

 investigation had its beginnings in studies made in our labora- 

 tory on the anaerobe Clostridium roseum-^. When we first began 

 the study on the anaerobe, we were unable to obtain a good 

 yield of spores. This was due to a recycling phenomenon 

 occurring in the culture. Spores that were produced early in the 

 growth cycle would germinate in the same stew in which they 

 were produced. This resulted in a culture having cells in all 

 stages of development including freshly germinated spores, 

 actively growing vegetative cells, sporulating cells, and spores 

 just released from their sporangia. In such a mixture it was 

 virtually impossible to isolate clean spores. To overcome this 

 difficulty we developed a technique of growing the organisms 

 under semi-synchronous conditions. This was done by inocu- 

 lating the medium in which the spores were to be produced with 

 a very heavy inoculum from an actively growing synchronous 

 culture. The result was a population in which nearly all of the 

 cells began to produce spores at about the same time. We were 

 thus able to harvest clean spores containing a mJnimum of 

 vegetative cells and freshly germinated spores. In such cultures 

 we found sporulation set in very early; in fact, the process was 

 complete in about seven hours. By staining an hour or so before 



References p. 59 



