THE FORMATION OF SPORES BY BACTERIA 113 



broken up by mechanical means, the complex is again decomposed 

 through the same mechanism that occurs during germination. 



It is evident from the above that we have reached an impasse in 

 our attempts to isolate and study the structure of the complex involved 

 in the stabilization of the enzymes in the spores. It seems to us that 

 further attempts to get at this through a study of germination will not 

 be rewarding. We have, therefore, for the time being at least, sus- 

 pended our studies on germination and focused our attention on the 

 process of sporulation, hoping this may be more fruitful. 



The various factors that control sporulation have been studied by 

 a number of investigators, so I shall not attempt to give a comprehen- 

 sive review here; the topic has been reviewed by Grelet ( 1950, 1951, 

 and 1952), by Murrell (1955), and by Nakata (1959). Hardwick and 

 Foster ( 1952 ) studied the requirements for sporulation by removing 

 the vegetative cells from the growth medium and suspending them in 

 distilled water. They found that if the cells had been properly nour- 

 ished, they would sporulate normally when suspended in distilled 

 water. By this study they were able to gain some insight into the re- 

 quirements for sporulation. The techniqiie has been criticized, because 

 of the possibility that some cells may lyse or may otherwise release 

 materials which make growth possible for others in the medium— that 

 is, they may supply outside nutrients for the sporulation of those cells. 



Although Perry and Foster (1954) claim to have effectively an- 

 swered this criticism, we felt that it would be better to study the proc- 

 ess of sporulation in the growth medium by developing synchronous 

 cultures, so that the process of vegetative cell growth could be sepa- 

 rated in relation to the process of sporulation. 



Although most of our work is based upon studies of Bacillus cereus, 

 an aerobic sporeformer, the investigation had its beginnings in studies 

 made in our laboratory on the anaerobe Clostridium roseum (Collier, 

 1958 ) . When we began the study on this anaerobe, we were unable to 

 obtain a good yield of spores. This was due to a recycling phenomenon 

 in the culture. Spores produced early in the growth cycle would ger- 

 minate 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-syn- 

 chronous conditions. This was done by inoculating the medium 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 minimum of vegetative cells and freshly 



