110 MOLECULES, VIRUSES, AND BACTERLV 



that inhibited the process. Upon the addition of versene, the toxic 

 metal ions were removed and germination proceeded. Dr. Riemann of 

 Denmark, working in Ordal's laboratory, has made an even more inter- 

 esting observation. He has been able to germinate most spores with an 

 equimolecular mixture of calcium and dipicolinic acid (personal com- 

 munication ) . He observed this while making some studies on the effect 

 of chelating agents on germination. Knowing that dipicolinic acid is a 

 fairly effective chelating agent, he tried to use this in place of versene 

 and found that it would bring about germination only in the presence 

 of calcium ions. The most rapid change was obtained when he had an 

 equimolecular mixture of the two. 



It is apparent from the above that the discoveries made by Hills 

 can be looked upon as forerunners of many important advances in our 

 knowledge of the germination of spores. 



The second observation that I mentioned above— namely, the dem- 

 onstration of active enzymes in intact spores— was made in connection 

 with some of our studies on germination. We observed, as others have, 

 that when spores germinate in the presence of L-alanine and adenosine, 

 or, as a matter of fact, with any of the other combinations of germina- 

 tion ingredients, not all of the spores in a suspension will do so. From 

 90 to 98 per cent germinate, but the rest remain heat-stable and un- 

 changed. The question naturally arises: Why do these remaining spores 

 fail to behave like the rest? Is it because they are different, or because 

 something has happened to the menstruum? 



To answer this question, we examined the solution in which the 

 spores had germinated to see whether the L-alanine or the adenosine, 

 or both, had been used up. We found no apparent change in the con- 

 centration of either during the germination process. If either of these 

 chemicals was used for germination, the amount was too small to be 

 detected by our methods of analysis. It appeared that nothing had 

 happened to the solution. One would be tempted to assume, therefore, 

 that the few spores that failed to germinate might be different from 

 the great majority that did germinate. In order to throw light upon this 

 problem, we centrifuged the suspension at the conclusion of the ger- 

 mination and added to the supernatant some fresh spores. None of 

 these germinated. This clearly demonstrated that something had hap- 

 pended to the menstruum. Since there was no change in the total 

 amount of alanine present, we examined the L-alanine to see if some 

 had been converted to D-alanine. This appeared reasonable because 

 Hills (1950) had previously demonstrated a D-alanine interference 

 with germination brought about by L-alanine. A racemic mixture of L 

 and D was found after less than one hour's contact with the spores. 

 This prompted us to look for the enzyme racemase; fortunately, this 

 was very easily found (Stewart and Halvorson, 1954). It proved to be 



