CHEMICAL CHANGES DURING SPORE GERMINATION 79 



the sole source of sulfur. Aliquots of well washed spores were analyzed for 

 free S^'^-methionine in the following treatments: (Aj ungerminated spores 

 (at zero time), (B) after shaking 15 minutes in a germination solution, (C) 

 after shaking 50 minutes in the germination solution, (D) after shaking 50 

 minutes in plain buffer. Previous experiments showed that about 90 percent 

 of the spores had lost heat resistance in 50 minutes, but that nothing sug- 

 gesting growth or change in morphology took place in the germination 

 medium. All the treatments had 0.5 mg unlabelled DL-melhionine per mi as 

 a pool to trap labelled methionine. Total radioactivity was measured in the 

 isolated methionine after boiling the cells in 50 percent ethanol. Residual 

 labelled sulfate was eliminated by addition of carrier Na2S04 and prepara- 

 tion with Ba(OH)o. The methionine was purified as a band by paper 

 chromatography, and the counts made on eluted material corresponding to 

 methionine (Rf=0.8) as judged by reactions with ninhydrin and with 

 platinic iodide. The methionine from the respective treatments had the fol- 

 lowing counts per minute (corrected for background I : (A) 2.6; (B) 17.4; 

 (C) 62.1; (D) 4.4. Thus, after 50 minutes in the germination solution, 

 there was 24 times as much free methionine as existed in ungerminated 

 spores, and 14 times as much as controls incubated in the non-germination 

 (buffer) medium. This experiment proves that a pool of free amino acids 

 is formed from spore proteins very early in the germination process. Un- 

 questionably this pool is reutilized for subsequent syntheses during germina- 

 tion. Thus, there is evidence that intraspore turnover may occur very early 

 in the germination process and I would like to leave this phase of the dis- 

 cussion with the reflection that most of the phenomena that we have observed 

 up to now are probably peripheral to the key phenomenon of activation of 

 the intraspore turnover. 



Further in connection with the proposal to subdivide germination termi- 

 nology is the matter of heat resistance, or resistance in general, of the spore. 

 Dr. Stumbo has already touched on an important point in this relation. I 

 already had something in mind to submit to your consideration. That heat 

 resistance is universally regarded as the distinguishing characteristic of a 

 spore is evidenced by its reiteration time and again during this conference. 

 I have a feeling we would be much closer to being able to provide a true 

 definition of germination— to provide a legal definition of the phenomenon 

 as it were — if we really knew what a spore means to a bacterium in nature. 

 I can understand how the measurement of heat resistance has evolved to 

 become the stereotyped criterion. It obviously is a manageable technique 

 andf gives reproducible results. However, from the standpoint of character- 

 izing the spore functionally, and that is what we accomplish when we define 

 a cell in terms of heat resistance or lability, we ought not overlook asking 



