ENZYMES ACTIVE IN THE INTACT SPORE 97 



Foster, J. W. 1956. Morphogenesis in bacteria: some aspects of spore for- 

 mation. Quart. Rev. Biol. 31: 102-118. 

 Lawrence, N. L. 1955. The cleavage of adenosine by spores of Bacillus 



cereus. J. Bact. 70: 577-582. 

 Lawrence, N. L. and H. Orin Halvorson. 1954. Studies on the spores of 



aerobic bacteria. IV. A heat resistant catalase from spores of Bacillus 



terminalis. J. Bact. 68: 334-337. 

 Murrell, W. G. 1952. Ph.D. thesis at the University of Oxford. 

 Powell, J. F. and J. R. Hunter. 1956. Adenosine deaminase and ribosidase 



in spores of B. cereus. Biochem. J. 62: 381. 

 Stewart, B. T. and H. Orin Halvorson. 1954. Studies on the spores of 



aerobic bacteria. II. The properties of an extracted heat-stable enzyme. 



Arch. Biochem. and Biophys. 49: 168-178. 

 Stewart, B. T, and H. Orin Halvorson. 1953. Studies on the spores of aerobic 



bacteria. I. The occurrence of alanine racemase. J. Bact. 65: 160-166. 



Discussion 



Herbert M. Nakata 



The importance of using clean spore suspensions and the necessity of 

 specifying the age and history of the spores used in germination studies 

 have been emphasized and discussed. Similar emphasis is encouraged when 

 using spore suspensions for enzymatic investigations. With the increased 

 number of publications in recent years regarding spore enzymes, it has 

 become apparent that some general criteria must be founded and accepted 

 to minimize misinterpretation of data, results, and conclusions by others. 

 Therefore, it is my intention to suggest and discuss briefly some aspects 

 that we feel lead toward the realization of such useful criteria whereby 

 enzymes classified as being active in the dormant intact spore can be dif- 

 ferentiated from the other categories listed in the program. 



{A) The first of these suggested criteria deals with the need for con- 

 trolling both the physical and chemical environments of the experimental 

 spore suspensions during the time measurements are made. That is, the 

 environment must be such that the enzyme activity occurs under conditions 

 precluding the possibility of spore germination. The term germination, as 

 I will use it, refers to the loss of heat resistance, refractility, and the sensi- 

 tivity to stains. In recent years, much emphasis has been placed on this 

 criterion and several methods, such as the use of (1) high temperatures, 

 (2) pH's unfavorable to germination, (3) substrates that are not conducive 

 to germination, and (4) germination inhibitors, have been suggested as some 

 ways in which complications due to undesirable germination can be avoided. 



(1) Use of high temperatures. Among these methods, the use of high 

 temperatures has been proven satisfactory by Dr. Lawrence (1955 a, b) and 

 in our laboratory for the study of the heat-resistant nucleoside ribosidase 



