MUTATIONS OF BACTERIA 509 



days. The proportion of resistant bacteria did not change, even when the 

 sensitive bacteria began to die, showing that the resistant bacteria have the 

 same death rate in aging cultures as the sensitive bacteria. 



DISCUSSION 



We consider the above results as proof that in our case the resistance to 

 virus is due to a heritable change of the bacterial cell which occurs independ- 

 ently of the action of the virus. It remains to be seen whether or not this is 

 the general rule. There is reason to suspect that the mechanism is more com- 

 plex in cases where the resistant culture develops only several days after lysis 

 of the sensitive bacteria. 



The proportion of mutant organisms in a culture and the mutation rate are 

 far smaller in our case than in other studied cases of heritable bacterial varia- 

 tion. The possibility of investigation of such rare mutations is in our case 

 merely the result of the method of detecting the mutant organisms. In other 

 cases, the variants are detected by changes in the colony type which is pro- 

 duced by the mutant organism, either in the pigmentation or in the character 

 of the surface or the edge of the colony. Often, colonies of intermediate charac- 

 ter occur, and it is difficult to decide whether they are mixed colonies or stem 

 from bacteria with intermediate character. This is particularly true of cases 

 where the mutation rate is high and where reverse mutation occurs. Fairly 

 high mutation rates, however, are a prerequisite of any study of colony vari- 

 ants, since the number of colonies that can be examined is limited by practical 

 reasons. 



The study of mutations causing virus resistance is free of these difficulties. 

 The segregation of the mutant from the normal organisms occurs in the one- 

 cell stage by elimination of the normal individuals, and the character of the 

 colony which develops from a mutant organism is of secondary importance. 

 Owing to the total elimination of the normal individuals, the number of organ- 

 isms which may be examined is very much higher than for any other method; 

 more than io 8 bacteria may be tested on a single plate. Since the mutations 

 to virus resistance are often associated with other significant characters, the 

 method may well assume importance with regard to the general problems of 

 bacterial variation. 



It must not be supposed that the peculiar statistical difficulties encountered 

 in our case are restricted to cases of very low mutation rates. The essential 

 condition for the occurrence of the peculiar distribution studied in the theoreti- 

 cal part of this paper is the following: the initial number of bacteria in a culture 

 must be so small that the number of mutations which occur during the first division 

 cycle of the bacteria is a small number. This will always be true, however great 

 the mutation rate, if one studies cultures containing initially a small number of 

 organisms. 



In a series of very interesting studies of the color variants of Serratia mar- 

 cescens, Bunting (1940a, 1940b, 1942; Bunting and Ingraham 1942) suc- 

 ceeded to some extent in obviating the statistical difficulties by always using 



