82 M. R. Pollock 



strain 5 mutated to 5/B "spontaneously" (i.e. in the absence 

 of penicillin). The demonstration in this instance was par- 

 ticularly satisfying because it was possible to show the pro- 

 duction of penicillinase (acid-formation — due to production 

 of penicilloic acid — in nutrient agar containing a pH indicator, 

 after addition of a high concentration of penicillin) by 5/B 

 cells which had never been in contact with penicillin, even 

 for the test itself. This is possible simply because the enzyme 

 diffuses out from the colony on or in nutrient agar and can 

 be demonstrated in the medium after the colony itself has 

 been removed. This completely disposes of any objection that 

 the mutation, in the first place, might be a relatively non- 

 specific event resulting in susceptibility to a subsequent 

 specifically induced change. 



Subsequently, it was discovered that spore suspensions of 

 strain 5 contained, as well as 5/B mutants, another type (5/P) 

 of cell, with intermediate penicillin resistance, which formed 

 penicillinase at approximately one-eighth of the rate of 5/B. 

 Its "spontaneous" mutative origin was never formally proved, 

 but it seems likely that it arose in the same way as 5/B. The 

 specific penicillinase activities (units enzyme/mg. dry bac- 

 terial weight) were measured on whole cultures growing 

 logarithmically in 1 per cent gelatin-broth and found not to 

 vary much within a single strain. All 5/P cells were able to 

 form colonies in agar containing penicillin up to a concentra- 

 tion of 0-1 units/ml. but not higher; whereas 5/B cells could 

 develop into colonies in penicillin up to 1 • units/ml. It was 

 thus possible to make accurate viable counts of mixtures of 

 5, 5/P and 5/B cells by plating out in suitable dilutions into 

 agar containing different concentrations of penicillin. The 

 proportions of these three types found in the spore suspension 

 studied were 1,000,000 : 6 : 4. Cultures of 5 from single spore 

 inocula were regularly found to contain this 1 : 250,000 pro- 

 portion of 5/B mutants, but the 5/P strain has so far only 

 been found in one spore suspension and must presumably 

 therefore result from a very rare mutation. On plates con- 

 taining 0-1 units penicillin/ml. there was no sure way of 



