Penicillin-induced Penicillin Resistance 85 



In this way it was established that cultures of strain 5 

 produce penicillinase consistently at approximately one five- 

 thousandth the rate shown by strain 5/B. Although this 

 activity was from 25 to 250 times (in different cultures) that 

 expected from the rather variable content of 5/P and 5/B 

 cells, it seemed essential to eliminate the possibility that these 

 mutants might, when growing in the presence of large numbers 

 of 5, produce much more enzyme than that expected from 

 activities measured in pure culture, and thus themselves be 

 responsible for the activity observed. 



The effect on penicillinase production of supplementing 

 the "natural" 5/P and 5/B content of 5 cultures by a known 

 number of added cells and subsequently growing the mixed 

 culture for 5 hours at 35°, with careful differential counts of 

 all 3 types, is illustrated in Table II. It can be seen that only 

 when the 5/P and 5/B content of the culture reached propor- 

 tions approximately 200 times the normal level, was there any 

 significant increase in the amount of penicillinase produced; 

 and the extent of this increase corresponds to the value ex- 

 pected from the additional number of 5/P and 5/B cells present. 



The penicillinase activity of strain 5 cultures cannot there- 

 fore be ascribed to the presence of 5/P and 5/B mutants. It 

 is not, however, certain whether this low penicillinase pro- 

 duction is a property distributed evenly among the rest of the 

 population. A search was made, without success, for any 

 evidence of further heterogeneity. But it is not known 

 whether colonies consisting of cells producing the enzyme, say 

 at one-fifth the rate of 5/P, would necessarily be detected by 

 the penicillin-sensitivity or penicillinase production tests used, 

 though this seems likely. Such a hypothetical strain would 

 have to form 1 per cent of the total population of strain 5 in 

 order to account for the observed activity. Direct testing of 

 cultures by manometric assay of penicillinase from indi- 

 vidual colonies has yielded only 5/P and 5/B types and nega- 

 tives. We have therefore provisionally concluded that the 

 wild type forms penicillinase, constitutively, at a rate corre- 

 sponding to the production of about 15 molecules per cell — 



