462 HOWARD B. NEWCOMBE 



of the phenomenon is uncertain. In the absence of evidence to the contrary 

 it has been assumed that mutations do occur at the normal rate, but that they 

 fail to develop visible colonies owing to the presence of large numbers of sensi- 

 tive bacteria that are not lysed by the phage — a situation which occurs if phage 

 is applied after bacterial growth has passed the logarithmic phase. 



Tests showed that this apparent reduction in mutation rate occurred only 

 if the end number of bacteria exceeded 5 X 10 9 . 



(3) It is known that bacteria which are infected during rapid growth have a 

 latent period of 13 minutes — at the end of which time they burst, liberating 

 on the average 180 phage particles (Delbruck and Luria 1942). If some of 

 the bacteria fail to be infected at the time of spraying, it is unlikely that they 

 will escape infection once lysis of the others starts. Any uninfected individuals 

 would, on the average, pass through somewhat less than one division during the 

 13-minute latent period. Thus, with a large proportion of the bacteria unin- 

 fected at the time of spraying, a somewhat less than twofold increase in popu- 

 lation would be expected before all the bacteria became infected, and the ap- 

 parent mutation rate from such an experiment would be increased propor- 

 tionally. Where all but a small proportion of the bacteria are infected at the 

 time of spraying, the apparent mutation rate would not be appreciably greater 

 than in the case of 100 percent infection. 



In the present experiments the number of phage particles applied was equal 

 to the number of bacteria, or slightly in excess. Large excesses were not used 

 since these involve long periods of spraying, with resultant wetting of the sur- 

 face of the agar and a tendency for the bacteria to be moved about by the mois- 

 ture. Larger numbers of phage particles have been used, however, by Beale 

 (1948), who concentrated the phage by centrifuging. The estimates of muta- 

 tion rate that he obtained in this manner do not differ appreciably from those 

 obtained in the present experiments, and it may be assumed that the quantities 

 used in the latter were adequate. 



By the method described in this section it was possible to determine the 

 rate with which resistant clones appear during the period of rapid growth 

 from the resting stage onward. 



The results of four separate experiments, each with eight independent repli- 

 cates, are given in table 6. In these experiments the bacteria were grown 

 over a period of approximately eight generations; and the mutation rate 

 obtained is that for the whole growth period. 



It will be seen that the average of estimates of rate from all experiments is 

 low (.59 X 10~ 8 ) and is in close agreement with the low estimates from method 1 

 (average .40X10 -8 ). As pointed out earlier, this constitutes evidence that re- 

 sistant clones arise from mutations occurring one or more generations prior to 

 their first becoming detectable. 



It should be mentioned at this point that subsequent experiments, described 

 in the next section and summarized in figure 1, have enabled mutation rate to 

 be calculated from a tenfold increase in bacterial titer onward. This eliminates 

 the contribution of the first few divisions, during which an excessive number of 



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