DELAYED EXPRESSION OF MUTATIONS 451 



present, some not having developed resistance by the time growth stopped. 

 Thus the mutation rates calculated by methods 1 and 2 would both be under- 

 estimated, and the extent of the underestimate would depend upon the magni- 

 tude of the delay. 



The information available so far sets no limit on the suspected delay or its 

 variability within clones of mutants, and it is even possible that an extreme 

 situation exists in which both are considerable. If this is true, mutation rate is 

 greatly underestimated by the methods outlined. 



It should be noted at this point that a delay similar to that suspected in 

 the case of spontaneous mutations does in fact occur in irradiated material 

 (Demerec 1946; and Demerec and Latarjet 1946). Although there is at 

 present no certainty that spontaneous and induced mutations behave in pre- 

 cisely the same manner, it is of interest to consider the nature of the delay in 

 the one case in which it has been established, that is, in induced mutations. 



To determine the time of appearance of induced mutations, Demerec ir- 

 radiated bacteria in liquid suspension, using ultraviolet radiation in some ex- 

 periments and X-rays in others. These treated bacteria were spread on agar, 

 incubated for varying periods of time to permit cell reproduction, sprayed 

 with phage Tl, and incubated again until colonies appeared. The spraying 

 caused all susceptible bacteria to be infected and lysed; but where a mutation 

 to resistance had occurred and had been expressed phenotypically, the result- 

 ing bacteria continued to grow after phaging, giving rise to one visible colony 

 for each such mutation. 



Irradiation caused an enormous increase in the number of resistant clones 

 that appeared during growth, over the number that appeared during the same 

 number of generations in untreated bacteria. The delay between irradiation 

 and phenotypic change was such that less than one percent of the induced 

 mutants appeared prior to the first division, 50 percent appeared after about 

 five divisions, and some did not appear until after 11 or 12 divisions. 



These observations give some support to the possibility that there is also a 

 delay in the expression of spontaneous mutations. 



The experiments described here were designed to distinguish between this 

 and the alternative possibility of a change in the rate of mutation during 

 growth. They show that there is in fact a delay, and that it is the cause of the 

 discrepant estimates of rate obtained by using the Luria and Debruck 

 methods 1 and 2. Some indication of the extent and variability of the delay 

 has been sought, and an attempt has been made to obtain a more accurate es- 

 timate of mutation rate. 



materials 



The bacterium Escherichia coli strain B/r was used in these investigations. 

 This is a mutant derived from strain B (Witkin 1946, 1947), and is more re- 

 sistant than B to the action of radiations. 



The mutations studied are those resulting in resistance to phage Tl. There 

 are at least two different categories of mutant: those resistant to Tl but 

 not to any other of the known phages, and those resistant to phages Tl and 



53 



