350 



CHAPTER 38 



following respects. Three lack B, Pa, and C 

 besides T, or L, or Bi; the other three lack T, 

 L, and Bi besides B, or Pa, or C. Individuals 

 of the T~L~Bc strain cannot grow on the first 

 three replicas mentioned because single re- 

 quired nutrients are missing, and cannot 

 grow in the last three because all three are 

 missing. Individuals of the B,Pa~C~ strain 

 cannot grow on the first three replicas because 

 all three required nutrients are missing and 

 cannot grow on the last three because one of 

 these is absent. If the master plate contains 

 a mutant to nutritional independence for one 

 of the nutritionally dependent loci only one 

 replica will show growth. For example, if 

 on the master plate there was a r+ mutant 

 among the individuals of the TL-Bc strain, 

 a clone will grow only on the replica lacking 

 B, Pa, C, and T. Actually, about 1 00 different 

 positions on the master plate show growth 

 on the replicas. This is a very much larger 



number than is detected in the six replicas 

 that test spontaneous mutation rate when the 

 two fines are plated separately. Some posi- 

 tions show growth only on one of the six 

 replicas. However, there are many positions 

 that show growth on two replicas; these 

 must have gained nutritional independence at 

 two loci. Finally, there are also many posi- 

 tions which grow on all six replicas, each 

 representing the occurrence of complete 

 prototrophs (T+L+Bi+B+Pa+C+). One can 

 use the clones on the replicas, or return to 

 the master plate, and demonstrate that these 

 changes are transmissible and preadaptive. 

 When tested, these clones prove to be pure, 

 that is, the nutritional independence gained 

 is not attributable to some kind of physical 

 association between two or more different 

 auxotrophs. The large number of clones 

 growing on the replicas, plus the fact that 

 many are complete prototrophs or are auxo- 



FIGURE 38-1. Use of replica-plating (shown diagrammatically) to detect spontaneous 

 mutations in E. coli. Replica 1 detects one mutant to T+, replica 3 detects one mutant 

 to Bi+, and replica 2' detects one mutant to Pa+. 



MASTER 

 PLATE 



REPLICAS 



T L B, 



Medium 

 Contains 



^ o o o 



, -r -r -r 



^ TLB, 



T L B, 



+ - + + + - 



TLB, TLB, 



B Pa C 



^ o o o 



Medium ^ B+p^+^H 

 Contains ~ 



B Pa C 



B Pa C 



B Pa C 



