Bacterial Mutation and Conjugation 



311 



terial lawn, about 10 s bacteria from one line 

 are plated onto agar containing complete 

 medium. Then three replica plates are made 

 for the TLB X - line (Figure 23-4), each 

 plate contains complete medium deficient in 

 a different single nutrient (T, L, and B t , 

 respectively). Occasionally, a replica shows 

 a clone that is able to grow because a pre- 

 adaptive mutant produces prototrophy for 

 the nutrient missing from the medium. 

 However, such clonal growth is not found 

 in the corresponding position on all three 

 replicas (or even on two) with greater than 

 chance frequency. The same results are ob- 

 tained when an equal number of bacteria of 

 the B~Pa~C~ line are plated on complete 

 medium and tested on appropriate replicas. 

 We may conclude, therefore, that on rela- 

 tively rare occasions mutants to prototrophy 

 for one nutrient do occur singly, but double 

 or triple mutants do not occur with detect- 

 able frequency. 



In another test, the preceding experiment 

 is repeated exactly, with the exception that 

 the same numbers of the two triply-mutant 

 strains are mixed in the liquid medium before 

 being plated on agar containing complete 

 medium. In this case (Figure 23-5), six 

 replicas are made with medium which is 

 complete except that three lack B, Pa, and 

 C in addition to lacking T, L, or B x ; the 

 other three lack T, L, and B, and also B, 

 Pa, or C. Individuals of the T~L~B X ~ 

 strain cannot grow on the first three replicas 

 mentioned because a single required nutrient 

 is missing; they cannot grow in the last three 

 because all three required nutrients are miss- 

 ing. Individuals of the B~Pa~C~ strain 

 cannot grow on the first three replicas be- 

 cause all three required nutrients are miss- 

 ing; they cannot grow on the last three be- 

 cause one of the three is absent. If the 

 master plate contains a mutant preadaptive 

 to nutritional independence for one of the 

 nutritionally dependent loci, in only one of 

 the six replicas will the mutant form a colony. 



For example, if a T+ mutant occurs among 

 the individuals of the T~L-Bi~ strain on 

 the master plate, a colony will grow only 

 on the replica lacking B, Pa, C, and T. 

 Actually, about a hundred different positions 

 on the master plate show growth on the 

 replicas. This number is very much larger 

 than that found in the two groups of three 

 replicas made after plating the two lines 

 separately. In the present case, some posi- 

 tions show growth only on one of the six 



+++ ++ + ___ 



TLBBPaC T L B, B Pa C 



\ / 



Mixed 



MASTER PLATE 



++++++ ^ 

 T L B,B Pa C •Medium Contains 



SIX REPLICAS 



2 3 



.9 .<? P 



T L + B,B Pa C T L B,B Pa C T L B,B Pa C 



o.. O. Q.. 



T L B,B Pa + C + T L B,B Pa C T L B,B Pa C 



figure 23-5. Replica-plating (shown diagram- 

 matically ) to detect genetic recombination in 

 E. coli. A completely prototrophic recombi- 

 nant is found at 12 o'clock in all replicas. A 

 recombinant for both Pa and C is found at 

 3 o'clock on replicas 5 and 6. Replica 1 has 

 a clone growing at 9 o'clock which may be 

 due either to recombination or to mutation to 

 T + . 



