30S 



CHAPTER 23 



positions of the resistant colonies on differ- 

 ent replicas arc random. II' the mutants are 

 preadaptive, however, all replicas probably 

 will be resistant in the same position (al- 

 though some exceptions can occur if the 

 velvet tails to place a portion o\' the same 

 colony on every replica plate). Of course, 

 one also readily finds resistant bacteria in 

 the corresponding position on the master 

 plate. Although this clone-sampling tech- 

 nique is advantageous for many other pur- 

 poses, it is still too laborious for testing the 

 preadaptation or postadaptation hypothesis. 

 since replicas of about ten thousand master 

 plates are required to be reasonably sure of 

 rinding one clonal streptomycin-resistant 

 mutant. 



This difficulty can be avoided by using a 

 third method for clone-sampling that in- 

 volves replica-plating contiguous colonies. 

 A billion or so bacteria (from a streptomy- 

 cin-sensitive clone) plated on drug-free agar 

 will produce small clones so closely spaced 

 that they grow together and form a bacterial 

 lawn (Figure 23-3A). Nevertheless, rep- 

 licas of this growth can be made on strepto- 



mycin-containing agar and will show growth 

 wherever drug-resistant mutants occur (Fig- 

 ure 23-3B, CD). One can then turn to 

 the corresponding regions on the master 

 plate to obtain samples to be tested for re- 

 sistance to the drug. If such samples are 

 no richer in resistant mutants than samples 

 from randomly-chosen sites on the master 

 plate corresponding to those which are not 

 mutant on any replica, the postadaptive view 

 is proved. When the experiment is actually 

 performed, the master plate is found to be 

 much richer in mutants at replica sites that 

 are mutant than at those that are nonmutant. 

 Moreover, replicas tend to have mutant 

 clones at corresponding positions on all 

 replica plates (Figure 23-3B-D). Accord- 

 ingly, most mutants are clearly preadaptive. 

 Other experiments show conclusively, in the 

 case of streptomycin, that almost all, if not 

 all, mutants resistant to the drug are pre- 

 adaptive — that is, streptomycin does not in- 

 duce a detectable number of resistant mu- 

 tants. Since the same results are obtained 

 with the drug chloramphenicol, one can 

 extrapolate and conclude that, in general, the 



figure 23-2. Separate colonies replica-plated (right) from a master plate 

 (left). (Courtesy of N. E. Melechen.) 



