Bacterial Mutation and Conjugation 



307 



available for testing the preadaptive or post- 

 adaptive origin of mutants. The first method 

 starts with growing a single (presumably) 

 streptomycin-sensitive clone in liquid me- 

 dium and then plating it on an agar medium 

 to produce a large number of separate col- 

 onies. A sample of each colony is then 

 streaked across an agar medium perpendicu- 

 lar to a strip in the agar containing strepto- 

 mycin. Where there is no streptomycin, 

 each streak of bacteria will grow on the agar 

 and, if enough clones are tested, at least one 

 streak will also grow in the streptomycin 

 region (Figure 23-1). If streptomycin- 

 resistant mutants are postadaptive in origin, 

 the growth on the streptomycin-containing 

 area will be sharply discontinuous, since the 

 members of the clone streaked across the 

 streptomycin were originally sensitives, and 

 only rarely will more than one of these 

 bacteria respond to streptomycin by post- 

 adaptive mutation. Moreover, other sam- 

 ples from the original clone will succeed in 

 growing on streptomycin only to the same 

 limited degree as did the first sample. If, 

 on the other hand, the mutation is preadap- 

 tive, the growth across the streptomycin will 

 be practically as continuous as after streak- 



BAND OF STREPTOMYCIN 



RESISTANT 

 CLONE 



SENSITIVE 

 CLONE 



figure 23-1. Streptomycin-sensitive and 

 streptomycin-resistant E. coli as determined 

 by streaking individual clones. 



ing the drug with a pure clone of resistant 

 bacteria or with a mixture of bacteria rich 

 in resistant individuals. The proof that the 

 parental clone contained a spontaneous, pre- 

 adaptive, streptomycin-resistant mutant will 

 be complete if other samples of that clone 

 also grow readily when streaked across this 

 drug. 



Considering the rarity of mutation from 

 streptomycin-sensitivity to -resistance in this 

 strain (one per 10 7 cells), the labor involved 

 in testing the preadaptive or postadaptive 

 nature of streptomycin-resistant mutants by 

 this clone-sampling technique is prohibitive. 

 (Nevertheless, the method has numerous 

 uses in other genetic studies of bacteria.) 



The second method which can be used to 

 sample clones involves replica plating. 1 As 

 before, this procedure starts by spreading 

 the members of a single clone on streptomy- 

 cin-free agar in a petri dish. As many as 

 a thousand separate colonies can form after 

 the plate is incubated and by pressing this 

 master plate on the top of a sheet of velvet, 

 a sample of almost every colony can be ob- 

 tained simultaneously. The velvet — whose 

 fibers pick up 10 to 30% of each colony — 

 is then used to plant a corresponding pattern 

 of growth on a series of additional replica 

 plates (Figure 23-2). Preliminary control 

 tests show that the velvet makes several 

 excellent replicas of the master plate, and 

 that both streptomycin-resistant and -sensi- 

 tive clones can be replicated this way. The 

 first replica is made on drug-free medium, 

 whereas the second and later ones are made 

 with streptomycin-containing plates on 

 which, obviously, only streptomycin-resist- 

 ant bacteria can grow into colonies. If the 

 postadaptive view is correct, the chance that 

 cells from one colony will grow on two 

 replicas in the presence of streptomycin is 

 the same as it is for two colonies to grow 

 on the same replica. In other words, the 



1 Based upon work of J. Lederberg and E. M. 

 Lederberg. 



