334 



CHAPTER 36 



cin-sensitive clone grown in liquid medium 

 which is plated on agar to produce a large 

 number of separate colonies. A sample of 

 each colony is then taken and streaked across 

 agar which has a strip containing streptomy- 

 cin. All clones will grow on the agar, except 

 in the streptomycin region. But if enough 

 clones are tested, one will be found to grow 

 there also (Figure 36-5). If streptomycin- 

 resistant mutations are postadaptive in origin, 

 the growth found on the streptomycin should 

 be sharply discontinuous, since the members 

 of the clone streaked across the streptomycin 

 were originally sensitives, and only rarely 

 would more than one of these bacteria be 

 expected to respond to streptomycin by post- 

 adaptive mutation. Moreover, return to the 

 original clone would furnish other samples 

 which would succeed in growing on strepto- 

 mycin only rarely, and then only to the same 

 limited degree as did the first positive sample. 

 If, on the other hand, the mutation is pre- 

 adaptive, the growth across the streptomycin 

 should be just about as continuous as one 

 would find after the drug was streaked with 

 a pure clone of resistant bacteria, or a mixture 

 of bacteria rich in resistant individuals. The 

 demonstration that the parental clone repre- 

 sented a spontaneous, preadaptive, strepto- 

 mycin-resistant mutant would be completed, 

 if other samples of that clone also grew readily 

 when streaked across this drug. 



FIGURE 36-5. Streptomycin-sensitive and strepto- 

 mycin-resistant E. coli as determined by streaking 

 individual clones. 



BAND OF STREPTOMYCIN 



RESISTANT 

 CLONE 



SENSITIVE 

 CLONE 



In view of the rarity in this strain of muta- 

 tion from streptomycin-sensitivity to re- 

 sistance (1 per 10^ cells), the labor involved is 

 so prohibitive that this clone-sampling tech- 

 nique is impractical for testing the pre- or 

 postadaptive nature of streptomycin-resistant 

 mutants. (Nevertheless, this method has 

 numerous uses in other genetic studies of 

 bacteria.) 



The second method which can be used to 

 sample clones involves replica plating} One 

 starts the procedure, as before, with a single 

 clone which has been spread on streptomycin- 

 free agar in a petri dish. After the plate is 

 incubated, there may be as many as a thou- 

 sand separate colonies. A sample of almost 

 every colony can be obtained simultaneously 

 by pressing this master plate on the top of a 

 sheet of velvet whose fibers pick up 10-30% 

 of each colony. The velvet is then used to 

 plant a corresponding pattern of growth on 

 a series of additional plates. The first copy 

 is made on drug-free medium also, whereas 

 the second and later copies are made with 

 plates containing streptomycin. (Of course, 

 preliminary control tests show that the velvet 

 makes a number of very good replicas of the 

 master plate — Figure 36-6 shows one replica 

 — and that both streptomycin-resistant and 

 sensitive clones are replicable this way.) On 

 the second and later replicas, only strepto- 

 mycin-resistant bacteria will grow into colo- 

 nies. If the postadaptive view is correct, a 

 colony that grows in the presence of strepto- 

 mycin on one replica will not show any greater 

 likelihood of growing on other replicas, made 

 from the master plate at the same time or 

 subsequently, than will a colony located in a 

 diff'erent position. In other words, the posi- 

 tions of the resistant colonies on different 

 replicas should be at random to each other. 

 If the mutants are preadaptive, however, 

 there should be a very good likelihood that all 

 replicas will be resistant in the same position 



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

 berg. 



