202 W. Hayes 



suited to the study of phenotypic expression. Moreover, in 

 control experiments, exposure of sensitive recipient cells in 

 which the zygotes are formed to any one of these drugs pre- 

 vents further cell division. The assumption can therefore be 

 made that a resistance gene present in such a phenotypically 

 sensitive cell is unlikely to be able to express itself after the 

 appropriate drug has been applied. 



The technique used is as follows (Hayes, 1957): Young 

 broth cultures of donor and recipient cells are mixed and kept 

 at 37° for 30 minutes to allow zygotes to form. The donor 

 cells have then fulfilled their fertilizing function and are 

 killed by adding to the mixture a high multiplicity of the 

 virulent phage, T6, to which the donor parent is sensitive but 

 the recipient cells, and the zygotes resulting from their 

 fertilization, are resistant. In this way, further mating is 

 prevented and we are left with what is termed a "zygote sus- 

 pension". To assess the kinetics of segregation and expres- 

 sion on synthetic minimal agar, two identical series of plates, 

 warmed to 37°, are inoculated with diluted zygote suspension 

 so as to yield, after incubation, about 20-30 recombinant 

 colonies per plate, each colony being composed of the progeny 

 of a T+L-j- recombinant segregant issuing from a single 

 zygote. At intervals after inoculation and incubation, the 

 surfaces of one series of plates are vigorously rubbed, in turn, 

 with distilled water by means of a glass spreader. This has the 

 effect of separating the progeny of any T+L+ recombinants 

 that may have already divided at the time of rubbing so that 

 the subsequent colony count is doubled for each generation. 

 Prior to division, of course, the colony count remains con- 

 stant since the effect of rubbing is simply to alter the position 

 of the zygotes or segregant s on the plates. At the same time 

 as plates of the first series are rubbed with distilled water, 

 plates of the second series are similarly rubbed with an appro- 

 priate concentration of valine or sodium azide, or with a 

 washed, high titre suspension of phage Tl. Since these agents 

 prevent any further division of sensitive organisms, only those 

 cells, whether zygotes or T+L-1- recombinant segregants. 



