510 JOSHUA LEDERBERG 



at intervals of 6-8 weeks. They are inoculated separately into test-tubes con- 

 taining about ten ml of liquid comj lete medium and incubated overnight at 

 30°C with gentle shaking. The following morning, an additional ten ml of the 

 same medium is added to each culture, and the tubes are incubated in the same 

 manner for an additional three to five hours. These cultures contain from 1-4X 

 io 9 cells per ml. They are then washed in the following manner: the cotton 

 plugs are replaced with sterile corks which have been kept in 95 percent alcohol 

 and the alcohol flamed off just before using. The cultures are then centrifuged 

 at about 2500 r.p.m. for 20 minutes, which suffices to jack the cells in the 

 bottom of the test tubes. The supernatant medium is carefully poured off, and 

 the tube is rinsed with about 10 ml sterile distilled water, care being taken not 

 to disturb the pellet. The cells are then resuspended in an additional 15-20 ml 

 sterile water, and recentrifuged. The supernatant wash water is decanted and 

 replaced with an equal volume of fresh sterile water, in which the cells are 

 suspended. In the meantime, minimal agar plates are prepared. A bottom layer 

 of about 15 ml minimal agar is poured into each Petri plate and allowed to 

 solidify. Cell suspensions of different mutant stocks are mixed at this time and 

 measured quantities (usually about io 8 -io 9 cells) are pipetted onto the agar 

 surface. At this time also, one may add such growth factor supplements as are 

 desired to permit the growth of recombination types other than prototrophs. 

 The cell suspensions are then mixed into a layer of about- ten ml molten mini- 

 mal agar (at 45-5o°C) which is poured onto the plates. After the agar hardens, 

 the plates are incubated at 30°C for a period of 48 hours. At this time proto- 

 troph colonies will be found distributed throughout the plate, many of them 

 at or near the surface and accessible to picking for further characterization. 



The procedure may be varied in several ways. It is important however that 

 the inoculum consist of "young" cells, since cultures of 24 hours or older have 

 given quite inconsistent results. It is possible to store the inoculum in distilled 

 water for at least twenty-four hours without appreciably affecting the yield, 

 which suggests that the aggregation of genetic types leading to the recombi- 

 nation process occurs in the molten or the solidified agar. This occurrence 

 must, however, take place within a few hours, since the recombinant proto- 

 trophs are not appreciably slower to appear than wild type cells in a similar 

 physiological state which may be streaked on the surface of the plates. Pre- 

 sumably, therefore, one could increase the yield of prototrophs by making 

 conditions more favorable for the free contact of the cells, as by packing them 

 together in a centrifuge tube in minimal liquid medium. However the compli- 

 cation of proliferation of prototrophs already formed would interfere with the 

 interpretation of such an experiment. Many physiological factors may inter- 

 fere with the recombination process, and, for example, the yield may be re- 

 duced markedly by inoculating too heavily, or by omitting an under-layer of 

 agar into which, presumably, deleterious metabolic products may diffuse. In- 

 stead of mixing the cells in semisolid agar, it is possible to streak the mixture on 

 the surface of slightly dried minimal agar plates. Under these conditions, how- 

 ever, the prototroph colonies are likely to be more heavily contaminated with 

 the residual parental mutant types. 



