SEGREGATIONS IN ESCHERICHIA COLI 509 



the metabolism of pyruvic and acetic acids, which will be described in more 

 detail elsewhere. Independent mutations to other inhibitors, including iodo- 

 acetate, azide, streptomycin, streptothricin, mercuric chloride, and Brilliant 

 Green, can be secured in a similar fashion, but genetic analysis of these muta- 

 tions has not been completed. 



Morphological variation has occasionally been noted (exceedingly rough or 

 very mucoid colonial form) but is relatively unsuitable for genetic work be- 

 cause the presumably random choice of prototroph recombinants may be 

 influenced. 



In addition to the EMB agar already described, a number of other natural or 

 "complete" media have been used. The Difco product "Penassay Broth" has 

 been used most extensively, and is satisfactory for the preparation of inocula, 

 except that it must be supplemented with cystine for the growth of cystineless 

 organisms, such as strain Y-24. Other satisfactory media include a broth con- 

 sisting of: peptone 5, glucose 5, yeast extract 3, g/1, as well as Difco Nutrient 

 Broth, and diverse concoctions containing peptone or casein hydrolysates and 

 meat or yeast extract. 



The synthetic or minimal medium contains, in g/1: NH 4 C1 5, NH4NO3 1, 

 Na 2 S0 4 2, K2HPO4 3, KH2PO4 i, glucose 5, asparagine 1.5, MgS0 4 0.1, trace 

 elements (Gray and Tatum 1944), and CaCl 2 , a trace. The medium is made 

 solid by the addition of agar in a concentration of 1.5 percent. 



To avoid flocculation when used with agar, the glucose and agar in solution 

 should be autoclaved separately, and mixed with the other components just 

 before using. Unwashed agar (Difco) is sufficiently free of the growth factors 

 under consideration to be satisfactory for many experiments; the use of washed 

 agar, however, is recommended for the cleanest results. 



The detection of recombinants is based upon the inability of biochemical 

 mutant bacteria to proliferate in the absence of their specific growth sub- 

 stances. Plating in minimal agar, therefore, has the effect of a sieve for proto- 

 troph cells. To insure against contamination with prototrophs derived by re- 

 verse mutation, which has been noticed at certain loci, it has been desirable to 

 use multiple biochemical mutants as the parental stocks in recombination 

 studies. Coincidental reversion at two or more loci is theoretically improbable, 

 and experimentally undemonstrable (Ryan, 1946, Tatum and Lederberg, 

 1947) . For example, plating either B'M-T+L+BS or B + M + T~L- Brsepa.ra.tely 

 into minimal agar did not lead to the appearance of prototrophs, B+M+T+L+- 

 Bi + . When, however, a mixture of these cell types was so "sieved," one proto- 

 troph was found for about each io 7 cells inoculated. These have been assumed 

 to arise from the recombination of "+" alleles to form the prototroph. 



In previous experiments, the two multiple mutants were inoculated together 

 into a complete medium and allowed to grow in mixed culture before plating 

 into minimal agar. This method is not satisfactory for present purposes be- 

 cause it allows possible selective differentials to alter the relative frequencies 

 of different recombination classes. A modified procedure has been developed, 

 which will now be described in detail. 



The mutant stocks are maintained on "complete" agar slants, transferred 



M7 



