GENETIC IMPLICATIONS OF MUTATIONS IN S. TYPHIMURIUM 269 



METHODS OF INDUCING AUXOTROPHIC MUTATIONS 



The strains of Salmonella typhimuriimi which we have used are two: 519 

 received from the New York Salmonella center at Beth Israel Hospital, and 

 533 (lie) from Gowen. 



Our method for isolating mutations to specific food or growth factor re- 

 quirements by penicillin screening is that of Lederberg and Zinder (1948) 

 and of Davis (1949) with some additions of our own. S. typhimurium is a 

 heterotrophic organism of the least exacting sort. Cultures will grow on a 

 basic medium containing ammonium sulphate, sodium chloride, potassium 

 phosphate buffers, with traces of other metallic ions, and glucose added as 

 an energy source. Better growth is obtained with a supplementary nitrogen 

 source, such as asparagin, and a further energy source, citrate, but these are 

 not essential. Thus the organism synthesizes all its own food comj)onents, 

 coenzymes, and growth factors, as well as the enzymes necessary for food 

 and energy tranformations. 



Suspensions are subjected to radiation by X-rays (up to 100,000 roent- 

 gens) or ultraviolet light (up to 3,600 ergs per mm.-), and are then transferred 

 to an enriched nutrient broth for 24 hours. The broth stimulates active 

 division of all organisms. These are centrifuged off, washed, and reinoculated 

 for 24 hours into the basic or minimal medium containing 100 units per ml. 

 of penicillin. This stops the divisions, and progressively kills the organisms 

 which divide actively. 



These organisms which penicillin screens out are called prototrophic 

 (Lederberg), and they are, of course, the unchanged originals. Any mutated 

 organisms w^hich now require some specific nutrilite will not divide on the 

 basic medium, and so they are not affected by penicillin. These are now 

 auxotrophic organisms (Davis), and they are isolated by plating on complete 

 agar, and identified by paper disc inoculations on successive plates of basic 

 medium with single nutrilites added — amino acids, nucleic acid fractions, or 

 vitamins, as shown in Figures 17.1 and 17.2. These methods are described 

 in more detail by Plough, Young, and Grimm (1950). 



AUXOTROPHIC MUTATIONS FROM RADIATED LINES 

 I shall cite only one set of isolations from such a radiation experiment, the 

 data for which are given in Table 17.1. Suspensions from an unradiated con- 

 trol and from seven successively increased X-radiation dosages were run 

 through the penicillin screening, and 500 auxotrophic mutants isolated. Of 

 these a total of 459 were recovered and their specific requirements deter- 

 mined. Although the control had been derived from successive single colony 

 isolations within 3 days of the tests, still 5 per cent of the isolated strains were 

 mutants — indicating that spontaneous mutation occurs and accumulates in 

 stock strains. 



From the major strain used (#533), 234 strains out of the 459 isolated 



