50 M. Demerec 



membrane filters, upon which bacterial colonies could be 

 transferred from one medium to another without disturbing 

 their position, this technique was adapted for studies of drug 

 resistance (Bornschein, Dittrich and Hohne, 1951; Dittrich, 

 1951). 



A third method of testing the origin of resistant variants 

 is the replica-plating method originated by Lederberg and 

 Lederberg (1952), in which samples of colonies grown on an 

 agar surface can be transferred to a drug-containing medium, 

 without serious disturbance of their spatial arrangement, by 

 stamping consecutive plates with a piece of sterile velveteen. 

 With this method it has been possible to show the drug- 

 independent origin of mutants resistant to streptomycin 

 (Lederberg and Lederberg, 1952) and isoniazid (Bryson and 

 Szybalski, 1952). 



A fourth method, which like the replica-plating method 

 permits "indirect" selection of resistant variants in the 

 absence of the drug, was described recently by Cavalli-Sforza 

 and Lederberg (1956), and its further application has been 

 discussed by Cavalli-Sforza (this symposium, p. 30). It is 

 carried out by making subcultures in liquid medium and 

 choosing the "best" subcultures by testing samples on agar 

 medium containing the drug. Using this method, Cavalli- 

 Sforza and Lederberg have supplied additional evidence of 

 the spontaneous origin of variants in Escherichia coli that are 

 resistant to streptomycin or to chloramphenicol. 



Mutability 



Two features usually associated with bacterial resistance to 

 streptomycin, namely, the appearance of highly resistant 

 variants in a single step and the appearance of streptomycin- 

 dependent variants, make it especially suitable for quantita- 

 tive studies of the occurrence of changes from sensitivity to 

 resistance or dependence, of reverse changes from dependence 

 to sensitivity, and of changes from dependence to resistance. 

 Such quantitative studies have been carried out extensively 

 with Esch. coli. It has been found that whenever any such 



