Indirect Selection and Drug Resistance 35 



When statistical fluctuations are considered, as shown by 

 Dr. J. Pfanzagl of Vienna (personal communication), the 

 enrichment expected is : 



l_e-'» _A 



l-e-""" /o 



where E is the ratio between the relative frequency of mutant 

 cells in tubes which have received at least one of them (/j), 

 and that one in the original suspension (/o); while m is the 

 expected number of mutants in the sample which has been 

 subdivided into n tubes. 



One such experiment, e.g. on 10 tubes, will give at best a 

 tenfold enrichment; but on repeating the indirect selection 

 experiment it is possible to isolate in a predictable number of 

 cycles a pure culture of resistant mutants, which will never 

 have experienced direct contact with the drug. 



This experiment was successfully carried out for high 

 degree resistance to streptomycin and for low degree resist- 

 ance to chloramphenicol in Escherichia coli. The speed of 

 selection observed was comparable to that predicted, as is 

 shown in greater detail in the paper by Cavalli-Sforza and 

 Lederberg (1956). 



A short report is given here of data in which this experi- 

 ment was amplified, considering that every experiment of 

 indirect selection tests just one culture, and that the most 

 informative stage is the first cycle (or, occasionally, the first 

 two cycles) of indirect selection. This usually permits the 

 counting of spontaneous mutants, and the comparison of 

 their number with that of the resistant cells counted by direct 

 selection, i.e. by plating in presence of the drug. In view of 

 the greater simplicity of the system, the experiment was 

 made on streptomycin resistance. 



Samples from a number of independent saturated cultures 

 were tested for streptomycin resistance and concentrations 

 of resistant cells per ml. ranging from to 105 resistants 

 were found. The tests on two cultures will be considered in 

 detail. 



