34 L. L. Cavalli-Sforza 



also be sensitive. Therefore, the test on one of the two 

 daughters will permit us to formulate with good probability 

 the prediction that also the other daughter is sensitive. If 

 the mother cell was resistant, both daughters should be 

 resistant; this situation being revealed by the test carried out 

 on one of the two daughter cells, it will be possible to verify 

 the prediction that the untested sister should give rise to a 

 pure colony of resistant cells. 



This method has been made technically possible, for the 

 first time, by the use of replica plating on solid media; and 

 it has permitted the indirect isolation of streptomycin- and 

 phage-resistant mutants. Later (Cavalli-Sforza and Leder- 

 berg, 1956), a method was developed for carrying out indirect 

 selection in liquid cultures — which has the advantage over 

 replica plating of leading more easily to quantitative analysis 

 — with a view to answering the question: Have all resistant 

 cells arisen by spontaneous mutations, or have some arisen 

 by other mechanisms, such as mutation induced by the drug, 

 or adaptation not controlled by nuclear determinants? 



Concentration by limiting dilution 



The principle of this method (Cavalli-Sforza and Leder- 

 berg, 1956) is to concentrate spontaneous mutants to resist- 

 ance by using a sample which contains few resistant cells, 

 possibly only one, and subdividing it further. If there is in 

 the sample before subdivision just one resistant mutant and 

 (N— 1) normal sensitives, i.e. if the relative frequency of 



mutant cells is ^j^, after subdivision in n tubes the frequency 



n , 

 of mutants will be :j^ in the tube which happened to receive 



the single mutant, and zero in the other tubes. Each tube will, 

 in fact, receive 'Njn bacteria but only one of them contains 

 the resistant mutant. This tube will therefore show an 

 enrichment of mutants of n times. It will be possible to 

 identify it by incubating all tubes after addition of fresh 

 broth, and testing samples from them for drug resistance. 



