72 Discussion 



for one can show that in some systems there is heterogeneity within a 

 clone which is due neither to genetic differences nor to detectable 

 differences in the environments of the differing individuals. For in- 

 stance, in some Salmonella strains only a characteristic proportion 

 (e.g. 10-^) of the cells of a clone can synthesize locomotor apparatus, 

 even when the clone has been grown in a shaken flask of broth, so that 

 all the cells grew in an identical environment (so far as it is possible to 

 provide one). A direct comparison of the progeny of synthesizing and of 

 non-synthesizing individuals (isolated by micromanipulation) shows no 

 difference : therefore the two kinds of cell do not differ genetically. The 

 difference between them can therefore be attributed neither to differ- 

 ences in their genotypes, nor to (detectable) differences in their environ- 

 ments. Thus, the genotype and the environment determine only the 

 relative probabilities of the two alternative phenotypes in any particular 

 individual. This is presumably true also in the case of drug resistance ; a 

 common example is that under some conditions of selection (for instance, 

 an antibiotic concentration which permits colony formation by say one 

 per cent of the cells plated) it is not infrequent to find that the anti- 

 biotic resistance of the population of these colonies is indistinguishable 

 from that of the parent strain ; all one can say is that the original pro- 

 genitors of these colonies differed in their phenotype from the mode, but 

 that particular variation of phenotype is not heritable. 



Dr. Hughes, would you please explain in more detail the actual 

 procedure by which you observed these small steps with apparent 

 increasing resistance to the drug. Did these cells, for instance, go through 

 an intermediate period of cultivation in the absence of the drug to 

 show that something other than a phenotypic adaptation had occurred? 



Hughes: Yes ; you take a single cell or microcolony, grow up a manage- 

 able amount of culture, about 500-1000 cells, then transfer to broth. 

 From this you plant out again in the test medium, which in this case is 

 agar with a given level of streptomycin. You weed the cells out so that 

 there is a clear area to each. You then cultivate for a standard length 

 of time at a standard temperature and measure the colony diameter. 

 Having measured up the whole of the diameters, you pick the smallest 

 and the largest colonies, make new emulsions and repeat the procedure. 



In the technique for testing two daughter cells separately, you take a 

 single cell and put it into a drop of broth. You wait until it has divided, 

 then transfer each individual into a drop of broth. You then transfer the 

 drop to a marked position on a small block of agar containing the in- 

 hibitor (I always put one on each corner of the block). Then you can 

 follow them at any interval ; you recover them after your standard time 

 of exposure and put them into normal nutrient medium, or if you wish, 

 into a penicillinase medium if you are working with penicillin, to see 

 whether they recover and give living organisms or not. You can repeat 

 this as often as your patience will allow. 



Davis: In the experiments where drug resistance was building up, 

 were they always cultivated in that concentration of the drug ? 



Hughes: No, they had one subculture in between each test in normal 

 broth. 



