Discussion 29 



In this instance, precisely as with the Lac~ and Lac+, we are deahng 

 with the selective multiplication, in a large population, of the rare 

 resistant organism. 



A more interesting experiment was that with chloramphenicol and 

 Strep, faecalis in a sealed agar plate. If the plate was poured with no 

 more than 50-100 organisms, then at threshold concentrations of chloram- 

 phenicol, after periods of as long as 50 days, every organism inoculated 

 ultimately grew out to form a visible colony. At this point we thought 

 we had demonstrated adaptation, because each colony which appeared 

 proved to be relatively resistant to chloramphenicol. However, if during 

 this period of supposed adaptation, and before visible colonies had ap- 

 peared, the agar were cut into e.g. 100 sectors, and the number of 

 organisms in each sector determined, we found that all through this 

 period there had been an extremely slow but progressive multiplication 

 of the organisins. Initially, these organisms were not resistant. It was 

 only after the population of the microcolony reached fairly large pro- 

 portions that resistant organisms began to appear; and those resistant 

 organisms then multiplied at a relatively rapid rate. These results are 

 again consistent with mutation and selection, and do not prove adapta- 

 tion. At least with penicillin and chloramphenicol, we have therefore 

 found no need to invoke physiological adaptation as the basis for the 

 development of increased resistance. All our data are consistent with 

 the rare appearance of mutation, followed by selection. 



Hinshehvood: Dr. Eagle, what is the criterion of a resistant organism? 



Eagle: An organism which is capable of growing rapidly to form a 

 colony at a concentration of penicillin or chloramphenicol at which 

 normally no colony appears. 



Hinshelwood: Under what conditions? 



Eagle: In the case of penicillin, plating out in agar and watching the 

 plates for about 5 or 6 days. The proportion of organisms which develop 

 colonies is of course a function of the concentration used. With our 

 particular strain of staphylococcus, for instance, at 0-02 [jig./ml. 100 

 per cent will grow up in time ; at • 03 (ig. the number begins to fall off 

 very sharply ; at • 04 \Lg. one gets down to a very small fraction of viable 

 organisms. 



Hinshelwood: In the curv^e you showed, the cells are dying off in one 

 part, obviously. There is a race between dying and adaptation. At a 

 given stage you plate, and the surviving cells will partially adapt. Sup- 

 pose that slow adaptation is going on, in competition with the dying off; 

 now you plate your samples at intervals, and naturally a larger and 

 larger number of cells can survive on the plate as the adaptation in the 

 liquid approaches completion. The number that can survive is steadily 

 increasing as the adaptation approaches more nearly to perfection. But 

 if you have too small an inoculum all the cells will be dead before any- 

 thing like adaptation has set in, so on the adaptive hypothesis you would 

 expect precisely the result found. I would suggest that the experiment 

 is not unambiguous and is made uncertain by your very criterion of what 

 is a resistant form. It is a form which has achieved some measure of 

 adaptation by the time the sample was taken. 



