Discussion 261 



again washed in phosphate buffer and centrifuged ; then resuspended in 

 phosphate buffer in tubes in a series containing 12, 16, 24, 50 and 100 [ig. 

 of streptomycin per ml. In another series we did just the same but 

 instead of phosphate buffer we used sahne. The tubes were kept at room 

 temperature. Samples were taken at 5- or 6-day intervals, and the screen 

 level was the same as the incubation concentration of streptomycin. 

 Over a 2-week period the number of cells remained practically the same 

 in the tubes containing 12, 16 and 24 [ig. of streptomycin per ml. A 

 slight but steady killing was observed in the tubes with 50 [j.g./ml. and a 

 quite marked decrease in the fraction surviving occurred in the tubes 

 with phosphate buffer containing 16 (jig. streptomycin per ml. after 5 

 days, and also in the tubes containing 24 [j,g./ml. after 10 days; but no 

 increase in the survival fraction was observed in the series where saline 

 was used instead of phosphate buffer. We did not retest, and so we do 

 not know whether these survivors are stable variants with a low degree 

 of resistance or only persistors. It may be that this survivor fraction 

 was only an accumulation of persistors, i.e. the plus variants on the 

 range up to the upper limit of the phenotypic variability of the original 

 sensitive genotype were increased. Some multiplication may have 

 occurred also in the tubes where we used phosphate buffer, because after 

 autolysis of some cells phosphate was present also, and perhaps such an 

 equilibration resulted in no change in the total number of cells. No 

 multiplication could occur, however, in saline, and so we could observe 

 no increase in the survival fraction. A tendency of the survival fraction 

 to increase also occurred in some of the tubes containing 50 and 100 

 [Jig. /ml., both in phosphate buffer and in saline. I think, however, that 

 this increase is deceptive, because there was a decrease in the total 

 number of the cells and therefore we got a proportionately higher survival 

 fraction. It may be concluded from these observations that in no case 

 did we obtain such a high proportion of induced resistants as was 

 reported by Akiba and by Szybalski ; and even before Szybalski published 

 a correction of his early conclusions we were of the opinion that, at 

 least in the case of Serratia marcescens, under our experimental conditions 

 there is no "Lamarckian" inheritance of streptomycin resistance. 



Kunicki-Goldjinger : I would suggest trying a shorter incubation period. 

 You have incubated your bacteria for 7 or 14 days, whereas 24-25 hours 

 incubation should be enough. After so long incubation more cells are 

 dying and the number of surviving cells is greatly diminished. 



Slonimski: Rizet and Marcou (1957, Microbiology, in press), working 

 with the filamentous fungus Podospora anserina, obtained interesting 

 results on the effect of cooling. Several strains of this fungus are bound 

 to die if reproduced vegetatively. It is necessary to outcross them to 

 keep them. However, if a strain having a vegetative life expectancy of 

 say 2 days at 25° is maintained for a while at 2°, its life expectancy 

 (at 25°) is increased to some 60 days. The kinetics of rejuvenation have 

 been partially worked out ; it depends on the duration, temperature and 

 genetic constitution of the strain. 



