ADAPTATION TO PANTOTHENATE DEFICIENCY 22-26 



It is important to note that no rapidly-growing mutant cells were 

 detected until the culture neared completion of growth and the cell 

 population was very high. Of course, since only a small amount of 

 the culture was sampled, it is unlikely that the adapted cells would 

 be detected when they first occurred, but only after they had multi- 

 plied sufficiently to be fairly numerous in the population. The mu- 

 tants maintained their rapid growth rate on transfer to pantothenate - 

 free liquid medium and reached their maximum growth in about a 

 week.' 



In addition to these two rapidly growing colonies some of the 

 cells plated on pantothenate -free agar grew slowly and formed visi- 

 ble colonies (around 0.25 mm.) in about two weeks. Thirty of these 

 were tested by transfer into pantothenate -free liquid medium, where, 

 except for five colonies which did not grow, they showed considerable 

 growth (a turbidity reading of between 60 and 270) in about nine days. 

 Three such colonies were detected in about 850 cells plated on the 

 83rd day after inoculation. The population at this time was about 

 6 X 105 cells per ml. Fifteen colonies were noted six days later 

 (from about five times that number of cells); the cell population had 

 increased to 1 x 10^ per ml. Because of their slow growth, a good 

 many of the colonies were probably overlooked. Apparently panto- 

 thenate -independent "mutants" with intermediate growth rates may 

 occur. 



Another culture (HI) similarly studied was incubated for 41 days 

 at 30^ C. until the population was slightly less than 1 x 106 cells 

 per ml. and was then held at 7^ C. until completion of growth; under 

 these conditions growth takes place more slowly and any change in 

 the population can be more easily detected. The development was 

 comparable to that obtained at 30° C. The growth rate was constant 

 (generation time 124 hours at 7°C.) and no mutants were picked up 

 in the samples plated until again the culture had reached its maxi- 

 mum growth. At this point, 76 days after inoculation, one small 

 colony appeared after several days on a pantothenate -free agar plate 

 in about 90,000 cells plated. No such colonies appeared in about 

 120,000 cells plated the next day, but 2 days later 3 colonies out of 

 90,000 plated grew more rapidly. On the next day 1 out of 100,000, 

 4 days later, 3 out of 80,000, 5 days later, 3 out of 120,000, 6 days 

 after this, 4 appeared, and in the last sample taken at 104 days, 

 there were 5 colonies. 



As before, the population was almost at its maximum point be- 

 fore the first rapidly growing colony was detected, so there was no 

 increase in population size or in number of adapted cells. 



These data show that the adaptation is not a simple one -step 

 process. First, there is a primary adaptation which enables the 

 culture to grow slowly in the pantothenate -free medium. When 

 the cells are transferred to another tube of pantothenate -free me- 



