ADAPTATION TO PANTOTHENATE DEFICIENCY 22-20 



The numbers designate the numbers of colonies appearing on 

 the plate. The letters "s" and "vs" designate small and very small 

 colonies. A number alone designates a large colony, i.e., 0.4 to 

 0.6 mm. Three large and three small colonies is designated 3,3s. 



This method does not determine whether the mutations take 

 place in the tubes prior to plating or whether they take place on 

 the plate, particularly since a time elapses before any colonies 

 appear. A slight film develops from the approximately 5 x 10 * to 

 5 X 10° non- synthesizing cells which are seeded on each plate. 

 Microscopic examination showed that each cell had increased on 

 the average to about 30 cells during several weeks on the plate. 

 The film appeared within two or three days after plating, after 

 which there was no further apparent macroscopic change until in- 

 dividual colonies began to appear (fig. 22-6). Microscopic exam- 

 ination of the minute colonies after several weeks on the plate 

 showed that the cells were vacuolated, as is characteristic of old 

 cells, and staining with dilute methylene blue showed that with few 

 exceptions the cells were all dead. 



The fact that the cell population seems to multiply very little 

 after the first few days suggests that the mutations do not take 

 place on the plates, but occur in the culture tubes prior to plating. 

 It appears further that the crowding of the cells may actually slow 

 down the growth rate of the mutant cells which are plated out, and 

 thus account for the long delay before their appearance, since 

 crowding generally slows down the growth of individual colonies. 

 As may be observed from table 22-2 there were a number of in- 

 stances in which 50 or 100, or even more colonies appeared simul- 

 taneously on one plate. It seems unlikely that so large a number 

 of mutants would occur simultaneously, as would have to be the case 

 if they occurred on the agar plate. 



In addition to the variation in numbers of colonies there is also 

 variation in the size and the time of appearance of the colonies, 

 which suggests that all the mutants are not identical. Further test- 

 ing will be required to determine how many classes of mutants are 

 involved. 



A few hundred cells of some of the colonies appearing on the 

 test plates were inoculated into liquid test*medium. Some colonies 

 which required about two weeks to develop into small colonies on 

 the test agar plates, on transfer to liquid medium reached maximum 

 growth within four to eight days. Colonies requiring a longer time 

 to develop on the plates, also grew more rapidly in liquid test medi- 

 um than they had on the agar plates, though still not as rapidly as 

 the faster-growing colonies. The more rapid growth rates in liquid 

 test medium may have been due to the fact that the crowding on the 

 plates inhibited the growth of the mutant cells on the plates as point- 

 ed out above. On the other hand, it is possible that secondary mu- 



