504 BACTERIOPHAGES 



rise to stocks of T4 which were not cofactor requiring. Stocks of 

 T4 phage prepared from cofactor-requiring plaques retained the 

 cofactor requirement, so that these characteristics were heredi- 

 tary. 



Broth stocks of T4 phage generally contain a mixture of dif- 

 ferent variants with respect to cofactor requirements. Delbriick 

 (1948) separated these variants from each other by adsorption 

 techniques. 



Example: The broth stock was diluted in a tryptophan-free chemically 

 defined medium until the tryptophan concentration was below the threshold 

 for adsorption. Then adsorption of the diluted phage stock with a suspension 

 of B grown in the tryptophan-free medium would remove all non-cofactor- 

 requiring variants. The infected bacteria could be removed by centrifuga- 

 tion and be used to produce a stock lacking a cofactor requirement. L-Trypto- 

 phan was then added to the supernatant to a concentration of 4 yug./ml. and 

 a suspension of B grown in tryptophan-free medium added. After sufficient 

 time for adsorption the infected bacteria were removed by centrifugation and a 

 sample of the supernatant plated on nutrient broth agar. Presumably all of 

 the tryptophan-requiring variants should have been removed in this adsorp- 

 tion, yet some phage particles capable of forming plaques on nutrient broth 

 agar remained. One such plaque was picked and used to inoculate a broth 

 culture of B to prepare a stock. This stock gave very low adsorption with 

 tryptophan but high adsorption with broth. Apparently this stock required a 

 cofactor other than tryptophan. Eventually it was found that the adsorption 

 requirement was tryptophan />/mj calcium ion. 



This adsorption technique using chemically defined media plus 

 various additions is a good method for separating different co- 

 factor-requiring variants present in a broth stock. A further 

 complication was introduced by Delbriick's (1948) observation 

 that the adsorption of some of the tryptophan-requiring variants 

 was strongly inhibited by indole. This inhibition was competi- 

 tive and the dissociation constant was less for the phage-indole 

 complex. Since E. coli rapidly converts tryptophan into indole, 

 this inhibition by indole presents a real difficulty in studying the 

 role of tryptophan in adsorption. 



An observation of T, F. Anderson's (1948c) which has greatly 

 simplified study of the cofactor requirement is that a bacterium 



