CULTURAL REQUIREMENTS OF BACTERIA 335 



well as tryptophane, tyrosine and cystine have been tested out 

 individually and collectively against known X and Y prepara- 

 tions and found to have no influence in producing growth under 

 these conditions. The constancy of pigment in the X fraction, 

 together with the fact of the disappearance of pigment from the 

 heart infusion on boiling with charcoal, may be suggestive. 

 However, the results obtained by the precipitation of this frac- 

 tion with phosphotungstic acid, indicate that the pigment is not 

 concerned in the action of the X fraction. By the addition of 

 phosphotungstic acid to this fraction, in the presence of 5 per cent 

 H«S04, it was possible in several experiments to obtain a filtrate 

 quite free from pigment, which when freed from phosphotung- 

 stic acid with Ba(OH)? and the excess of Ba removed, contained 

 an active X factor. Such a solution, evaporated on a watch 

 glass j-ielded a semi-crystalline residue. The phosphotungstic 

 precipitate also contains a small amount of the X, but in the 

 single experiment in which it was attempted to learn quantita- 

 tively how it was distributed by using diminishing quantities 

 against a constant amount of Y, the filtrate seemed to have about 

 75 per cent of all the X in the fraction. Unfortunately, these 

 observations were made with a single solution of phosphotungstic 

 acid, and all subsequent preparations of the reagent have de- 

 stroyed the activity of the X fraction entirely. Up to the pres- 

 ent, therefore, all that can be said of the X fraction is that it is 

 apparently not in any way connected wdth the pigment nor with 

 the histidine which it contains. It is hoped that further work 

 will throw more Ught on this factor. 



Further ■purification of Y fraction 



The Ag2S04 filtrate, or Y fraction has proved to be somewhat 

 simpler to work with than the X fraction. When evaporated, 

 after freeing quantitatively from Ag and Ba, it is semi-crystalline. 

 It contains a varjdng quantity of tjTosine and perhaps some 

 trjTitophane. By precipitation with a small quantity' of HgS04 

 and allowing the material to stand over night, any tryptophane 

 is thrown out, together with part of the tyrosine. The result- 

 ing filtrate, after remo\'ing Hg and H2SO4, contains active Y. 



