318 J. HOWARD MUELLER 



is complicated by tliis fact, since it is necessary to separate by 

 some means the accessory substances from the nitrogen supply 

 before either one can be studied separatelj'. 



The need for an accessory factor 



Before describing the various methods by which a separation 

 of the accessory factors of meat infusion was attempted, an 

 experiment will be described wliich strengthens the probability 

 that such substances, other than protein degradation products, 

 are necessary. It is possible that only amino acids or peptones 

 might be required for growth, but that in the preparation of com- 

 mercial peptone some essential amino acid, as, for example, 

 tryptophane, is wholly, or for the most part, altered or destroyed. 

 It is quite conceivable that unstable groupings other than trj^)- 

 tophane may be present in the original protein molecule, which 

 may withstand moderate heating in neutral solution and thus 

 be present in meat infusion among the anuno acids or peptones 

 in a soluble form, and yet be almost or quite lacking in commer- 

 cial peptone. If such were the case, a whole protein hydrolyzed 

 by trjT)sin or erepsin, together with salts and glucose, would 

 probably serve as a complete culture medium. A specimen of 

 commercial" casern after several day's digestion with trypsin was, 

 as a matter of fact, found to be quite satisfactory without the 

 presence of infusion, for the pneumococcus and streptococcus. 

 However, when the casein was purified by three precipitations 

 from Na2C03 solution by acetic acid, washed with alcohol and 

 ether and then digested as before, growth was negative. The 

 following protocol shows the results of such an experiment, in 

 which casein was prepared directlj* from milk. The "crude" 

 casein is the first precipitate obtained by acetic acid, the "pure" 

 casein has been three times reprecipitated and finally washed in 

 alcohol and ether. 



The two preparations of casein were dissolved in 0.5 per cent 

 Na2C03 and digested with a small quantity of trypsin (Fair- 

 child) at 37° under toluol for two weeks. At the end of this time, 

 the two solutions were boiled and filtered. Twelve cubic centi- 



