CULTURAL REQUIREMENTS OF BACTERIA 331 



tatod more easily by that reagent, than tryptophane, in purify- 

 ing the latter. It was, therefore, a logical procedure to attempt a 

 separation of the active fraction of the mercuric sulphate precipi- 

 tate by fractional precipitation with the same reagent. The 

 results of a number of preliminary experiments in this direction 

 gave evidence that the first crude mercuric sulphate precipitate 

 contained two substances, both of which, together, were required 

 for reactivation. One of these was easily reprecipitated by the 

 addition of mercuric sulphate solution to the solution obtained 

 from the first crude precipitate. The second was not completely 

 reprecipitated even by the use of a considerable excess of the 

 precipitant. 



The following experiment will illustrate these facts. It will 

 be observed that in place of an acid hydrolysate of casein, a 

 commercial enzjine digest of milk proteins to the amino acid 

 stage, called "aminoids" has been used. This was done for 

 two reasons. In the first place, the process is less troublesome 

 than the long continued acid hydrolysis, and in the second place 

 the preparations obtained are somewhat more active than those 

 from an acid hj'drolysate. The latter, after a certain amount 

 of chemical manipulation will often give rather weak growth in 

 test media, corresponding to only a "2" or a "3" on the BaS04 

 turbidity scale. It is reaUzed that such a procedure is not be- 

 yond criticism, and that it perhaps strengthens the possibiUty 

 that one is not dealing with amino acids, but with adsorbed non- 

 protein material. However, it is equally possible that these 

 physiologically active substances are partially destroyed or 

 altered by long treatment with acid. At any rate, the main 

 facts of each point established with aminoids have been checked 

 with an acid hydrolysate of casein, and like results obtained. 



Ten grams aminoids were dissolved in 100 cc. water and pre- 

 cipitated with 100 cc. of a 10 per cent solution of HgS04 in 5 

 per cent HjSOj. After standing over night the bulky precipitate 

 was filtered on a Buchner funnel, and washed with water. It 

 was then suspended in water, made sUghtly alkaUne with Ba 

 (0H)2 and decomposed with US, allowing the reaction to take 

 place for some time, with occasional warming. The precipi- 



