320 J. HOWARD MUELLER 



ments with the pneumococcus and streptococcus have shown that 

 "protein-free milk," which does contain the water soluble vita- 

 mine, is almost without activity when substituted for meat in- 

 fusion in media. To economize space, protocols of these ex- 

 periments are omitted. 



It is not impossible that physiological extracts other than meat 

 infusion might supply the accessory factors in a form more free 

 from other nitrogenous compounds such as amino-acids, than 

 the latter, and in a few experiments it has been possible to show 

 that some other preparations, notably one from blood, and one 

 from spinach leaves, gave growth when mixed with peptone and 

 only scant growth in its absence. The blood was diluted with 

 water, acidified, boiled and filtered. The spinach leaves were 

 dried, ground fine and extracted with water. In the case of the 

 latter, initial extraction of the dried powder with ether did not 

 remove the accessory substances, and boiUng with repeated 

 changes of alcohol for several hours extracted only a small part. 

 It would perhaps be possible to develop a technic along either 

 line for the preparation of a solution of the accessory factors 

 sufficiently free from protein nitrogen to investigate the nature 

 of the requirements of the test organisms by the addition of pure 

 amino acids, but is has seemed more satisfactory first to exhaust 

 as far as possible the more obvious methods for a separation of 

 the meat infusion. 



Attempts to separate the growth accessory factors from the amino 

 acids of meat infusion 



1. Repeated extraction of meat. While carrjdng out some ex- 

 periments along another fine, it was observed that the test or- 

 ganisms grew as well on a trypsin digest of the insoluble meat 

 residue remaining after the preparation of meat infusion which 

 had first been thoroughly boiled out in three changes of water, 

 without the addition of any meat infusion, as upon the usual 

 peptone broth. This suggested the possibiUty that the growth 

 accessory material might be extracted from the coagulated 

 protein with some difficulty, and might be partially separated 

 in this way from amino acids, etc. Chopped beef was, therefore, 



