O MELICK 



acidity. The medium as finally standardized is prepared by 

 the following formula: 



Redistilled Water 1000. 



Sodium Chloride 5. 



Asparagin 3.4 



Di-Sodium Hydrogen Phosphate 2. 



Magnesium Sulphate .5 



Ammonium Lactate 6.3 



After dissolving, the reaction of the medium is made +0.2 

 acid by the addition of sodium carbonate, then autoclaved for 

 ten minutes at ten pounds pressure, tubed and again sterilized 

 as before. Any tubes showing the presence of a precipitate 

 are discarded. 



Employing this medium I made daily transfers for from one 

 to two weeks. At the end of this time, all (except a very few) 

 of the one hundred strains with which I worked grew suffi- 

 ciently in forty-eight to seventy-two hours to be used in the 

 preparation of an antigen. (The few cultures which grew 

 sparingly or not at all when first inoculated in this medium 

 later grew sufficiently by first inoculating them into tubes 

 which contained 1/20 part broth in addition to the protein free 

 medium. These cultures were transferred daily to tubes con- 

 taining decreasing amounts of the broth and finally after 

 several transfers the cultures grew well in the medium without 

 broth addition.) 



With the fact established that an extensive cultivation of the 

 organism could be accomplished in this protein-free medium, 

 a series of experiments was next instituted to determine 

 whether or not the antigenic properties of the bacteria differed 

 when grown in the protein-free and in the protein-containing 

 media. 



For this determination twelve rabbits were used, six of which 

 were injected individually with B. typhosus, B. paratyphosus 

 A, B. paratyphosus B, B. enteritidis, B. suipestifer, and B. coli 

 communis, grown on plain agar. The other six were also in- 

 jected with the same organisms which had been grown in the 

 protein-free medium. For injection, the organisms were killed 

 by heating at 60 C. for one hour, control tests being made as 

 to non-viability. An equal number of organisms from each 



