4 MELICK 



The experimental work which is the basis of these studies 

 was performed in the differentiation of strains of the colon- 

 typhoid-intermediate group of bacteria by means of the spe- 

 cific antibody-antigen reaction as displayed in the fixation of 

 the complement of a hemolytic complex. 



The results to be presented in detail have to do with the pro- 

 duction of standard antigens, and fall under three general 

 headings: first, the elimination of admixed culture medium 

 proteins; second, the disintegration of the bacterial cells with 

 a minimal chemical modification and a maximal liberation of 

 their antigenic proteins; third, the conservation of antigens 

 without the addition of chemical preservatives. 



I. The elimination of admixed culture medium proteins. 

 Commonly, the bacteria used in the preparation of antigens 

 have been grown either in protein containing liquid media, 

 such as meat infusion broth, or on the surface of solid media 

 impregnated with protein constituents. For members of the 

 intestinal group of bacteria, beef infusion agar has been most 

 commonly used. Whole blood, blood-serum, ascites fluid, egg, 

 and other substances have been added to the nutrient agar 

 when the organisms required special conditions for maximum 

 growth. In each case, protein substances have been intro- 

 duced into the medium, including in most instances bacterial 

 proteins derived from the massive number of organisms which 

 develop during the commercial preparation of the so-called 

 peptones. 



The removal of bacteria from such a medium is accompanied 

 by a transfer of considerable quantities of the culture medium 

 constituents which cannot be entirely removed from the bac- 

 teria even by combined nitration and centrifugalization. The 

 use of such an antigen is untrustworthy. Its injection may well 

 stimulate the production of immune bodies to the admixed 

 culture medium proteins as well as to the proteins of the bac- 

 teria themselves. The resulting immune serum therefore, when 

 tested with similarly prepared antigens, may react not only 

 with the bacterial proteins but also with a variety of others 

 derived from the medium itself. 



