384 SPECIFIC MICRO-ORGANISMS 



S- The antigen is an alcoholic extract of the heart muscle of 

 beef. The clean, finely chopped heart muscle, 10 grams, is ex- 

 tracted in 100 c.c. of absolute ethyl alcohol at 37° C, with fre- 

 quent shaking for two weeks. It is filtered through paper. The 

 filtrate is then refrigerated a day and again filtered. This clear 

 filtrate is the plain alcoholic antigen and it is sealed up in small 

 ampoules or stored in a tightly stoppered bottle. The strength of 

 antigen to be used must be ascertained by careful titration. 

 A dilution of i c.c. of the antigen in 9 c.c. of salt solution is first 

 prepared. Then various quantities, o.i c.c, 0.2 c.c, 0.3 c.c, 

 0.4 c.c. and 0.5 c.c. of this suspension are placed in separate tubes. 

 To each tube is added 2 units of complement and sufficient salt 

 solution to bring the total volume to 0.6 c.c. The tubes are placed 

 in the ice-box overnight. Then one unit of corpuscles (0.2 c.c) 

 and two units of hemolytic amboceptor (0.2 c.c.) are added and the 

 tubes are incubated an hour at 37° C. Of those tubes in which 

 hemolysis is not complete, the one containing the least antigen 

 marks the concentration at which the antigen is distinctly anti- 

 complementary. The second test of the antigen is now under- 

 taken. Various amounts of a i to 100 dilution, o.oi c.c, 0.03 c.c, 

 0.05 c.c, 0.1 c.c and 0.2 c.c, are measured into tubes. To each 

 tube is then added 2 units of complement, 0.02 c.c. of -serum from 

 an active untreated case of syphilis and sufficient salt solution to 

 make a total volume of 0.6 c.c. The tubes are left overnight in 

 the ice-box. Then i unit of corpuscles (0.2 c.c.) and 2 units of 

 hem.olytic amboceptor (0.2 c.c) are added and the tubes are 

 iiicubated one hour at 37° C. Of the tubes showing no hemolysis 

 (complete fixation), that one which contains the least antigen 

 marks the lowest effective concentration of the antigen. This 

 amount of antigen should be very much less than the anti-corii- 

 plementary amount ascertained in the first test. Ordinarily 

 it is about }ioo of this amount. The unit of antigen to be em- 

 ployed should be chosen so that it is several times greater than 

 the least effective quantity but still not more than one-fifth to 

 one-fourth the least anti-complementary amount. Having chosen 



