REACTIONS DEPENDING UPON COMPLEMENT FIXATION 307 



pared in absolute methyl alcohol, which is conveniently put up in 

 glass beads or ampoules, in quantities of about 1 c.c. each. 



(6) Cholesterinized Alcoholic Extracts. Since cholesterinized 

 extracts of syphilitic fetal liver are the most sensitive of all, such 

 material would naturally be the ideal antigen. But unfortunately 

 syphilitic livers are relativly scarce. Under such conditions beef 

 heart or guinea-pig heart will be found to represent a satisfactory 

 material to start with. A few grams of this, freed from fat and 

 connective tissue as far as possible, are finely hashed and extracted 

 with absolute alcohol in the proportion of 10 c.c. for every gram. 

 To this end it is advised to shake the mixture for several hours and 

 then to heat it for an hour at 60 C. Simply standing at room tem- 

 perature for a week or ten days with an occasional shaking will, 

 however, answer the same purpose. The supernatant fluid is then 

 poured off, filtered, and treated with a 1 per cent, solution of choles- 

 terin in absolute ethyl alcohol, in the proportion of five parts of the 

 alcoholic extract to four parts of the cholesterin solution. The 

 resultant product may be kept in a dark bottle at room temperature. 



Prepared according to either one of these methods the antigen 

 keeps for many months without losing strength, but should be 

 tested once a month as a matter of routine and discarded if any 

 loss in strength is discovered. To this end emulsions of varying 

 dilution are prepared with 0.9 per cent, saline, treated with con- 

 stant amounts of complement, incubated for thirty minutes in a 

 water-bath at 37 to 40 C., and then combined with the hemo- 

 lytic system that has been chosen, to ascertain whether complement 

 fixation has taken place or not. The general plan to be followed 

 is shown in the accompanying table: 



