REACTIONS DEPENDING UPON COMPLEMENT FIXATION 273 



grams of beef heart, liver or kidney are passed through a meat- 

 hashing machine and extracted with ten times the amount of abso- 

 lute alcohol by standing for several days at incubator temperature. 

 The resultant mixture is filtered through ordinary filter paper, the 

 filtrate evaporated to dryness with the aid of an electric fan, the 

 residue taken up with as little ether as possible, and the ethereal 

 solution treated with five times its volume of acetone. A precipitate 

 forms, which is allowed to settle to the bottom, when the supernatant 

 fluid is poured off. From the remaining brown, sticky material 

 a saturated solution is prepared in absolute methyl alcohol, which 

 is conveniently put up in glass beads or ampoules in quantities of 

 about 1 c.c. each. 



Prepared in this manner the antigen keeps for many months 

 without losing in strength, but should be tested from time to time 

 nevertheless. To this end emulsions of varying strength are pre- 

 pared with 0.9 per cent, saline, treated with constant amounts of 

 complement, incubated for thirty minutes in a water-bath at 37 to 

 40 C., and then combined with the hemolytic 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 accompany- 

 ing table: 



Hemolytic am- 



Washed sheep boceptor (2^ 

 corpuscles times the titer 

 (5% emulsion). strength). 



Result. 



c.c. 

 0.5 



0.5 



3:3 0-5 



0.5 



0.5 



0.5 



c.c. 

 0.5 



0.5 



0.5 



0.5 



0.5 



0.5 



As the antigen in itself is capable of absorbing a certain amount of 

 complement it will be found that with the stronger emulsions either 

 no hemolysis at all occurs, or partial hemolysis only takes place. 

 For the actual experiment two-thirds of that strength is chosen which 

 first gives complete hemolysis. In the above example it will be 

 18 



