LYSINS 159 



example cited, 1.0 c.c. of the antigen was found to be anticomple- 

 mentary, consequently 0.25 to 0.3 c.c. would be the proper amount 

 of antigen to employ in the test. 



Complement. Fresh guinea-pig serum is the usual source of com- 

 plement for fixation reactions. The animal should be healthy and 

 not previously injected with protein of any nature. The serum of 

 pregnant pigs is not trustworthy. Blood may be obtained directly 

 from the heart of the living animal by aspiration through a hypo- 

 dermic needle, from a severed carotid artery, or, more expeditiously 

 by cutting the throat of the animal, avoiding the esophagus, and 

 collecting the blood in sterile Petri dishes. The freshly drawn blood 

 is allowed to stand for a few hours at a low temperature and the serum 

 is pipetted off. Complement must be kept cold (below 16 C.) and in 

 the dark. It must be used fresh, for it deteriorates rapidly. In a 

 frozen condition, however, it will remain active for two or three 

 weeks. Both the "activating" and combining properties of normal 

 fresh guinea-pig serum are sufficiently constant for the reaction of 

 complement fixation. 



Hemolytic System. (a) Hemolytic Serum (Hemolysiri). Hemolytic 

 serum is obtained from rabbits which have been injected with 2 c.c., 

 4 c.c., and finally 6 c.c. of a 50 per cent, solution of washed sheep 

 red blood cells 1 at intervals of two or three days. The injections 

 may be made intraperitoneally or intravenously, the latter being 

 preferable. Not less than nine days after the injection the animal 

 is bled to death from the carotid artery under anesthesia, the blood 

 being received in sterile test-tubes, which are placed in an inclined 

 position in the ice-box. The serum is removed, centrifugalized if not 

 wholly free from blood corpuscles, and placed in small amber bottles 

 with aseptic precautions. These are heated to 56 C. for half an hour 

 to effect inactivation (to destroy complement). 



(6) Red Blood Cells. Erythrocytes of the sheep are used. The 

 blood of a sheep is collected either in small sterile flasks containing 

 one volume of 0.85 per cent, salt solution and 0.5 per cent, sodium 

 citrate, or in sterile centrifuge tubes. If the former is used, nine 

 volumes of blood are allowed to flow into the flask and immediately 

 mixed intimately with the citrate solution, which prevents clotting. 

 This method is applicable if the blood cannot be centrifuged imme- 



1 Fresh red blood cells of the sheep are freed from serum by repeated washings with 

 physiological salt solution usually five washings "suffice. The corpuscles are then 

 suspended in a volume of salt solution twice that of the corpuscles themselves. 



