REACTIONS DEPENDING UPON COMPLEMENT FIXATION 275 



that is added. Finally, the corpuscles are suspended in such an 

 amount of saline, that the volume injected equals that of the full blood 

 which was originally used. From nine to eleven days later, according 

 to the amboceptor content, w r hich can be readily ascertained by a 

 preliminary test of a few drops of blood, the animal is bled to death, 

 the blood being collected under aseptic precautions. To this end 

 it is convenient to use a test-tube which has been drawn out into a 

 capillary near its closed end, at an angle of about 115 degrees. This 

 is sealed, the open end closed with cotton, and the whole sterilized. 

 After the animal has been anesthetized, the neck is shaved, scrubbed 

 with soap and alcohol, and the carotid dissected out through a median 

 incision. The tip of the capillary is broken off and the tube, moistened 

 with sterile saline, introduced into the vessel, when the blood will 

 rise into the collecting tube. The capillary is quickly sealed in a 

 flame and the tube then placed on ice for the serum to separate out. 

 Subsequently, the serum is pipetted off with a sterile pipette, heated 

 for thirty minutes at 56 C., treated with carbolic acid to the extent 

 of 0.5 per cent., and may then be kept in a dark colored bottle, 

 well corked, on ice. Instead of doing this I find it more convenient 

 to fill small glass beads with about 0.5 c.c. of the serum each, to seal 

 these, and to keep them in an ice-box. The addition of carbolic 

 acid is then not necessary. 



The titer of the amboceptor should be at least such that 0.5 c.c. 

 of a 1 to 2000 dilution (in 0.9 per cent, saline) will completely 

 hemolyze 0.5 c.c. of a 5 per cent, emulsion of washed sheep corpus- 

 cles (see below), in the presence of 0.5 c.c. of a 1 in 10 dilution of 

 guinea-pig complement (see below), within thirty minutes at 37 C. 

 With the two injections of 30 c.c. of sheep's blood, each, one may 

 at times obtain a serum which will still hemolyze this quantity 

 of corpuscles in a dilution of 1 to 6000. At other times better results 

 are obtained by giving the rabbit four or five injections of 5, 10, 15, 

 and 20 c.c. of washed corpuscles, in succession, five days apart, 

 the animal being killed when the desired titer has been reached. 



Using one of the little beads just mentioned, I make up a 1 to 100 

 stock dilution which, when kept on ice, will usually retain its titer 

 for many weeks, and is used to make up the higher dilutions on the 

 days when these are wanted. It is best, however, to test it against 

 the complement anew at least once a w r eek, as the activity of the 

 complement varies considerably in different guinea-pigs. In the 



