DIAGNOSTIC REACTIONS 295 



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

 according to the amboceptor content, which can be readily ascer- 

 tained 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 capil- 

 lary 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 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. 



Not every animal can be brought to a satisfactory titer, however, 

 and during the winter months especially it is not unusual to find 

 but one animal of perhaps a dozen that will furnish a satisfactory 

 amboceptor. 



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 



