REACTIONS DEPENDING UPON COMPLEMENT FIXATION 309 



and pipetting off the washings, to stir up the corpuscles in the new 

 portion of saline that is added. Finally, the corpuscles are sus- 

 pended 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, which 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 anes- 

 thetized, 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. 



Even more satisfactory is the use of a large Keidel tube (holding 

 about 30 c.c.), which after anesthetizing the animal is thrust into 

 the heart and then fills itself automatically, after pinching off the 

 drawn-out end inside of the rubber tubing (see Fig. 19 and text 

 on p. 312). 



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

 heated for thirty minutes at 56 C., and may then be treated 

 with carbolic acid to the extent of 0.5 per cent. I find it more 

 convenient, however, to fill small sterile glass beads w r ith 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 hemo- 

 lyze 0.5 c.c. of a 5 per cent, emulsion of washed sheep corpuscles 

 (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 w r ill 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. 



