276 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



actual experiment, viz., in the study of the patient's sera, from 2| 

 to 3 times the completely hemolyzing dose is used. 



3. The Washed Corpuscles. The necessary amount of sheep's 

 blood is readily procured from a slaughtering house. If this is not 

 available, a sheep may be kept near the laboratory and is bled from 

 the ear as occasion demands. In the hemolytic experiment, it is 

 not essential to work aseptically. After separation of the serum 

 the corpuscles are washed three times with saline, as mentioned 

 above. At last all the fluid is carefully pipetted off; from the 

 remaining corpuscles a 2.5 per cent, emulsion is prepared in saline, 

 which corresponds to a 5 per cent, emulsion of the native blood. 



We use the corpuscles only on the day on which they are procured 

 and on the one following. They should be kept in the ice-box while 

 not in use. If the supernatant fluid shows the least discoloration 

 they should be discarded. 1 



4. The Complement. Guinea-pig serum is used as complement. 

 As this is supposedly derived from disintegrating leukocytes, it is 

 recommended to obtain the blood some hours before use. We 

 usually kill the guinea-pig the evening before, by cutting the ves- 

 sels of the neck, after anesthetizing the animal with ether. The 

 blood is received in Petri dishes and is kept over night on ice. The 

 following morning the serum is pipetted off; if desired one can then 

 place the clotted blood in centrifuge tubes and obtain still more 

 serum by centrifugation. If it is not practical to kill the animal the 

 evening before, this may be done in the morning of the day on which 

 it is used; it is then placed on ice for two or three hours and the 

 serum obtained by centrifugalizing the clot. Before use the serum is 

 diluted 1 in 10. The unused portion of the concentrated serum may 

 be kept frozen, for one or two days, but before further use it must 

 be tested and adjusted to the hemolytic amboceptor as described. 

 Very often it will be found to be inert. In my laboratory, we have 

 set aside special days of the week for complement fixation work, 

 and we then make no attempt to preserve any of the complement. 



Where only a few specimens are to be examined at one time it 

 is not necessary to kill the animal. A few c.c. of blood can be 

 obtained by puncturing the heart with an antitoxin syringe, under 

 anesthesia. My own preference, however, is to kill the animal. 



1 For washing purposes, as well as for diluting the various reagents, it is 

 essential to use chemically pure sodium chloride. Some of the tablets furnished 

 by dealers will cause hemolysis in themselves. 



