296 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



the complement anew at least once a week, as the activity of the 

 complement varies considerably in different guinea-pigs. In the 

 actual experiment, viz., in the study of the patient's serum, from two 

 and one-half to three 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 remain- 

 ing 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 vessels 

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

 is received in Petri dishes and is kept overnight on ice. The follow- 

 ing 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; 

 the blood 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 



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. 



