310 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



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 

 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. 

 So long as this was supposedly derived from disintegrating leuko- 

 cytes, it was recommended to obtain the blood some hours before 

 use. We then killed the guinea-pig the evening before, by cutting 

 the vessles of the neck, after anesthetizing the animal with ether. 

 The blood was received in Petri dishes and kept overnight on ice. 

 The following morning the serum was then pipetted off and ready 

 for use. Subsequent studies have shown, however, that a satis- 

 factory complement may be obtained from perfectly fresh blood, 

 and that it is not necessary to wait for its supposed liberation from 



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

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

 dealers will cause hemolysis in themselves. 



