FIXATION OF COMPLEMENT 183 



to be tested in such a manner that separate, well-defined colonies 

 are obtained. After twenty-four hours' incubation at 37 C., 

 colonies when haemolytic are surrounded with a clear, well-defined 

 halo contrasting sharply with the dark opaque colour of the agar. 

 If blood-agar is not available, a substitute may be devised by smear- 

 ing some sterile human or rabbits' blood on a sterile agar plate. 

 (b) A young agar culture is emulsified in 4-5 c.c. of physiological 

 salt solution ; 0-1 c.c. of this suspension is mixed in a tiny test-tube 

 with O9 c.c. of sterile salt solution and one drop of a sterile suspen- 

 sion of well- washed rabbit or other corpuscles. After twelve to 

 twenty -four hours haemolysis will be apparent if the organism forms 

 haemolysins. 



2. Fixation or absorption test. 1 A haemolytic serum may be used 

 as a delicate reagent for complement, and may thus serve as a 

 test for an organism or an immune serum. As an example take the 

 case of a supposed cholera vibrio. If an immune serum (previously 

 heated to 56 C. so as to destroy complement) haemolytic for the 

 corpuscles of an animal, or bacteriolytic for a given micro-organism, 

 e.g. cholera vibrio be mixed with the red corpuscles of the same 

 animal, or with the cholera vibrio, the corpuscles or the vibrios 

 respectively absorb the corresponding amboceptor or immune body. 



Bordet showed that if corpuscles or microbes that have absorbed 

 the corresponding amboceptor be added to fresh non-heated comple- 

 ment (e.g. fresh guinea-pig serum), the corpuscles or the microbes 

 absorb the complement, so that none remains free in the liquid. 



But if fresh guinea-pigs' serum be added to cholera vibrios which 

 have not absorbed any cholera amboceptor, the complement will 

 not be absorbed and remains free in the liquid. The proof of this 

 is that if " sensitised " corpuscles (i.e. corpuscles which have taken 

 up haemolytic amboceptor) be added to such a mixture, the globules 

 are quickly haemolysed. If, on the other hand, vibrios which have 

 already taken up the cholera amboceptor be added to the same 

 quantity of fresh serum, the microbe-amboceptor complex absorbs 

 the complement ; and, provided the amount of fresh serum is not 

 too great, the complement is absorbed so completely that " sensi- 

 tised " corpuscles when added to the mixture are not dissolved. 

 If vibrios other than cholera be added to cholera serum, the ambo- 

 ceptor is not fixed, the complement added remains free, and the 

 sensitised corpuscles are dissolved. These facts constitute the 

 " Bordet-Gengou phenomenon." The mixture of an inactivated 

 haemolytic serum (i.e. heated to 56 C.) with the homologous 

 corpuscles (i.e. those with which the haemolytic serum was prepared) 



1 Often termed " deviation of complement " test. 



