

FIXATION OF THE COMPLEMENT 



235 



From an examination of the table it follows that the amount of haemolytic 

 serum added to tube No. 3 will, in this particular instance, be the most 

 suitable for subsequent experiments : in this tube haemolysis is complete 

 in half an hour. Tubes Nos. 1 and 2 contain too much serum, and in Nos. 4 

 and 5 haemolysis is not complete and they therefore contain too little serum. 



Further, examination of the control tubes shows : 



1. That the heated haemolytic serum only haemolyzes when complement 

 is added. 



2. That complement alone does not haemolyze the red cells. 



In carrying out the reaction of complement fixation then, the quantity of 

 Ol c.c. of this particular hsemolytic serum per 1 c.c. of the dilution of red 

 cells will be used. 



Complement. Normal guinea-pig serum collected aseptically will be used 

 as complement. The amount of complement to be added is of the greatest 

 importance : if there be an excess of complement the whole of it will not be 

 absorbed by the antigen-antibody mixture and the excess remaining in 

 solution will haemolyze the red cell-serum mixture and give an erroneous 

 result. The smallest quantity of guinea-pig serum which will haemolyze 

 1 c.c. of the sensitized red cell emulsion must therefore be determined. 



If fresh guinea-pig serum be used it will be found to be very rich in com- 

 plement but the amount rapidly diminishes in the first few hours. It is 

 preferable therefore to use serum collected 8-10 days before [and stored in an 

 ice chest] ; it is not so active but its titre remains constant for several days 

 (Nicolle and Pozerski). The following table illustrates the method of titration. 

 Incubate the tubes for half an hour at 37 C. and then note the results. 



Tubes Nos. 1, 2 and 3 alone contain enough complement to produce com- 

 plete haemolysis. The quantity in tube 3 will be used because it is the 

 smallest quantity which produces complete haemolysis. 



Antigen. The organisms to be used as antigen should be prepared as 

 follows : Take a young agar culture (in the case of the cholera vibrio or 

 typhoid bacillus, for instance, a 24 or 48-hour culture) and emulsify one 

 loopful in 2 c.c. of sterile normal saline solution. In carrying out the experi- 

 ment small quantities only of this emulsion are used because large quantities 

 of albuminoid substances may produce, in the absence of a specific reaction, 

 a mechanical deviation of the complement and so give fallacious results. 



