viii-rlS MANUAL OF METHODS FOR PURE CULTURE STUDY 



PROCEDURE FOR COMPLEMENT FIXATION TEST 



The amounts of reagents used in the final test for complement 

 fixation are those which have been found to be appropriate from 

 the preliminary titrations described above. Stated in the form of 

 a general protocol, the test should be set up as follows : 



Tube 1. Amount 1 of immune serum plus complement plus antigen. 



Tube 2. Amount 2 of immune serum plus complement plus antigen. 



Tube 3. Amount 3 of immune serum plus complement plus antigen. 



These tubes constitute the test for complement fixation. A careful 

 series of controls is necessary, as follows: 



Tube 4. (Anticomplementary serum control) : Double the largest 

 amount of antiserum plus complement. 



Tube 5. (Anticomplementary antigen control) : Twice the amount 

 of antigen used in test plus complement. 



Tube 6. (Hemolytic system control) : Complement alone. 



Tube 7. (Saline control) : Saline alone. 



Add sufiicient saline so that the total volume of fluid, when all in- 

 gredients are in the tubes, will be 1.25 ml. 



TABLE 6 



COMPLETE COMPLEMENT FIXATION TEST — WITH RESULTS IN A TYPICAL INSTANCE 



The optimum temperature of incubation of the mixtures for com- 

 plement fixation varies under different conditions. The test is in 

 some cases more sensitive when these mixtures are kept in the refrig- 

 erator at 5-10°C. for 4 hours. For most purposes incubation in a wa- 

 ter bath at 37°C. for 1 hour, as given in the protocols, is satisfactory. 



After this incubation, add to all tubes except tube 7, 3 units of 

 amboceptor contained in 0.25 ml. of the diluted amboceptor scrum 

 and to all tubes add 0.25 ml. of 2.5% sheep cells. Shake well, and 

 incubate them again, for 15-30 minutes, depending upon the rate 

 of hemolysis in the control tubes. 



At the end of the second period of incubation, note the results. 



The partial or complete absence of hemolysis in any of the first 

 3 tubes denotes fixation of complement, indicating union between the 

 antigen and antiserum. Hemolysis in these tubes indicates lack of 

 fixation or a negative reaction. 



