VIII47-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



ment and in the final test, use 3 units of amboceptor (hemolysin). Example: If, in the 

 above titration,' 0.25 ml. of a 1-1600 dilution of the amboceptor produced complete 

 hemolysis of 0.25 ml. of the 2.5% suspension of sheep cells, 3 units of amboceptor would 

 be contained in 0.25 ml. of a 1-533 dilution of the stock amboceptor hemolysin serum, 

 or a 1-265 dilution of amboceptor serum which has been put up with an equal part of 

 glycerin. 



Titration of Complement. Since the activity of complement in the serum of different 

 guinea pigs varies, and as the activity of any sample of complement changes on stand- 

 ing, this reagent must be titrated at least once daily. The activity of the sample to 

 be used is, therefore, titrated in terms of the arbitrarily established unit of hemolytic 

 amboceptor. In general, there is a reciprocal relationship between complement and 

 amboceptor. Within certain limits, hemolysis of a given amount of red corpuscles 

 can be produced by mixtures containing more of complement and less of amboceptor, 

 and vice versa. The purpose of the following titration is to determine by dilution the 

 smallest amount of complement which will cause complete hemolysis of 0.25 ml. of 

 2.5% sheep red cells in the presence of 3 units of amboceptor. After having obtained 

 and mixed the serum from at least 3 guinea pigs, dilute the complement 1-10 and 

 proceed as in Table 4. 



TABLE 4 



TITRATION OF COMPLEMENT WITH RESULTS IN A TYPICAL INSTANCE 



Saline is added to the tubes in this series to bring the volume of fluid in each tube 

 up to 1.25 ml., the amount of fluid used in the final test. Tube 9 is the control for the 

 hemolytic activity of the complement alone; tube 10 serves a similar purpose as an 

 amboceptor control, and tube 11 is a control for the isotonicity of the saline solution. 



At the end of the 15 min. period of incubation, note the last tube showing complete 

 hemolysis. This gives the smallest amount of the 1-10 dilution of complement which 

 will cause the hemolysis of 0.25 ml. of a 2.5% suspension of sheep cells in the presence 

 of 3 units of amboceptor. In the final test, use 1.5 times as much complement as in 

 this tube. Example: If, as illustrated in the table, the smallest amount of complement 

 causing hemolysis were 0.1 ml. of the 1-10 dilution, use 0.15 ml. of a 1-10 dilution of 

 complement in the final test. With diff'erent specimens of complement, it may be 

 necessary to use a different series of amounts to arrive at the exact titer of the comple- 

 ment. This method of titration is devised to permit the use of minimal amounts of 

 complement in the final test. 



Titration of the Antigen. After the bacterial antigen has been prepared by emulsify- 

 ing the culture in saline, it is necessary to find out by titration three of its properties. 

 These are: (a) the ability of the antigen alone to inhibit the action of comi)lement, 

 called the anticomplementary action of the antigen, (b) the hemolytic properties of 

 the antigen, and (c) the capacity of the antigen to fix complement in the presence of 

 its specific antiserum, called the binding ])ower of the antigen. These properties can 

 be determined by the procedure outlined in Table 5. 



Interpretation of Results. It will probably be found that most bacterial suspensions 

 are anticomplementary, and some are slightly hemolytic. In the first series of tubes 



