GENERAL TECHNIC 413 



centrifuge tubes and allowed to coagulate; it is then broken up with a 

 glass rod, centrifuged, and the serum secured at once; such complement, 

 however, is likely to be unduly hypersensitive to the anticomplement 

 action of organic extract and of mixtures of extract with normal se- 

 rums. As a general rule, therefore, it is good practice to bleed the animal 

 late in the afternoon preceding the day on which the experiment is to be 

 made, or at least some hours before the regular work of the day begins. The 

 serum should be clear and contain no corpuscles. ) 



Titration of Complement. In the original Wassermann reaction 

 fresh guinea-pig serum is used in a constant dose of 0.1 c.c. I have used 

 for several years just half the amount of corpuscles and complement 

 directed in the original technic, and find that the reactions are somewhat 

 sharper and clearer, besides being more economic. Using the corpuscles 

 and amboceptor as constants, the amount of complement necessary to 

 produce hemolysis may be determined after the manner described in 

 a previous chapter. To a series of test-tubes add increasing amounts of 

 complement serum diluted 1:20 as follows: 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 

 0.8, 0.9, 1, and 1.2 c.c. Add 1 c.c. of a 2.5 per cent, suspension of sheep's 

 cells and an amount of hemolytic amboceptor equal to two units. To 

 this add sufficient salt solution to bring the total volume of each tube up 

 to about 3 c.c.; shake gently and incubate for one hour at 37 C. That 

 tube showing just complete hemolysis contains one unit of complement 

 (Fig. 107). 



And now we come to a very important question, namely, the amount 

 of complement that is to be used in conducting complement-fixation tests. 

 Many present-day observers use exactly one unit of complement and 

 one unit of amboceptor. This is permissible, providing the complement 

 is titrated in the presence of a constant dose of antigen and a constant 

 dose of serum, in order that due allowance for the anticomplementary 

 action of these may be made in the titration. Under these circum- 

 stances, however, it is necessary to titrate each patient's serum with the 

 complement, because one serum or even the pooled serums of different 

 persons should not be taken as a standard in the titration, for two impor- 

 tant reasons: (1) The patient's serum which we are about to test may 

 be more anticomplementary than the serum used in the complement 

 titration, and hence when used in the main test, with exactly one unit 

 of complement, mild degrees of inhibition of hemolysis will be secured 

 that may be interpreted as slightly positive reactions; or (2) the serum 

 used in the complement titration may contain more or less natural 

 hemolytic amboceptor than the patient's serum, and this factor exerts 



