176 
Dourine 
In either case a necessary preliminary is the titration of complement 
{vide, p. 166). This established, sufficient complement dilution is made 
up—0-5 c.c. of the dilution to contain the smallest amount indicated 
by titration—to do for the titration of antigen and for as many serum 
tests and controls as are to be made. It is advisable to make up an 
excess of complement rather than have a deficit, so as to use one stock 
uniform dilution throughout and avoid having to make up fresh stocks 
during the testing. 
The trypanosome antigen is then titrated against a known positive 
dourine serum and a known negative serum {vide, p. 167). 
First method of procedure—-for one or several tests. 
Four tubes and one pipette of 1-0 c.c. capacity, graduated 1-100, are 
needed for each serum to be tested. TO c.c. salt solution is measured 
into each tube. In each set of four tubes 0-2, 0-15, 0-1 and 0-2 c.c. of 
the inactivated test serum is added. Antigen in the amount already 
decided by titration is now added to the first three tubes in each set, 
omitting it from the fourth tube which serves as a serum control. 
Complement, 0-5 c.c. of the dilution required, is then added to all tubes. 
Sets of positive and negative sera are included with the above, and, in 
addition, controls for the various reagents. For the reagent controls 
five tubes are needed: (1) Antigen control, omitting the test serum, 
(2) haemolytic control, omitting serum and antigen, (3) haemolytic 
serum control, omitting test serum, antigen and complement, (4) com¬ 
plement control, omitting test serum, haemolytic serum and antigen, 
(5) red cells control, containing only red cells and salt solution. The 
controls are made up to a uniform volume of 2-5 c.c. by adding salt 
solution as required. 
When the test serum, antigen and complement have been mixed 
together, the tubes are incubated at 38-39° C. for 70 minutes. 
Equal quantities of the haemolytic serum dilution and the red cell 
suspension (4 per cent.) are mixed together and TO c.c. of the mixture 
added to every tube excepting the last two controls, Nos. 4 and 5, to 
which 0-5 c.c. red cells only are added. 
The tubes are again shaken and incubated for another two hours 
when the reactions may be read, a second reading being made the 
following morning, about twelve hours later, the racks being left at 
a cool room temperature meanwhile. 
The above procedure is indicated in the following table: 
