i8o 



TECHNIQUE OF THE COMPLEMENT FIXATION METHOD 



tube 1/2 c.cm<, the third tube nothing, the antigen tube (tube 19) 2 c.cm. Physiolog- 

 ical salt solution is added to make up i c.cm. in each tube; first tube nothing; second 

 tube 1/2 c.cm.; third tube i c.cm. of saline. 



The normal extract required for the tubes in the second rack is similarly estimated, 

 0.2 c.cm. is needed for each test, 5 (tests) Xo.2 = 1.0+0.4 fc> r the antigen control tubes 

 = 1.4 or 1.5 c.cm. in round numbers. For purposes of dilution i : 5, 6 c.cm. of salt 

 solution are added and i c.cm. ( = 0.2 of extract) placed into each of the tubes (20 to 

 26) and 2 c.cm. into the normal extract control test-tube (tube 27). In this series 

 also, salt solution is added to make up equal quantities of i c.cm. : first tube nothing, 

 second tube 1/2 c.cm., third tube i c.cm.; antigen tube (27), nothing; system (28), 

 complement (29) and blood (30) control tubes each i c.cm. 



The second ingredient of the test is next added, i.e., the respective serum. This is 

 not diluted but added directly; 0.2 c.cm. into the first tube of each test; o.i c.cm. into 

 the second; 0.2 c.cm. into the third tube; also 0.2 into the control series of tubes labelled 

 with the patient's names in the second rack. Salt solution is again added to make 

 up to the equal quantity of 2 c.cm. in each tube, thus: 0.8 c.cm. into first, 0.9 c.cm. into 

 the second, 0.8 c.cm. into the third tube, and 0.8 c.cm. into the control series; nothing 

 into the antigen tubes, i c.cm. into system, complement, and blood control tubes. 



The addition of complement follows next. Each tube, except the blood tube, 

 receives o.i c.cm. of complement. Thus the tubes are counted and if, for example, 

 nineteen tubes are present 19X0.1 c.cm. complement is taken, or in round numbers 

 2 c.cm. 



Complement is always diluted i : 10, or 2 c.cm. complement + 18 c.cm. saline, so 

 that each tube except the blood tube (30) receives i c.cm. of this diluted complement. 

 Tube (30) receives i c.cm. of saline instead. All tubes are then carefully shaken and 

 the racks placed in the incubator for one hour. 



During this time, the hemolysin and washed red blood cells are properly diluted. 

 The red blood cells are made up in a 5 per cent, suspension of which each tube will 

 receive i c.cm. Thus in the present test there are thirty tubes, requiring 30 c.cm. of 

 blood suspension; since i c.cm. of washed blood when diluted i : 20 will supply twenty 

 tubes, for 30 c.cm. about i 1/2 c.cm. of blood will be required, or 2 c.cm. will make 

 40 c.cm. of a 5 per cent, blood suspension. 



