220 A MANUAL OF BACTERIOLOGY 



0-1 c.c. as our unit volume, and quantities in the test of 5 volumes 

 of antigen dilution ; 5 vohjmes of complement dilution ; 1 volume 

 of the various dilutions of anti-serum, or of patient's serum ; and 

 5 volumes of hsemolytic system ( = corpuscles + haemolytic 

 serum). 



In order to titrate the antigen a series of dilutions of it is pre- 

 pared, e.g. 1 in 5, 1 in 10, 1 in 20, 1 in 30, ... 1 in 50. Five volumes 

 of each dilution are taken in a series of tubes ; to each tube are 

 added a unit volume of normal inactivated serum and a unit 

 volume of complement dilution, containing 2-3 minimal hsemolytic 

 doses per unit volume as determined by a previous titration of 

 the complement serum. The mixtures are heated at 37 C. for 

 half an hour, and to each are then added 5 volumes of the hsemolytic 

 system. The mixtures are again heated at 37 C. for half an hour. 

 At the end of this period the tubes are examined and the highest 

 dilution of antigen giving no haemolysis is noted. A second 

 similar test may then be performed using dilutions of antigen 

 above and below this dilution, and the dilution of antigen yielding 

 complete inhibition of haemolysis more closely determined. Thus, 

 if in the first test antigen dilution 1 in 20 showed complete inhibi- 

 tion and dilution 1 in 30 showed a good deal of haemolysis, in the 

 second test antigen dilutions of 1 in 20, 1 in 22, 1 in 24, and 1 in 

 26 might be tried. Having found the weakest dilution of antigen 

 which just completely inhibits haemolysis, half this strength of 

 antigen should be used in the actual test. Complement and 

 haemolytic serum are titrated in the same manner as described 

 under the Wassermann test, and 35 minimal haemolytic doses 

 of the haemolytic serum are employed. Serum haemolytic for 

 sheep's corpuscles is generally employed, and the sheep's cor- 

 puscles used in the test must be well washed and a 4-5 per cent, 

 suspension employed. 



The haemolytic serum may be obtained by injecting rabbits with 

 a 10 per cent, suspension of well-washed sheep's red corpuscles. 

 The sheep's blood should be obtained as aseptically as possible 

 from the slaughter-house ; the blood, as it runs, is caught in a 

 sterile wide-mouth bottle containing a coil of fine wire with which 

 it is defibrinated by shaking. The fluid blood is then mixed with 

 sterile physiological salt solution (0-85 per cent.) and centri- 

 fuged, and the deposited corpuscles are again washed with salt 

 solution two or three times. Three doses of 1 c.c., 2 c.c., and 



