260 INFECTION AND IMMUNITY 



The corpuscles are concentrated by centrifugalization, the citrate solu- 

 tion is decanted, and corpuscles are washed with salt solution, as before, 

 to remove the serum. 



D. When large quantities of blood are desired, either from man or 

 from an animal, the blood may be received directly into a flask into 

 which a dozen or more glass beads or short pieces of glass tubing have 

 been placed. The flask is shaken for five or ten minutes, immediately 

 after the blood has been taken and, in this way, defibrination is accom- 

 plished. 



Since, for comparative tests, it is necessary to establish some stand- 

 ard concentration of red blood cells, it is customary in these tests to 

 employ a five per cent emulsion of corpuscles in salt solution. To 

 obtain this, one volume of sediment of washed red blood cells is mixed 

 with nineteen parts of 0.85 per cent salt solution. 1 Such an emulsion, if 

 kept sterile and in the refrigerator, will serve for hemolytic tests for 

 from one to three days. An emulsion should not be used if the super- 

 natant salt solution shows any transparent redness, as this indicates 

 hemolysis. 



If the substance in which hemolysins are to be determined is serum, 

 this should be inactivated by exposure to 56 C. in a water bath, and 

 to each test, complement may be added in the form of fresh guinea-pig 

 or rabbit's serum. No absolute rule for the quantity of complement 

 to be used in these tests can be given. In each case the particular 

 complement used should be titrated to determine the minimum quan- 

 tity which will produce hemolysis of 1 c.c. of the sensitized cell sus- 

 pension. 



In the actual test, mixtures are made of the corpuscle emulsion, the 

 inactivated immune serum, and complement in small test tubes and the 

 volumes of the various tubes made equal by the addition of definite 

 quantities of salt solution. The contents of the tubes are thoroughly 

 mixed and the tubes put in the incubator or in a water bath at 37.5 C. 

 If complete hemolysis occurs, the fluid in the tube will assume a deep 

 Burgundy red. If no hemolysis occurs, the fluid will remain uncolored 

 and the corpuscles will settle out. Incomplete hemolysis will be evi- 

 denced by a lighter tinge of red in the tube and by the settling out of a 

 varying quantity of blood corpuscles. 



1 The method here given was formerly much employed. It is now the general 

 practice, however, to use one volume of the actual sediment to nineteen volumes of 

 salt solution. 



