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, the thoroughly washed corpuscles from one volume of the 

 original blood are mixed with nineteen parts of 0.85 per cent salt solu- 

 tion.' 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 supernatant salt solution shows any transpa- 

 rent redness, as this indicates hemolysis. 



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

 this may be used either as such or it may 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. As 

 a starting-point, however, when 1 c.c. of a 5 per cent emulsion of red 

 corpuscles is used, it is best to use about 0.1 to 0.2 c.c. of fresh guinea- 

 pig serum as complement. 



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 as- 

 sume a deep Burgundy red. If no hemolysis occurs, the fluid will 

 remain uncolored and the corpuscles will settle out. Incomplete hemo- 

 lysis will be evidenced by a lighter tinge of red in the tube and by the 

 settling out of a varying quantity of blood corpuscles. 



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

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

 salt solution. 



