208 ACTIVE IMMUNIZATION 



this single serum may suffice for ordinary purposes. Women during 

 menstruation, hard workers, and individuals who are pale and below 

 weight, even if otherwise healthy, should not be taken as controls, 

 nor even included in a pool. Occasionally, apparently normal 

 individuals are also encountered, who habitually have a higher 

 opsonic content than normal, and such must, of course, also be 

 excluded. The process of digestion further tends to increase the 

 opsonic content of the blood, so that it is advisable to take the blood 

 of the patient and the pool approximately at the same hour of the 

 day. As with the patient's blood the control serum also should 

 not be more than twenty-four hours old. 



Preparation of Washed Corpuscles (Leukocytes) . The blood is most 

 conveniently collected from the ear and received in a tube containing 

 1.5 per cent, sodium citrate in 0.9 per cent, salt solution. The 

 amount will depend upon the number of specimens that are to be 

 prepared; 1 c.c. is sufficient for at least a dozen mounts. Small 

 test-tubes of 5 c.c. capacity are very convenient. Clots must be 

 avoided and the specimen promptly discarded if the slightest coagu- 

 lum forms. Wright lets the blood drop directly (from the finger) 

 into the citrate solution, while I use the small tube a (Plate III) 

 to make the transfer. To prevent clotting I use a little beaker 

 with citrate-saline, and between transfers always rinse the pipette in 

 this and keep some of the solution in the end, so that the blood 

 immediately comes in contact with this; a number of drops of 

 blood are allowed to enter by capillary attraction and are then blown 

 out into the little test-tube; after every addition the citrate tube is 

 closed with the finger and inverted so as to secure uniform dilution. 

 The corpuscles are then thrown down by centrifugation, the super- 

 natant fluid pipetted off and replaced with 0.9 per cent, saline, the 

 corpuscles brought into suspension and again thrown down, when the 

 saline is carefully withdrawn with a capillary pipette. Wright then 

 uses the superficial layer of corpuscles only, as this is especially rich 

 in leukocytes (the leukocytic cream). 



If large quantities of leukocytes are required, rabbits are injected 

 into the pleural cavity with 5 to 10 c.c. of an emulsion of aleuronat 

 mush in bouillon, or 0.9 percent, saline, the mixture being sterilized; 

 killed cultures (at 120 C.) of staphylococci (albus or aureus) may 

 be used for the same purpose. The needle for injection should be 

 somewhat dull, so that bloodvessels are not injured and hemor- 



