222 ACTIVE IMMUNIZATION 



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 saline is carefully withdrawn with a capillary pipette.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 per cent, 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- 

 rhage is prevented. After twenty hours the pleural cavity will 

 contain an exudate rich in leukocytes, which is pipetted off, placed 

 in citrate-saline, and washed as described above. 



Ordinarily the leukocytes should not be kept longer than five or 

 six hours. 



Preparation of Bacterial Emulsion. As the W T right technique neces- 

 sitates working with uniform emulsions, i. e., with emulsions in 



