216 ACTIVE IMMUNIZATION 



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 saline is carefully withdrawn with a capillary pipette.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 Wright technique neces- 

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

 which the bacteria ,re evenly distributed, this step is really the crux 

 of the whole process. With certain organisms, such as the staphy- 

 lococci, the difficulty is not so great, but with others, notably the 

 tubercle bacillus, it is almost impossible to obtain uniform results. 



Staphylococci and streptococci may be grown on plain agar, while 

 gonococci, pneumococci, and meningococci are cultivated on blood 

 agar or hydrocele agar. Small tubes, like the one pictured at b (Plate 

 III) are charged with a little saline (0.85 to 1.2 per cent.). A bit 

 of the culture is removed with a platinum loop and gently rubbed 

 against the wall of the tube, at the surface of the liquid, until a 

 uniform turbidity results throughout the specimen. This is then 

 centrifugalized for a minute or two, so as to remove clumps as 

 far as possible, and to obtain the desired degree of density of the 

 bacterial emulsion. This point can only be learned by experience. 

 For convenience' sake, small glass capsules may be prepared con- 

 taining emulsions of barium sulphate of varying degrees of turbidity, 

 and corresponding to bacterial emulsions of standard strength. 

 With these the centrifugalized specimen may be compared before 

 use in the actual experiment. Wright advocates an emulsion of 

 cocci of such strength that with normal serum the average number 

 of organisms per leukocyte (see below) is about four or five. 



