VACCINES 189 



red cells (of course, containing a large admixture of red cells) to be used as a leuko- 

 cyte cream or by slanting the centrifuge tube we can pipette off the proportion 

 of the leukocyte mixture needed from the bottom, sides or top of the slanted layer 

 of blood cells. 



Having prepared our leukocyte j;mulsion. and the serum from the normal indi- 

 vidual as well as that from the patient, it only remains to prepare our bacterial 

 emulsion. For bacteria in general, with the exception of tubercle bacilli, we simply 

 take up a small loopful of a young agar culture (eighteen hours or less), and emulsify 

 it uniformly with salt solution, added by degrees until the suspension amounts to 

 Yz to i c.c., and giving a faint turbidity. To thoroughly distribute and especially 

 to break up clumps repeated suction and ejection with a capillary pipette provided 

 with a rubber nipple is satisfactory. 



The presence of clumps in a bacterial emulsion invalidates the estimation of 

 phagocytosis, for the reason that a leukocyte will take up a clump of twenty or more 

 bacilli as readily as one separate organism. 



Having at hand (i) the suspension of leukocytes, (2) the bacterial emulsion, 

 and (3) the sera of the patient and the normal individual, we are ready to proceed 

 with the test. 



Using a capillary bulb pipette with a pencil mark to indicate i volume we draw 

 up to the mark (i) the leukocyte cream. Then wiping off the tip of the pipette we 

 draw up this volume of leukocyte emulsion about % inch to make an air break 

 between this and (2) i volume of the bacillary emulsion. Again making an air 

 space we draw up (3) the serum of the normal individual. This gives three columns 

 in the capillary tube with intervening breaks of air. We next eject the three con- 

 stituents into a watch-glass and thoroughly mix them by alternate suction and ejec- 

 tion with the tube and nipple. When mixed we draw the mixture up into the same 

 capillary tube, seal off the capillary end in the flame and put in an incubator for 

 exactly fifteen minutes. 



We next repeat the process identically except that the patient's serum is used in- 

 stead of that of the normal individual. 



These tubes having been kept at the same temperature for the same length of 

 time are then taken out, the contents blown into a watch-glass, mixed thoroughly 

 a second time, and then a smear is made a drop of the mixture being deposited 

 on a very clean slide and the smear made by a second narrower slide (by cutting off 

 the corner of the slide) which is drawn along in a zigzag way. The smears are then 

 stained (Leishman's or Wright's blood stain or Ziehl-Neelson's for tubercle bacilli) 

 and the number of the bacteria in from 50 to 100 leukocytes counted. This 

 number divided by the number of cells gives the phagocytic average. 



The phagocytic average of the patient's tube divided by that of the normal in- 

 dividual's tube gives the opsonic index. Thus, in counting 100 cells we find 500 

 phagocytized cocci in the patient's tube, giving an average of 5, and in the normal 

 individual's blood we get 1000, an average of 10. Then the opsonic index would be 

 5 -f- 10, or 0.5. 



PREPARATION or VACCINES 



It has been found satisfactory to make use of stock vaccines in- 

 gonorrhceal and tuberculous affections. In treatment of tuberculosis 



