158 PRACTICAL METHODS IN IMMUNITY 



from which it is derived indicating preparation of the food that is, 

 the opsonin so alters or sensitizes the bacteria that they can be engulfed 

 or phagocytized by the polymorphonuclear leukocytes (the microphages 

 of Metchnikoff). About the same time Neufeld and Rimpau noted 

 the presence of a substance in immune sera which so acted on bacteria 

 as to prepare them for phagocytosis. Their designation" bacterio- 

 tropic substance" is practically synonymous with opsonin. 



In 1902 Leishman introduced the method of determining the "phagocytic index." 

 By taking one part of blood and one part of an emulsion of the bacteria in question 

 and keeping the mixture in a moist chamber at body temperature for a standard 

 time, as 15 to 30 minutes, and then spreading the blood-bacteria mixture and 

 staining the film with Leishman or Wright's stain he counted the number of bacteria 

 in a certain number of polymorphonuclears, and by dividing obtained the average 

 number per leukocyte of bacteria phagocytized. 



The Wright technic for determining the phagocytic average, and 

 from this the opsonic index, is as follows: 



Blood is taken from the patient and at the same time from a normal individual, 

 or preferably the blood of several normal individuals is pooled. This blood is best 

 collected in a Wright's tube, although it may be received in a small test-tube. After 

 coagulation and separation of the serum, the serum is ready for use. 



The next step is to prepare the leukocyte emulsion. For this we fill a centri- 

 fuge tube with normal salt solution, to which has been added i% sodium citrate 

 the latter to prevent coagulation. Then having pricked a finger congested by a 

 constricting rubber band, from 15 to 20 drops of blood are added to the citrated 

 salt solution, and the mixture thoroughly shaken. After centrifugalization for about 

 5 minutes the red corpuscles will be thrown to the bottom of the tube with the leu- 

 kocytes forming a superimposed layer. In order to free the leukocytes entirely from 

 serum admixture, the supernatant citrated salt solution is pipetted off, and a fresh 

 tubeful of salt solution is added to the blood-cell sediment. Again shaking, we then 

 centrifuge, obtaining for a second time a sediment of blood cells with the leukocytes 

 in the superimposed layer. In some laboratories the washing in salt solution is 

 again repeated, but for all practical purposes two washings as described above suffice. 



The superimposed layer of white cells may now be pipetted off from the heavier 

 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 emulsion, and the serum from the normal in- 

 dividual 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 

 1/2 to i c.c., and giving a faint turbidity. To thoroughly distribute and especially 



