QUANTITATIVE ESTIMATION OF BACTERIOTROPINS 201 



potency, that of determining the bacteriotropic or opsonic index of the 

 serum is in most general use. 



NeufekTs technic is that generally employed, and is similar to the 

 serum dilution method employed by Simon. It varies from the 

 technic of Wright in several particulars: 



1. The immune serum is free from complement (thermolabile op- 

 sonin) . 



2. The actual number of bacteria .within the leukocytes are not 

 counted. Various dilutions of serum are used, and the highest dilution 

 in which the bacteria are ingested in great numbers is compared with a 

 normal serum in similar dilution as a control. The highest dilution that 

 still favors phagocytosis is then taken as the bacteriotropic liter of the serum. 



Serum. The serum is inactivated by heating it to 55 C. for one- 

 half hour. In old or carbolized serums this may be omitted, as they 

 are usually free from complement. Tuberculous serums also should 

 not be heated, as their bacteriotropins are very susceptible to heat. 



Normal serum from an animal of the same species as was used in the 

 preparation of the immune serum should be used as the normal control. 



An exactly parallel series of dilutions with normal salt solution are 

 made of the immune serum and pooled normal serums in a series of 

 small test-tubes. At least 0.5 c.c. of each dilution should be available 

 for the test; the following dilutions may be used: 1:10, 1:20, 1:50, 

 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000, 1:2000, etc. After working 

 for some time with normal serums one soon learns the dilution in which 

 the normal bacteriotropins are attenuated. It may not be necessary, 

 therefore, to use the whole series of dilutions with the normal serum. 



Leukocytes. Leukocytes may be obtained in several different ways : 

 (1) By injecting a guinea-pig intraperitoneally sixteen to twenty-four 

 hours previously with from 5 to 10 c.c. of sterile aleuronat solution (for 

 method of preparation see p. 339). Pipet the peritoneal exudate into 

 about 20 c.c. of sterile 1 per cent, sodium citrate in normal salt solution 

 in centrifuge tubes. Centrifugalize, and wash the leukocytes again 

 three times with sterile normal salt solution. The sodium citrate solu- 

 tion prevents the coagulation and formation of clumps of leukocytes. 



2. Instead of aleuronat, a sterile saturated solution of peptone may 

 be injected. 



3. If rabbit's leukocytes are preferred, 10 c.c. of aleuronat should be 

 injected into each pleural sac or 20 c.c. intraperitoneally. For mice, 

 an injection of 1 c.c. of aleuronat intraperitoneally is sufficient; human 

 leukocytes may be obtained after the method of Wright. 



