THE STREPTOCOCCI 420 



from the streptolysin, and the antibody against it did not neutralize 

 streptoleucocidin. 



Nakayama used the following method for the determination of leucocidin, 

 a method which is given because it can be applied to tests for similar 

 substances from many organisms. His procedure was as follows : Leucocytes 

 were obtained by injecting- aleuronat as usual. When rabbits were used the 

 aleuronat was injected into the pleura. After twelve hours, the animals 

 were bled, and the exudate withdrawn after opening the chest, and mixed 

 with equal amounts of 1.5 per cent sodium citrate solution to prevent coag- 

 gulation. 



A solution of methylene blue is made as follows: 1 c.c. methylene blue, 

 20 c.c. absolute alcohol, and 29 c.c. distilled water. 



Various leucocytic suspensions are made up with 0.9 per cent salt solution, 

 and the mixtures made up to 2 c.c. There are progressively fewer leucocytes 

 in the successive mixture. Two drops of methylene blue solution are then 

 added to each tube and the mixture covered with a layer of liquid paraffin. 

 The tube is then put in the incubator at 37 for two hours. If reduction 

 occurs, the solution becomes colorless ; if no reduction occurs, the color remains 

 green. This would make the minimum quantity of leucocytes which cause 

 a reduction of the methylene blue. Then, to test the leucocidin different 

 quantities of the leucocydal fluid are added to twice the minimum quantity 

 of leucocytic suspension which has caused reduction in the above test. These 

 mixtures are then incubated for one and one-half hours at 37, and at the 

 end of this period, two drops of methylene blue are added and the tubes 

 again covered with liquid paraffin. Again they are incubated for two hours 

 and readings are made. If the tube contains a green color, it indicates that 

 something has prevented reduction. 



Antibodes and Immunization. For reasons not wholly under- 

 stood at present, recovery from streptococcus infection does not to 

 any marked degree produce immunity against these bacteria. Active 

 immunity may, however, be produced in rabbits, goats, horses, and 

 other domestic animals by treatment with gradually increasing doses 

 of streptococcus cultures. 92 



In carrying out such immunizations it is necessary to use for the 

 first injection attenuated or dead bacteria. Attenuation may be 

 accomplished by moderate heating or by the addition of chemicals 

 (terchloride of iodin). Neufeld 93 advises, for the first injection in 

 immunizing rabbits, the use of ascitic-broth cultures killed by heat- 



92 Koch und Petruschky, Zeit. f . Hyg., xxiii, 1896. 

 Neufeld, Zeit, f. Hyg., xliv, 1903. 



