12 



been shown by Van de Velde,* and by Neisser and Wechsberg,** to be 

 the case with staphylococcus filtrates. The organism used in these experi- 

 ments was BiO4, which has been passed through 78 rabbits, and is very 

 virulent for rabbits and for guinea-pigs, and produces an active hemoly- 

 sin when grown in heated rabbit serum or in a mixture of equal parts 

 of rabbit serum and ascites fluid. The filtrates used were from iwenty- 

 four to thirty-six-hour cultures in a mixture of rabbit serum and ascites 

 fluid, which has been heated to 560. for one-half hour before inoculation. 



Neisser and Wechsberg have shown that living leucocytes reduce 

 methylene blue solution when the supply of oxygen is shut off, while 

 dead leucocytes no longer possess this property. In my experiments this 

 fact was made use of to determine whether or not the leucocytes had 

 been killed by the filtrate. 0.3 c. c. to 0.4 c. c. of leucocytic exudate from 

 a rabbit was added to 1.5 c. c. of ~the filtrate, incubated for two hours, 

 then two drops of methylene blue solution added and the contents of the 

 tube shaken up and covered with a layer of sterile olive oil. If there 

 was reduction in two hours the result was considered negative, but if 

 there was no reduction in that time this fact was taken as evidence that 

 the leucocytes were dead. Control tubes with a mixture of equal parts 

 of heated rabbit serum and ascites fluid were always made, and the 

 results recorded only when there was marked reduction in the control 

 in two hours. A large number of these experiments were carried out, 

 but it is not necessary to tabulate more than one of them here. 



Experiment. .3 c.- c. exudate + 1.5 c. c. 24-hour filtrate; no re- 

 duction in 2 hours, but a trace of reduction in 3 hours. 



.3 c. c. exudate -|- 1.5 c. c. 36-hour filtrate; no reduction in 3 hours. 



.3 c. c. exudate 4- 1.5 c. c ascites-serum ;* nearly complete reduction 

 in 2 hours. 



The leucocytic exudate was obtained from the pleura of a rabbit 

 which had been previously injected with aleuronat suspension The 

 methylene blue solution was made according to the formula given by 

 Neisser and Wechsberg, which is given below : 



Solution A. Methylene blue, i; absolute alcohol, 20; distilled 

 water, 29. 



Solution B. i c. c. of solution A ; 49 c. c. of physiologic salt solu- 

 tion. 



Two drops of solution B, which must be sterile, are added to each 

 tube in the experiments. 



*An. de 1'Inst. Pasteur, 389G, X, 580. 

 **Zcitschr. f. Hyg., 1001. XXX VI, 209. 



*The term "asdles-serum" as used here refers to a mixture of equal parts of 

 ascites fluid and rabbit serum, heated to 55 . for one-half hour. 



