HEMOLYTIC STAPHYLOCOCCI 25 



a maximum after 1 week; that leukocidin was produced by white and orange 

 strains; that the more virulent the strain the more leukocidin produced; that 

 leukocidin was destroyed by heating at 56 C. ; that normal horse and immune 

 serum possesses antileukocidin ; that leukocidin does not attack kidney cells. 



In making our tests the same strains used for hemolytic activity were 

 used. The cultures were inoculated each day into 10% serum broth 

 for 16 days so that on the 17th day we had 16 cultures of each strain 

 of from 1 day to 16 days old. The cultures were then centrifuged at 

 high speed for 5 minutes, and 1 c c of the supernatant fluid was used 

 for the test. 



Leukocytes were obtained by injecting 8-10 c c of sterile aleuronat 

 into the pleural cavity of guinea-pigs, and after 15 hours the animals 

 were bled to death and the pleural exudate removed with a capillary 

 pipet. An equal amount of 1.5% sterile sodium citrate was added to 

 the cells to prevent coagulation. 



The presence of leukocidin was determined by the methylene blue 

 reduction test. The methylene blue consisted of 1 c c saturated solution 

 of methylene blue, 20 c c absolute alcohol, and 29 c c distilled water. 

 The minimum quantity of leukocytes to reduce methylene blue was 

 first measured by using different amounts of leukocytes varying from 

 0.2 c c to 2 c c, the volume being made equal through the series with 

 sterile salt solution. Two drops of methylene blue were added, and 

 then the mixture was covered with a layer of sterile liquid paraffin to 

 prevent reoxidation from the air. The tubes were incubated at 37 C. 

 for 2 hours. 



To twice the minimum quantity of the leukocytes found necessary 

 to give reduction of methylene blue was added 1 c c of the super- 

 natant centrifuged culture. The tubes were incubated at 37 C. for 

 11/2 hours, when 2 drops of methylene blue and liquid paraffin were 

 added. Incubation was continued for 2 hours more when the readings 

 were made. In case of reduction, no leukocidins were present, since the 

 leukocytes had not been injured. 



It was found that leukocidin appeared on the 4th day and dis- 

 appeared on the 8th day ; and that only strains H2 and T9 produced 

 leukocidins. Thus it is seen that H2, which did not show hemolysin 

 production in broth cultures, produces most leukocidin, and A5, which 

 produced most hemolysins, does not produce leukocidins. Al is nega- 

 tive in both cases, while T9 is positive in both cases. However, strains 

 A5 and H2 indicate distinctly that hemolytic and leukocidin activity 

 are not dependent on each other. 



