370 



Scientific Proceedings (124). 



with the required amount of blood in the Petri dish. Identical 

 platings were made from the same suspension of each organism 

 in the study of the influence of medium reaction (P H 7.0 and Pr- 

 8.0) upon hemolysin production. For the icebox test, duplicate 

 series of plates, each consisting of both the special neutral and 

 alkaline media, were inoculated in parallel from the same bacterial 

 suspensions, and incubated at 37 0 C. for approximately 36 hours. 

 Then one series was removed and placed in the icebox at 8° C. 

 for three days. The plates were carefully examined daily, 

 and the results fully recorded. 



Only a brief summary of the results of this work can be con- 

 sidered, as the experimental data are too extensive to be recorded 

 in this place. Important factors influencing the clearing or the 

 degree of hemolysis about the colonies were found to be the thick- 

 ness of the medium layer, and the relative position of the colony 

 to the layer, i.e., on the surface, embedded in layer, or sublayer 

 (between medium and bottom of dish). Surface and sublayer 

 colonies usually were large, but if the medium was rather thick 

 (2 to 3 mm.), they ordinarily showed no surrounding hemolyzed 

 zone. Colonies embedded in the thick layer rarely showed any 

 clearing, but generally, after 36 to 48 hours' incubation, a colora- 

 tion varying from a deep green, brownish green to almost black. 

 In the thinner layers, they appeared pale green to grayish in color. 

 If a clearing actually occurred immediately in contact with the 

 deeply embedded colony, the outer deep green or brownish green 

 zone of methemogoblin would fully envelope and mask the re- 

 action. Colonies in the thinner layer which showed an immediate 

 inner zone of clearing frequently were surrounded by an outer 

 zone of coloration varying from a pale to a dark, or even brown- 

 ish green. In general, the colonies which best showed the hemo- 

 lyzing action were embedded in a blood -agar layer of one mm. or 

 slightly more, and as growth advanced, tended to cause a slight 

 uplifting of the medium. The hemolyzing action, when occur- 

 ring, usually appeared after 48 hours' incubation, and reached 

 its maximum extent in from 72 to 96 hours. The hemolyzed 

 zone varied from a barely visible clear surrounding ring to one 

 which was 1 or 2 mm. in diameter (occasionally greater), rarely 

 very broad, such as is seen with the hemolyzing streptococcus. 



