FURTHER BIOCHEMICAL METHODS vi,.-17 



intact, the erythrocytes are not hemolyzed. Later, when the bacteria 

 break down, substances are Hberated which have a more or less 

 pronounced hemolytic action. A third group of organisms are 

 "indifferent," producing no visible change in the hemoglobin or 

 erythrocytes. The production of hemolysins and changes occurring 

 in the hemoglobin under bacterial action are important in the dif- 

 ferentiation of streptococci, pneumococci and other bacteria. Strepto- 

 coccus pyogenes is the type of organism which produces an exohemo- 

 lysin; pneumococci and streptococci of the viridans group, are types 

 of organisms which produce methemoglobin. 



Method I. Blood Agar Plate Method. Either streak cultures on blood 

 agar plates or poured i)lates of blood agar mixed with bacteria can be 

 used for this purpose. The sharpest results are obtained with poured 

 plates. For the streak method, prepare blood agar plates by melting 

 100 ml. of 2% meat infusion agar, cooling the agar to 45° C, adding 

 5-10 ml. of sterile defibrinated blood (sheep, rabbit or horse blood) 

 and pouring this blood agar into Petri dishes. After the agar has 

 hardened, streak the surface with the organism. Incubate the plate for 

 24 hours or longer at 37° C. Also incubate uninoculated plates as 

 checks against contamination. A clear area under and beyond the 

 edge of the growth (beta hemolysis) indicates laking of the red cells 

 due to an hemolysin elaborated by the organism. Organisms which 

 produce methemoglobin cause a greenish coloration (alpha hemolysis) 

 in the blood adjacent to the growth. In using the poured plate 

 method, the blood agar is prepared in a tube or flask and inoculated 

 with a suspension of the organisms that will give 25 to 50 colonies 

 per plate. It is important that no sugar be added to the agar. The 

 temperature at the time of mixing the organisms with agar should 

 be approximately 45° C. The inoculated blood agar is poured into 

 Petri dishes, allowed to harden and incubated. After incubation, 

 clear areas, having varied significant characteristics, appear around 

 the colonies which produce hemolysin (beta). The colonies of "green 

 producing" streptococci and pneumococci appear surrounded by a 

 greenish zone of erythrocytes containing methemoglobin (alpha). 

 After continued incubation of this type of culture, a zone of hemolysis 

 occurs beyond the zone of greenish cells, and at times several rings of 

 alternate hemolysis and methemoglobin formation may be observed. 



Method II. Blood Broth Mixtures. To 0.5 ml. of a sterile 5% suspen- 

 sion of washed rabbit, sheep or horse blood cells in 0.85% NaCl 

 solution, add 0.5 ml. of a 12 to 18 hour sugar-free broth culture of the 

 organism to be tested. Incubate this mixture for 2 hours, at 37° C, 

 preferably in a water bath. The production of an hemolysin is shown 

 by the laking of the cells, giving a clear solution. Organisms which 

 form methemoglobin produce darkening of the cells, and do not 

 hemolyze them in this test. A tube containing 0.5 ml. each of the 

 blood suspension and of sterile broth should be inoculated as a 

 control. The corpuscles of rabbits blood are removed by centrifuging 

 and washed as described on p. viii4o-15 of Leaflet VIII. 



