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Objective: Some pathogens are able to produce exoenzymes called hemolysins which lyse red blood cells and thus their action can be demonstrated on a blood agar plate.
1. Using a sterile loop, inoculate a blood plate (SBA) with the pure culture of the organism to be tested using the quadrant method. Also stab the medium in the second quadrant with your loop. (Some hemolysins show their effects better under lower oxygen concentrations.)
2. Incubate for 48 hours at optimum temperature for the organism.
Interpret by noting the reaction around isolated colonies as follows:
Alpha (α) hemolysis: formation of a green or brown zone around the colonies (due to loss of potassium from the red cells).
Beta (β) hemolysis: complete lysis of cells and reduction of released hemoglobin; a clear zone appears around isolated colonies.
Gamma (γ) hemolysis: no hemolytic reaction (no change of the medium surrounding isolated colonies).
-- The reaction should be checked only around isolated colonies. If you do not have isolated colonies on the blood agar, a lighter inoculation should be streaked and the test repeated.