Hemolysis on Blood Agar


What is the purpose of the test?

This test provides information on what hemolytic enzymes a bacterium possesses. By providing a culture medium enriched with red blood cells, it is possible to determine whether a bacterium can destroy the cells and whether it can digest the hemoglobin inside.




How is hemolysis determined?


Bacteria possessing hemolysins will be able to discolor or clear blood agar in the vicinity of their colonies.




What medium is used? 


A nutrient medium augmented with the addition of 5% sheep blood routinely is used. Such a medium is called blood agar.




How is the test performed?  


Hemolysis is determined by streaking for isolation on a blood agar plate. In clinical settings, this might also include several stabs of the inoculum into the agar to encourage any anaerobic versions of the enzymes to digest blood cells. After incubation overnight, the medium is inspected for telltale signs of alpha- or beta-hemolysis. If the medium is discolored or darkened after growth, the organism has demonstrated alpha-hemolysis. If the medium has been cleared under growth, the organism is beta-hemolytic. No discernible change in the color of the medium constitutes gamma-hemolysis.




What reagents are added?





To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the called blood agar plate medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

5. Remove the cap from your inoculum and your lid from your sample plate.  

6. Flame the mouth of your inoculum tube.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.  

8. Immediately transfer the inoculum into the fresh, sterile medium. NOTE: you must streak the inoculum back and forth across the plate for several seconds to inoculate the plate. You will know it has been successfully done when streak marks appear on the agar surface.  

9. Flame the mouth of your inoculum tube once again.  

10. Replace the cap on the inoculum tube and the plate lid.  

11. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


12. Place the inoculated plate into the 35-37 C incubator.  

13. Press the New Day button to move forward 24 hours.  


Determination of Test Results


14. Incubate for the appropriate length of time. For this test 24 hours is sufficient.  

15. Retrieve desired incubated culture from the incubator.  

16. Observe the medium surrounding colonies in the plate. The culture showing a darkening or discoloration of the medium in the vicinity of growth demonstrates alpha-hemolysis. Cultures showing clear halos around colonies and under growth is exhibiting beta-hemolysis.

17. Record test result.  

18. Dispose of the culture.