Esculin Hydrolysis Test


What is the purpose of the test? 


The purpose is to see if the microbe can hydrolyze the compound esculin as a carbon source.



How is esculin hydrolysis determined?


When microbes metabolize esculin in a nutrient medium, the dark brown compound esculetin is produced and the medium is obviously discolored.



What medium is used? 


Usually, the medium used is bile esculin agar slant. This is a nutrient agar-based medium containing 0.1% esculin and 10% bile salts, and allowed to solidify at a slant. The bile salts inhibit some bacteria, and so the ability to grow in the presence of bile salts represents a second test use for the medium.



How is the test performed? 


An inoculum from a pure culture is transferred aseptically to a sterile tube of bile esculin agar and streaked along the slant. There is no need to stab the medium. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Abundant growth on the slant indicates a positive test for growth in the presence of bile. If growth is present, esculin hydrolysis can be observed if the medium has taken on an intense, chocolate brown coloration.



What reagents are added? 





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


Inoculation of Medium


1. Select the bile esculin agar slant medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

5. Remove the caps from your test tubes.  

6. Flame the mouths of your test tubes.  

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.  

9. Flame the mouths of your tubes once again.  

10. Replace the caps on the test tubes.  

11. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


12. Place the inoculated tube 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 for determining the result.  

15. Retrieve desired incubated culture from the incubator.  

16. Observe test result. The change of all or a significant portion of the medium to a chocolate brown indicates breakdown of esculin to esculetin, a positive test. Negative results would be indicated by growth on the slant but no change from the normal amber color of the medium.  

17. Record test result.  

18. Dispose of the culture.