Mannitol Fermentation Test


What is the purpose of the test? 


The purpose is to see if the microbe can ferment the carbohydrate (sugar) mannitol as a carbon source.



How is mannitol fermentation determined? 


If mannitol is fermented to produce acid end products, the pH of the medium will drop. A pH indicator in the medium changes color to indicate acid production.



What medium is used?  


Several media are available for this. Most commonly used is phenol red mannitol broth. The medium is a nutrient broth to which 0.5-1.0% mannitol is added. The pH indicator phenol red is red at neutral pH but turns yellow at pH <6.8. It also changes to magenta or hot pink at pH >8.4.



How is the test performed?  


An inoculum from a pure culture is transferred aseptically to a sterile tube of phenol red mannitol broth. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. A positive test consists of a color change from red to yellow, indicating a pH change to acidic.



What reagents are added?





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


Inoculation of Medium


1. Select the phenol red mannitol broth 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. If the test was followed as described above, the culture will have changed to yellow in the presence of acids (indicating a positive test) or magenta or hot pink in the presence of bases/alkali (indicating a negative test).  

17. Record test result.  

18. Dispose of the culture.