Methyl Red Test

 

What is the purpose of the test? 

 

This test determines whether the microbe performs mixed acids fermentation when supplied glucose. Mixed acids fermentation results in accumulation of a variety of acids and a significant drop in the pH of the medium.

 

 

How is the mixed acids fermentation determined? 

 

If the microbe performs mixed acids fermentation, the medium will acquire an acidic pH. Addition of a pH indicator which changes color below pH 4 indicates presence of the extreme acidity associated with mixed acids fermentation.

 

 

What medium is used? 

 

The medium used is MRVP broth, a nutrient medium with 0.5% glucose added. This medium is also used for the Voges-Proskauer test, which determines whether neutral fermentation products result from growth.

 

 

How is the test performed? 

 

An inoculum from a pure culture is transferred aseptically to a sterile tube of MRVP broth. The inoculated tube is incubated at 35-37 C for 24 hours.

 

 

What reagents are added? 

 

After incubation, five drops of methyl red are added. It is a pH indicator which is yellow at neutral pH but turns red at pH <4.0. Mixed acids fermentation results in a red color change.

 

 

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

 

Inoculation of Medium

 

1. Select the MRVP 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.  

14. Incubate for the appropriate length of time. This test is properly interpreted after 24 hours.  

15. Retrieve desired incubated culture from the incubator.  

 

Addition of Reagents

 

16. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, select methyl red.  

17. Remove the cap from the tube.  

18. Place the end of the dropper into the tube and add the reagent to the culture.  

 

Determination of Test Results

 

19. Observe test result. For this test, addition of the appropriate reagent makes the culture turn red if the test is positive. If negative, no color change is observed.  

20. Record test result.  

21. Dispose of the culture.