Introduction and Background

The Methyl Red (MR) and Voges-Proskauer (VP) tests are tests that detect different by-products of bacterial carbohydrate fermentation but use the same growth medium.  The tests are two parts of the important IMViC (Indole, MR, VP, and Citrate) family of tests that are commonly used to identify species of Gram-negative bacteria.  The two tests detect the following by-products of carbohydrate metabolism:

  1. MR – Acid production, as Methyl Red is a pH indicator
  2. VP – Acetoin, as the VP reagents detect this non-acidic product of fermentation

The results for the MR test are shown in the chart below:

Color of Tube After Adding Reagent



Positive for acid production

No color

Negative for acid production

The results for the VP test are shown in the chart below:

Color of Tube After Adding Reagents



Positive for acetoin production

No color

Negative for acetoin production

Laboratory Procedure

A typical procedure for this experiment would involve the following steps:

  1. Obtain the following materials:
    • 2 tubes of MR/VP medium
    • Methyl Red pH indicator
    • VP reagent A
    • VP reagent B
    • Inoculation loop
    • Bacterial culture
  2. Transfer a loopful of the bacterial culture to each of the tubes or MR/VP medium.
  3. Cap the tubes (if a screw-cap tube, loosen by ¼ turn) and incubate in the designated incubation area for 2-3 days.
  4. Make sure one tube is labeled for the MR test and the other labeled for the VP test.
  5. To the MR tube, add 5 drops of methyl red and examine the medium’s color.
  6. To the VP tube, add 12 drops of VP reagent A and 5 drops of VP reagent B. Mix the chemicals into the medium, remove the cap from the test tube because the reaction needs oxygen to occur, and examine the color of the liquid at the top of the medium after 30 minutes.

Perform the following data analysis and submit the answers to the following questions:

  1. For the VP test:
    • Why is oxygen required?
    • Why is the waiting time 30 minutes or longer?
  2. For the MR test:
    • If the red color disappears a few seconds after the addition of Methyl Red, is the test result positive or negative? Provide one reason supporting your answer.
  3. What is the role of acetoin in bacterial metabolism?

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