Methyl Red – Voges Proskauer (MRVP) tests

 

The methyl red (MR) and the Voges-Proskauer (VP) tests are used to differentiate two major types of facultative anaerobic enteric bacteria that produce large amounts of acid and those that produce the neutral product acetoin as end product.  MRVP tests are of value in the separation of Escherichia coli and Enterobacter aerogenes, both coliform bacteria, which appear virtually identical except for certain physiological differences that are used as indicators of the sanitary quality of water, foods, food-production and eating establishments.  Both these tests are performed simultaneously because they are physiologically related and are performed on the same medium MRVP broth. Opposite results are usually obtained for the MR and VP tests, ie., MR+, VP^_    or MR^_, VP+.

Aim

To find out whether an organism is MR-VP positive or negative.

Principle

The hexose monosaccharide glucose is the major substrate oxidized by all enteric organisms for energy production.  The end products of this process will vary depending on the specific enzymatic pathways present in the bacteria. In MR test the pH indicator methyl red detects the presence of large concentrations of acid end products.  Although all enteric microorganisms ferment glucose with the production of organic acids, this test is of value in the separation of    E. coli and E.  aerogenes.

Both of these organisms initially produce organic acid end products during the early incubation period.  The low acidic pH (4) is stabilized and maintained by E. coli at the end of incubation.  During the later incubation period, E.  aerogenes enzymatically converts these acids to nonacidic end products such as 2,3-butanediol and acetoin (acetyl methyl carbinol), resulting in the elevated pH of approximately 6.  The methyl red indicator in the pH range of 4 will remain red, which is indicative of a positive test.  At a pH of 6, still indicating the presence of acid but with a lower hydrogen ion concentration, the indicator turns yellow and is a negative test (Figure 1).

The VP test determines the capability of some organism to produce non acidic or neutral end products, such as acetyl methyl carbinol, from organic acids that result from glucose metabolism, which is characteristic of E.  aerogenes (Figure 2).  The reagent used in this test, Barritt’s reagent, consists of a mixture of alcoholic α-naphthol and 40% potassium hydroxide solution.  Detection of acetyl methyl carbinol requires this end product to be oxidized to a diacetyl compound.  This reaction will occur in the presence of the α-naphthol catalyst and a guanidine that is present in the peptone of the MRVP medium.  As a result, a pink complex is formed, imparting a rose color to the medium.  Development of a deep rose color in culture 15 minutes following the addition of  Barritt’s reagent is indicative of the presence of  acetyl methyl carbinol and represents a positive result.  The absence of rose coloration is a negative result (Figure 3).

Requirements

24 hr nutrient broth cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

MR-VP broth (5ml / tube), Methyl red indicator, Barritt’s reagent A and B, Bunsen burner, inoculating loop, glass marking pencil, dropper etc.

Procedure

1.  Divide the total MR-VP tubes into two sets.  Using sterile techniques, inoculate each set of   MR-VP broth with given cultures of organisms.  Uninoculated tubes serve as control.

2.  Incubate the tubes at 37^o C for 24-48 hrs.

3.  Add 5 drops of methyl red indicator to the first set of organisms and observe the color change for MR test.

4.  To the other set of tubes, add 12 drops of Barritt’s reagent A and 2-3 drops of Barritt’s reagent B.  Shake the tubes gently for 30 seconds with the caps off to expose the media to oxygen.

5.  Allow the reaction to complete for 15-30 min and observe the color change for VP test.

Observations

In the MR test, the MR-VP broth remained red color in the tubes inoculated with Bacillus, Streptococcus, Staphylococcus and E.coli. No change observed in tubes inoculated with  Pseudomonas sp.

In the VP test, no development of a crimson to ruby red color formed in any of the tubes inoculated.

 

Result

Among the given culture of organisms Bacillus, Streptococcus, Staphylococcus and E.coli are MP positive and VP negative whereas  Pseudomonas sp. is MR and VP negative.  

       Barritt’s reagent A and B

 Solution A

Alpha-naphthol                                   5.0 g

Absolute ethanol                                 95.0 ml

(Note: Dissolve the alpha- naphthol in the ethanol with constant stirring).

Solution B

Potassium hydroxide                          40.0 g

Creatine                                               0.3 g

Distilled water                                    100 ml

(Note: Dissolve the potassium hydroxide in 75 ml distilled water. The solution will become warm. Allow to cool to room temperature. Add the creatine and stir to dissolve. Add the remaining water. Store in a refrigerator). 

MR-VP broth                                            

Peptone                                               7.0 g

Dextrose                                              5.0 g

Potassium phosphate                           5.0 g

Distilled water                                    1 litre

pH                                                       6.9