Protocol for MR/VP tests

Protocol for MR/VP tests

1.     MR TEST:

Procedure for Methyl Red (MR) Test

MR-VP broth is used for both MR Test and VP test. Only the addition of reagent differs, and both tests are carried out consecutively.

1.      Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under investigation.

2.      Incubate at 28 °C for up to 4 days.

3.      Add about 5 drops of the methyl red indicator solution to the first tube

4.      A positive reaction is indicated, if the colour of the medium changes to red within a few minutes.

MR Positive: When the culture medium turns red after addition of methyl red, because of a pH at or below 4.4 from the fermentation of glucose.

MR Negative: When the culture medium remains yellow, which occurs when less acid is produced (pH is higher) from the fermentation of glucose.

 

MR test

2. VP TEST:

Procedure of Voges Proskauer Test

1.      Inoculate a tube of MR/VP broth with a pure culture of the test organism.

2.      Incubate for 24 hours at 35^oC

3.      At the end of this time, aliquot 1 mL of broth to clean test tube.

4.      Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH. (Note: It is essential that the reagents be added in this order.)

5.      Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

Results and Interpretation

A positive test is represented by the development of a red color 15 minutes or more after the addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin . The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper like color, potentially resulting in a false positive interpretation.

(for Voges-Proskauer test, Barrit’s reagent is added to another tube).

Media and Reagents

1. Media: The medium is MR/VP broth
2. Reagents: (Barrit’s Reagent)

A.    Alpha-naphthol, 5% color intensifier

                                        i.            Alpha Naphthol-5g

                                      ii.            Absolute ethyl alcohol- 100 mL

B.     Potassium Hydrooxide, 40%, oxidizing agent

                                        i.            Potassium hydroxide 40g

                                      ii.            Distilled water to: 100 mL

Vp test

Compostion of MRVP Broth:

Ingredients Gms / Litre Buffered peptone 7.000 , Dextrose 5.000,  Dipotassium phosphate 5.000 Final pH ( at 25°C) 6.9±0.2

Buffered peptone-glucose broth 
(commercially available as MR-VP broth)

Ø  Buffered peptone 7.0 g (can be replaced by 3.5 g of peptic digest of animal tissue and 3.5 g of pancreatic digest of casein) .

Ø  Dipotassium phosphate 5.0 g 

Ø  Dextrose 5.0 g.

Ø  Add 1 liter of deionized water for a final pH of 6.9 (± 0.2) at 25°C. 

Ø  Gently heat to completely dissolve the ingredients and distribute 5 ml aliquots into culture tubes.

Ø  Sterilize via autoclaving at 121°C and 15 psi for 15 minutes. After the medium cools down it can be store in a refrigerator until use.