Methyl Red (MR) and Voges-Proskauer (VP) Test principle, Method, Interpretation & QC #MR & VP Mallu Medicos Lounge As the channel name suggests, our channel will be a perfect lounge for the malayali medicos..we wil be covering videos which will be like lecture classes related to the subjects biochemistry and microbiology in which we are specialised.. It will be a better learning experience for the students especially for those who are not able to understand and follow the normal classes in college..we assure the students that you will get a basic idea regarding the topic and extra reading can be done from the reference textbooks.. Our Partner Channel Health & Voyage channel link - https://youtu.be/nzKqRVjlwc0 Qualification Maneesha M Joseph MSc MLT (Microbiology) Assistant Professor Baby memorial college of allied Health science Kozhikode #Methyl Red Test #Voges-Proskauer Test #MRVP Procedure #MRVP Tests #MR-VP Tests #VP (Voges Proskaeur) Test #MR/VP Test #Methyl red (MR) and Voges-Proskauer (VP) #Methyl red test in Microbiology #Medical #Microbiology #MR/VP Test malayalam lecturer #Mallu Medicos Lounge #MalluMedicosLounge #MLT #Channel introduction #HealthAndVoyage

1. By,
Maneesha M Joseph
Assistant Professor
Baby Memorial Collage
Kozhikode
Video Class is uploaded in YouTube Channel Mallu Medicos Lounge
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2. METHYL RED TEST
Ø The methyl red (MR) test detects the production of sufficient acid during the
fermentation of glucose and the maintenance of conditions such that the pH of an old
culture is sustained below a value of about 4.5, as shown by a change in the colour of
the methyl red indicator which is added at the end of the period of incubation.
Ø Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests to
be performed from the same inoculated medium by aliquoting portions to different
tubes.
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3. PRINCIPLE OF METHYL RED (MR) TEST
Ø Some bacteria have the ability to utilize glucose and convert it to a stable acid like
lactic acid, acetic acid or formic acid as the end product.
Ø These bacteria initially metabolise glucose to pyruvic acid, which is further
metabolized through the ‘mixed acid pathway to produce the stable acid.
Ø The type of acid produced differs from species to species and depends on the specific
enzymatic pathways present in the bacteria.
Ø The acid so produced decreases the pH to 4.5 or below, which is indicated by a change
in the colour of methyl red from yellow to red.
Ø In the methyl red test (MR test), the test bacteria is grown in a broth medium
containing glucose.
Ø If the bacteria has the ability to utilise glucose with production of a stable acid, the
colour of the methyl red changes from yellow to red, when added into the broth
culture.
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4. MEDIAAND REAGENTS USED IN METHYL RED (MR) TEST
MR VP broth (pH 6.9)
Ingredients per litre of deionized water:
Buffered peptone= 7.0 gm
Glucose= 5.0 gm
Dipotassium phosphate= 5.0 gm
Methyl red solution, 0.02%
a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
c. Store at 4 to 8 degree C in a brown bottle. Solution is stable for 1 year.
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5. PROCEDURE OF METHYL RED (MR) TEST
Ø Prior to inoculation, allow medium to equilibrate to room temperature.
Ø Using organisms taken from an 18-24 hour pure culture, lightly inoculate the
medium.
Ø Incubate aerobically at 37 degrees C. for 24 hours.
Ø Following 24 hours of incubation, aliquot 1ml of the broth to a clean test tube.
Ø Reincubate the remaining broth for an additional 24 hours.
Ø Add 2 to 3 drops of methyl red indicator to aliquot.
Ø Observe for red color immediately
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6. MANEESHA M JOSEPH 6

7. QUALITY CONTROL OF METHYL RED (MR) TEST
Klebsiella pneumoniae ATCC 13883—MR negative (yellow)
Escherichia coli ATCC 25922—MR positive (red)
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8. PRINCIPLE OF VP TEST
v Voges Proskauer test, commonly known as VP test is used to determine the ability of some
organisms to produce neutral end products (e.g., 2, 3-butanediol or acetoin) from glucose
fermentation.
v All members of the Enterobacteriaceae can convert glucose to pyruvic acid by the Embden-
Meyerhof pathway, but bacteria can further metabolize pyruvic acid by two different
pathways.
v Organisms metabolizing pyruvic acid by the mixed acid pathway will produce more acid
end products, such as lactic acid and acetic acid, and maintain an acidic environment.
v If the organism produces large amount of organic acids that includes formic acid, acetic acid,
lactic acid, and succinic acid from glucose fermentation, the broth medium will remain red
after the addition of methyl red, a pH indicator.
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9. v Bacteria fermenting sugars via the butanediol pathway produce acetoin (i.e.,
acetyl methyl carbinol or 3-hydroxybutanone) as an intermediate which can be
further reduced to 2,3-butanediol.
v 2 pyruvate = acetoin + 2CO 2
v acetoin + NADH + H + = 2,3-butanediol + NAD +
v In the presence of KOH the intermediate acetoin is oxidized to diacetyl, a reaction
which is catalyzed by alpha naphthol.
v Diacetyl reacts with the guanidine group associated with molecules contributed
by peptone in the medium, to form a pinkish-red-colored product.
v The alpha naphthol in the Barritt’s modification of the VP test serves as a color
intensifier.
v However, MR-negative organisms further metabolize the initial fermentation
products by decarboxylation to produce neutral acetyl methylcarbinol (acetoin),
which results in decreased acidity in the medium and raises the pH towards
neutrality (pH 6.0 or above)..
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10. MR VP broth
v Buffered peptone 7.0 gm/L
v Dextrose 5.0 gm/L
v Dipotassium phosphate 5.0 gm/L
Final pH ( at 25°C) 6.9±0.2
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11. PROCEDURE
v Inoculate MRVP broth with a pure culture of the organism.
v Incubate at 35°-37°C for a minimum of 48 hours in ambient air.
v Add 6 drops of VP reagent I (alpha napthol) and 2 drops of VP
reagent II(40% KOH).
v Observe for the color change in the broth medium.
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12. Positive: Crimson to ruby pink (red) color
Negative: No change in colouration
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13. QUALITY CONTROL OF VP TEST
VP positive: Enterobacter aerogenes (ATCC13048)
VP negative: Escherichia coli (ATCC25922)
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