Methyl Red (MR) and Voges-Proskauer (VP) Test

Methyl Red (MR) and Voges-Proskauer (VP) Test principle, Method, Interpretation & QC #MR & VP
Mallu Medicos Lounge
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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
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#HealthAndVoyage

1. By,
Maneesha M Joseph
Assistant Professor
Baby Memorial Collage
Kozhikode
Video Class is uploaded in YouTube Channel MalluMedicos Lounge
MANEESHA M JOSEPH1

2. METHYL RED TEST
ØThe 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.
ØClark and Lubsdeveloped 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.
MANEESHA M JOSEPH2

3. PRINCIPLE OF METHYL RED (MR) TEST
ØSome 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.
ØThese bacteria initially metaboliseglucose to pyruvic acid, which is further
metabolized through the ‘mixed acid pathwayto produce thestable acid.
ØThe type of acid produced differs from species to species and depends on the specific
enzymatic pathways present in the bacteria.
ØThe acid so produced decreases the pH to 4.5 or below, which is indicated by a change
in the colour of methyl red fromyellow to red.
ØIn the methyl red test (MR test), the test bacteria is grown in a broth medium
containingglucose.
ØIf the bacteria has the ability to utiliseglucosewith production of astable acid, the
colour of the methyl red changes fromyellow to red, when added into the broth
culture.
MANEESHA M JOSEPH3

4. MEDIA AND REAGENTS USED IN METHYL RED (MR) TEST
MR VP broth (pH 6.9)
Ingredients per litre of deionizedwater:
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 in300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water tomake 500 ml.
c. Store at 4 to 8 degree C in a brown bottle.Solution is stable for 1 year.
MANEESHA M JOSEPH4

5. PROCEDURE OF METHYL RED (MR) TEST
ØPrior to inoculation, allow medium to equilibrate to room temperature.
ØUsing organisms taken from an 18-24 hour pure culture, lightly inoculate the
medium.
ØIncubate aerobically at 37 degrees C. for 24 hours.
ØFollowing 24 hours of incubation, aliquot 1ml of the broth to a clean test tube.
ØReincubatethe remaining broth for an additional 24 hours.
ØAdd 2 to 3 drops of methyl red indicator to aliquot.
ØObserve for red colorimmediately
MANEESHA M JOSEPH5

6. MANEESHA M JOSEPH6

7. QUALITY CONTROL OF METHYL RED (MR) TEST
Klebsiella pneumoniaeATCC 13883—MR negative(yellow)
Escherichia coliATCC 25922—MR positive(red)
MANEESHA M JOSEPH7

8. PRINCIPLE OF VP TEST
vVoges 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.
vAll 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.
vOrganisms 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.
vIf 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.
MANEESHA M JOSEPH8

9. vBacteria 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.
v2 pyruvate = acetoin + 2CO2vacetoin + NADH + H+= 2,3-butanediol + NAD+
vIn the presence of KOH the intermediate acetoin is oxidized to diacetyl, a reaction
which is catalyzedby alpha naphthol.
vDiacetyl reacts with the guanidine group associated with molecules contributed
by peptone in the medium, to form a pinkish-red-coloredproduct.
vThe alpha naphthol in the Barritt’smodification of the VP test serves as a color
intensifier.
vHowever, 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)..
MANEESHA M JOSEPH9

10. MR VP broth
vBuffered peptone 7.0 gm/L
vDextrose 5.0 gm/L
vDipotassium phosphate 5.0 gm/L
Final pH ( at 25°C) 6.9±0.2
MANEESHA M JOSEPH10

11. PROCEDURE
vInoculate MRVP broth with a pure culture of the organism.
vIncubate at 35°-37°C for a minimum of 48 hours in ambient air.
vAdd 6 drops of VP reagent I (alpha napthol) and 2 drops of VP
reagent II(40% KOH).
vObserve for the colorchange in the broth medium.
MANEESHA M JOSEPH11

12. Positive:Crimson to ruby pink (red) color
Negative:No change in colouration
MANEESHA M JOSEPH12

13. QUALITY CONTROLOF VP TEST
VP positive:Enterobacter aerogenes(ATCC13048)
VP negative:Escherichia coli(ATCC25922)
MANEESHA M JOSEPH13