 Basis: metabolic action of microorganisms

on the culture media

 Used for the identification of enteric organisms/
gram negative bacilli
 One of the earliest sets of test used for the
identification of enteric bacilli
 includes such organisms as Klebsiella,
Enterobacter, Citrobacter and Escherichia coli

 This acronym stands for

• I - Indole
• M- Methyl red
• V - Voges – Proskauer
• ( i ) is inserted for euphony
• C - Citrate
 Indole, a benzyl pyrrole, is one of the metabolic
degradation product of amino acid tryptophan

 Indole positive bacteria produce tryptophanase, an

enzyme that is capable of hydrolyzing and
diaminating tryptophan, thus producing: - indole
- pyruvic acid
- ammonia
 Materials:
2% Peptone broth tube
Test organisms
Ether
indicator: Erlich/Kovac's reagent
(para-dimethyl-aminobenzaldehyde)
Procedure:

Inoculate 1 loopful of the test organism into the

tube of peptone broth.

Incubate at 370C for 24-48 hours.

Next laboratory period, add 1 ml. of ether.

Shake well and allow to stand for a few minutes
until the ether rises to the surface.

Gently add about, 1cc. of Kovac’s or Erlich’s

reagent down the side of the tube so that it forms
a ring between the medium and the ether layer.
 Positive result
• Bright red or purple ring
• If indole has been produced
by the organism it will, being
soluble in ether, it will be
concentrated in the ether layer
and upon the addition of Erlich’s reagent, a
positive result is the production of a
purple ring at the
junction of the medium and the ether layer

 Negative result – Yellow color

- no red or purple ring
 All enterics oxidize glucose for energy; however the
end products vary depending on bacterial
enzymes
 Both the MR and VP tests are used to determine
what end products result when the test organism
degrades glucose
 MR test is a quantitative test for acid production,
requiring positive organism to produce strong acids
(lactic, acetic, formic) from glucose via the mixed
acid fermentation pathway
 End result is based on the final pH reached
 only those organism that can maintain low ph of
about
ph 4-4.5 can be called methyl red – positive
Materials:

MR-VP broth medium (contains 10%

glucose)
Test organisms
Methyl red ph indicator
Procedure:

Inoculate 1 loopful of the test

organism into a tube MR-VP
medium.

Incubate for 24-48 hours at 370C.

Next laboratory period, add 5 to 10

drops of methyl red reagent.

Mix thoroughly and observe the

results.
 Positive result – cherry red/bright red color
• ph 4-4.5
• Ex. Salmonella, Escherichia,
Citrobacter, Proteus, Morganella
and Providencia

 Negative result – Yellow color

• At neutral pH the growth of the bacteria is not
inhibited
• The bacteria thus begin to attack the peptone in
the broth, causing the pH to rise above 4.5
• At this pH, methyl red indicator produce a yellow
color
 is a test for the detection of acetyl-methyl
carbinol (acetoin) which is also a degradation
product of glucose
 Materials:
MR-VP medium (contains 10% glucose)
Test organism
Potassium Hydroxide
Alpha-napthanol reagent
 When these reagents are added to a broth in
which acetyl methyl carbinol is present, they turn
a burgundy color/crimson red color (a positive VP
test)
 organisms that are VP (+) are always MR (-)
acetyl-methyl carbinol + Potassium Hydroxide
0xidized
dimethyl-carbinol
React with
guanidine compounds

Crimson red
 Inoculate MR-VP medium with 1 loopful of the test organis
Incubate for 48 hours at 370C.
Next laboratory period add 0.6 ml. 5% alpha-napthol reag
Mix and shake the mixture lightly.
Add 0.2 ml (5drops). of 40% potassium hydroxide reagent
(KOH).
Mix and shake the mixture lightly.
Shake the tube gently to expose the medium to atmosphe
oxygen and allow the tube to remain undisturbed for
10 to 15 minutes.
Positive result
• Crimson Red color
• Presence of Acety methyl carbinol
• Ex. Enterobacter and Klebsiella

Negative result
• Remains Yellow to Amber; no change in color
• Ex. E. coli
 a test depends upon the ability of the organism, to
utilize citrate as the sole source of carbon and
energy growth
 Materials:

• Solid media : Simmon’s Citrate Agar

• Liquid media: Kosher’s Media
• Test Organism
 Simmon's media contains bromthymol blue, a pH
indicator with a range of 6.0 to 7.6
• uninoculated Simmon's citrate agar has a pH of
6.9, so it is an intermediate green color (neutral
pH)
• Growth of bacteria in the media leads to
development of a Prussian blue color at more
Procedure:

Inoculate the test organism on the medium by stab streaking.

Incubate at 370C for 24 - 48 hours.

Observe.
Positive result
• Deep blue/ Prussian blue color
• indicating that the test organism
has been able to utilize citrate for energy source
• Ex. Enterobacter, Klebsiella, Salmonella,
Citrobacter
and Providencia

Negative result
• Retains its original color (Green)
• Ex. Escherichia, Shigella and Morganella
Microorganis Indole MR VP Citrate
E. coli m
Enterobacter
Citrobacter