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 organismsEtherindicator: 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 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 – 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 organisms that are MR (+) are always VP (-)

Materials:

MR-VP broth medium (contains 10% glucose)

Test organismsMethyl 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• Ex. Enterobacter and Klebsiella

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 organismPotassium 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 dimethyl-carbinol

guanidine compounds

Crimson red

0xidized

React with

Inoculate MR-VP medium with 1 loopful of the test organism

Incubate for 48 hours at 370C.

Next laboratory period add 0.6 ml. 5% alpha-napthol reagent. 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 atmospheric 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 alkaline pH's (around 7.6) (positive citrate)

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

Microorganism

Indole MR VP Citrate

E. coli

Enterobacter

Citrobacter