Imvic Test

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 E. coli m Enterobacter Citrobacter

Indole

MR

VP

Citrate