Practical IMVIC test

IMViC Test

IMViC is a series of tests that are useful in the identification of enteric bacteria Tests include: 1. I = Indole test 2. M = Methyl red test (MR) 3. Vi = Voges-Proskauer test (VP) 4. C = Citrate test

Indole Medium

Tryptone broth, contains extra tryptophanTrp. (tryptophan) can be utilized as a sole carbon and energy source by some bacteria that produce an enzyme tryptophanase (tryptophanase)Tryptophan Indole + pyruvic acid + NH3 (C-source) (N-source)We test the presence of the bi-product Indole with a chemical called Korvac’s reagentKorvac’s reagent reacts with Indole and turns solution redThe alcoholic layer concentrate the red color as a ring at the top.

Indole Procedure

Procedure: 1. Inoculate tryptone broth with young bacterial culture. 2. Incubate at 37 degrees for 48 hours

Indole Results

Tryptone Broth after addition of Kovacs(+) Indole test on left --- (-) Indole test on right

Methyl red

Strains of E. coli are mixed acid fermenters; they degrade carbohydrates into acidic end products such as: lactic acid, acetic acid, succinic acid, and formic acid

MR (continued)

The Methyl-Red tests for acidic products resulting from the ability of an organism to produce and maintain stable acid end products from glucose fermentation. These acidic products will drop the pH of the medium to pH 4.5 or below Methyl red is a pH indicator will turn red if mixed acid fermentation has occurred and remain yellow in pH over 6.2.

The principle: 

 Glucose Fermentation (acidic end products) MR+

Hydrogenlyase Glucose 2pyrovate (Lactic acid, CO2+H2 (MR+) formic acid, acetic acid)

Materials for methyl red test:

Media(MR-VP media ): Peptone ∕ Glucose ∕ K2HPO4 brothReagents: MR reagent: methyl red solution

The Procedure:

Incubate 5 ml of the broth with young bacterial culture for 24- 48 hrs at 37°C. Add methyl red reagent into the broth .The results:Red color solution : + ve ; e.g. E.coli ∕ faint yellow= -ve; Klebsiella

The Voges-Proskauer test= V test:

The principle:

This test is used to detect microorganism that ferment glucose via the butanediol pathway produce acetoin as an intermediate which can be further reduced to 2,3-butanediol.

The principle:

Glucose 2pyrovate AcetoIn 2,3 butanediol α -naphtholAcetoin + KOH Red color


Material VP test :
Media(MR-VP media ): Peptone ∕ Glucose ∕ K2HPO4 brothReagents: VP reagent: α –naphthol+ KOH which is called (Barritt's reagent).The procedure:Incubate 5 ml of the broth with young bacterial culture for 24- 48 hrs at 37°C.Add α –naphthol to the broth then add KOH .

The results:

Red color broth = +ve, e.g. Klebsiella∕ pale yellow= -ve ,e.g. E.coli

Citrate Test

Simmon’s Citrate agar is used to determine an organism’s ability to use citrate as a sole carbon source.

Citrate Test (continued)

The pH change is induced by CO2, which is given off as a bi-product of citrate utilization. When it reacts with Na and H2O in the agar it raises the pH above 7.6 The organism must contain the enzyme citrase to degrade citrate EX: (citrase) Citrate CO2 + Na HCO + H2O

(blue color change)

The materials:
Media : Simmon Citrate slant agar. The Procedure: The slant is streaked only with the required bacteria Incubate at 37 degrees for 48 hrs.

Citrate Results

1. Green color is (-) for Citrase: eg. E.coli 2. Blue color is (+) for Citrase: e.g. Klebsiella

Urease test

This test is used to detect if the m.o. possess the enzyme Urease .

The Principle:

Urease

The materials

Media: Urea agar base + Urea solution. The indicator: phenol red( pH indicator ) included within the media.

The Procedure

The results: Pink color = +ve; e.g.Proteus; orange color = -ve ,e.g. E.coli

Catalase Test

This test used to detect the ability of m.o. ( aerobic bacteria) produce the enzyme catalase that degrade H2O2.

The Principle:

catalase H2O2 H2O+ O2 (gas) The Procedure: Place a drop of distal water on a clean dry slide . Transfere a small amount of the bacterial growth to the slide using a wooden stick. 2-3 drops of 3%H2O2 solution is added to the bacterial growth and mix slightly .

The results:

Vigorous bubbling (due to O2 release )= + ve Staph.


No bubbling = -ve result (no catalase is produced) e.g. E.coli