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 tryptophan Trp. (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 reagent Korvac’s reagent reacts with Indole and turns solution red The alcoholic layer concentrate the red color as a ring at the top.

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

Indole Results Results: Red ring at the top : + ve ;E.coli Coloreless ring at the top: -ve; e.g. Staphylococcus aureuos,Klebsiella  

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 broth Reagents: 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 -naphthol Acetoin + KOH Red color

Material VP test : Media(MR-VP media ): Peptone ∕ Glucose ∕ K2HPO4 broth Reagents: 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: Urea+ H2O Ammonia + CO2 Urease pH 6.8 pH8.1 Dark Orange Dark pink (phenol red is a pH indicator) Urease

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

The Procedure Inculate the urea agar with the required m.o. Incubate at 37°C for 24-48 hrs.

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