Biochemical Test

Basic Microbiology laboratory for Dental Students

By LecturerYasemin Al-Shboul

Spring2011-2012

Biochemical Activities of Microorganisms (Part 2)

• IMViC Reaction: Indole Test, Methyl Red Test, Voges-Proskauer Test, and Citrate Test.

• Indole Test:• The Indole test is a biochemical test performed on bacterial species to determine the ability of the

organism to split Indole from the amino acid tryptophan. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as tryptophanase .

• Tryptophan

• Kovac's reagent (Yellow) + Indole --------- Red Color

Procedure.

Interpretation: Pink-red ring: Positive / Yellow ring: Negative

• Indole test

Biochemical Activities……

MR-VP Tests:Divide Enterobacteriaceae into two groups based on the way the

metabolize glucose.

Methyl Red (MR) TestDetermines the ability of an organism to produce acids as final products from glucose fermentation. Fermentation

Glucose Lactic Acid, Acetic Acid, Formic Acid

Red Color MR indicator + Acidic pH

fermentation

MR TEST• Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2

Biochemical Activities..……

Procedure.

Interpretation:

Red color: Positive / Yellow color: Negative

Voges-Proskauer (VP) Test

Determines the ability of an organism to produce Acetoin as a final product from glucose fermentation.

Glucose Acetoin

Acetoin + α-Naphthol Diacetyl

fermentation

40% KOH

Oxidation

• VP TEST

Biochemical Activities..……

Diacetyl + Peptone (from medium Pink Red

Procedure.

InterpretationPink Red color: Positive / Yellow color: Negative

Citrate Test:Determines the ability of an organism to use citrate as the only source of carbon.

Citrate Organic Acids + CO2

CO2 + Na (from conjugate base salt) Na2CO3

α-Naphthol

Citrate

Utilization

• Citrate test

Biochemical Activities..……

Bromothymol Blue Indicator (Green) Blue

Procedure InterpretationPink Red color: Positive / Yellow color: Negative

Urease Test:Determines the presence of Urease enzyme.

Urea NH4 (Ammonia) + CO2

Phenol Red Indicator (Yellow) Pink-Red

Na2CO3

Alkaline pH

Urease

NH4

Alkaline pH

• Urease test

Biochemical Activities..……

Procedure.InterpretationPink Red color: Positive.

Yellow color (no change): Negative.

Motility Test:

Many bacteria are capable of movement.Most motile bacteria use structures called Flagella.

Flagella rotate in a counterclockwise direction, thus giving motility.

Biochemical Activities..……

Flagella Types:Monotrichous: single polar flagellum.

Lophotrichous (tufts): multiple polar or bipolar flagella.

Peritrichous: flagella distributed over the entire cell.

Procedure.

Interpretation:

Turbid media: Positive / Clear around stab line: Negative.

Triple Sugar Iron (TSI) Agar: Differentiates bacteria based on their ability to ferment glucose, lactose, and

sucrose to acid end products, +/- gas and to hydrogen sulfide (H2S).

Kligler Iron Agar (KIA): similar to TSI Agar but without sucrose.

• Motility test

Biochemical Activities..……TSI agar media contents:

0.1% Glucose.

1% Lactose.

1% Sucrose .

Phenol Red pH Indicator: Yellow (Acid), Red (Base).

Sodium Thiosulfate: for hydrogen sulfide production.

Ferric Ammonium Sulfate: as H2S indicator, which forms ferric sulfide

(FeS) Black Precipitate.Procedure.

Interpretation.

Biochemical Activities..……

A=Acidic (Yellow color) / K-Alkaline (Red color)

Slant / Butt

K/A: Only Glucose fermentation

A/A: Glucose & Lactose fermentation.

K/K or K/No Change: No G. or L. Fermentation

A/K: Will never happen.

Black Precipitate: H2S Production.

Cracking in media: Gas Production (CO2, H2).

• KIA AGAR

Example KIA IMViC Urease Motility

Escherichia coli A/A/G - - + +-+Kelbsiella pneumonia

A/A/G+ + - -+-Enterobacter

aerogenes A/A/G+ + - --+

Salmoella typhi K/A/G/H2S + - + --+Proteus volgarisK/A/G/H2S+ - + -++Shigella sonneiK/A- - + +--Pseudomonas

aeruginosaK/K+ - - --+

GOOD LUCK