Basic Microbiology laboratory for Dental Students By Lecturer Yasemin 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 Procedure. Interpretation:

Pink-red ring: Positive / Yellow ring: Negative

---------

Red Color

 Indole test

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

. Methyl Red (MR) Test

metabolize glucose

Determines the ability of an organism to produce acids as final products from glucose fermentation. Fermentation
fermentation

Glucose

Lactic Acid, Acetic Acid, Formic Acid

MR indicator + Acidic pH

Red Color

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.
fermentation

Glucose

Acetoin

40% KOH

Acetoin + -Naphthol
Oxidation

Diacetyl

 VP TEST

Biochemical Activities..
-Naphthol Diacetyl + Peptone (from medium Pink Red

Procedure. Interpretation
Pink Red color: Positive / Yellow color: Negative

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

Citrate

Citrate Utilization

Organic Acids + CO2

CO2 + Na (from conjugate base salt)

Na2CO3

 Citrate test

Biochemical Activities..
Bromothymol Blue Indicator (Green) Na2CO3 Alkaline pH Blue

Procedure
Interpretation

Pink Red color: Positive / Yellow color: Negative

Urease Test:
Determines the presence of Urease enzyme. Urea Urease NH4 (Ammonia) + CO2 NH4 Phenol Red Indicator (Yellow) Alkaline pH Pink-Red

 Urease test

Biochemical Activities..
Procedure.

Interpretation
Pink 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 Escherichia coli Kelbsiella pneumonia

KIA A/A/G A/A/G

IMViC

Urease

Motility

++---++

+ -

Enterobacter aerogenes
Salmoella typhi

A/A/G
K/A/G/H2S

--++
-+-+

+
+

Proteus volgaris
Shigella sonnei Pseudomonas aeruginosa

K/A/G/H2S
K/A K/K

-+-+
++----+

+
-

+
+

GOOD LUCK