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 --------Red Color

Kovac's reagent (Yellow( + Indole .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( Test

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 pHRed 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

Acetoin + -Naphthol

KOH 40% Oxidation

Diacetyl

 VP TEST

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

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 .Glucose 0.1% .Lactose 1% . Sucrose 1% ).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 Enterobacter aerogenes Salmoella typhi Proteus volgaris Shigella sonnei Pseudomonas aeruginosa

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

IMViC

Urease

Motility

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+ + -

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GOOD LUCK