Experiment Name: Indole Test

Learning objective:

This test is used to determine the ability of microbes to degrade amino acid tryptophan.

Principle:

Certain microorganisms can metabolize tryptophan by tryptophanase enzyme. This enzymatic

degradation leads to the formation of indole, pyruvic acid and ammonia. The presence of indole
is detected by addition of kovac's reagent .

Tryptophanase

Tryptophan Indole + Pyruvic acid +NH3

HCL, alcohol

P-dimethylaminobenzaldehyde + Indole Quinoidal red-violet

Dehydration reaction Compound

Requirement:

SIM ( sulfide indole motility ) agar, bunsen burner, inoculating needle, test tube,test tube rack
and permanent glass marker, incubator

Reagent:

Kovac's reagent, for detection of indole :

P-dimethylaminobenzaldehyde : 5.0 gm

Amyl alcohol : 75.0 ml

Hydrochloric acid (concentrated) : 25.0 ml

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Procedure:

1. At first take a sterilized test tube containing 4 ml of tryptophan broth

2. Inoculate the tube aseptically by taking the growth from 18 to 24 hours pure culture

3. Incubate the tube for 24 to 48 hours at 37º c

4. After proper incubation, add 10 drops of kovac's reagent to the tube touching the wall of glass
tube

5. Observe for the presence or absence of ring

Result Interpretation of Indole Test:

Fig: Indole Test

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Positive: Formation of a pink to red color (cherry-red ring) in the reagent layer on top of the
medium within seconds of adding the reagent.

Example: E.coli , Proteus vulgaris

Negative: No color change even after the addition of appropriate reagent.

Example: Salmonella spp., Klebsiella spp.

Experiment Name: Catalase Test

Learning objective:

To determine the ability of an organism to produce catalase.

Principle:

Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water. An
organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
Bubbles of oxygen are released if the organisms is a catalase production.

Catalase

H2O2 H2O + O2

Hydrogen peroxide Water Oxygen gas

Requirement:

Bunsen burner, Wooden stick, / glass rod, 3% hydrogen peroxide, Glass slide, Permanent glass
marker, sample (organism).

Procedure:

1. At first take a clean grease free glass slide

2. Place a one drop of 3% hydrogen peroxide onto the glass slide

3.By using sterilized glass rod/ wooden stick take organisms and smeared with hydrogen
peroxide reagent

4. Observe for the evolution of oxygen bubbles

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Result Interpretation of Catalase Test:

Fig: Catalase Test

Positive: Presence of catalase producing bubbles

Example: Listeria monocytogenes, Vibrio cholera

Negative: Absence of catalase no-producing bubbles

Example: Clostridium sp. Streptococcus sp.

Experiment Name: Oxidase Test

Learning objective:

To determine the ability of microbes to produce Oxidase enzyme.

Principle:

A piece of filter paper is soaked with a few drops of oxidase reagent. A colony of the test
organisms is then smeared on the filter paper. When the organism is oxidase produce in the p—
phenyl diamine in the reagent will be oxidized to a deep purple color.

Requirement:

Bunsen burner, Filter paper, Sterilized platinum loop, petridish, sample (organism)

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Reagent:

Oxidase reagent (Tetramethyl p-phenyle diamine)

Procedure:

1. At first take a clean petridish

2. Place a piece of filter paper onto the petridish

3. Place one drop of oxidase reagent onto the filter paper

4. By using sterilized platinum loop take colony of the organisms and smeared with oxidase
reagent gently

5. Then wait 20-30 seconds for result

Result Interpretation of Oxidase Test:

Fig: Oxidase Test

Positive: Blue purple color

Example: Pseudomonas spp. Neisseria spp.

Negative: No color change

Example: Acenitobacter spp. Enterobacteriaceae