General General MicrobiologyMicrobiologyLaboratoryLaboratory

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The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria.

TryptophanTryptophan, for example, is

hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase.tryptophanase.

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The pyruvic acid can be further metabolized to produce large produce large amounts of energyamounts of energy.

The ammonia is available for use in use in synthesis of new amino acidssynthesis of new amino acids.

Indole can be detected by reaction with Kovac's reagent (Para- Para- dimethylaminobenzaldehyde in dimethylaminobenzaldehyde in alcoholalcohol) to produce a red color.

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POSITIVE CONTROL: E.coli.

NEGATIVE CONTROL: Enterobacter aerogenes

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Trypton Broth 1%.Kovac’s Reagent.Test Tube.Culture from bacteria.

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1. Inoculate tryptone broth (1%) with the organism.

2. Incubate at 37 oC for 48 hours.3. Add 10 drops of Kovac's reagent.

A red color in the alcohol (upper) layer is a positive result.

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MR-VP medium/broth MR-VP medium/broth is two tests in one, thethe Methyl Red TestMethyl Red Test and the Voges-Proskauer the Voges-Proskauer testtest.

It is very useful in separating members of the

family EnterobacteriaceaeEnterobacteriaceae and some StreptococcusStreptococcus.

Most members of the EnterobacteriaceaeEnterobacteriaceae give either a positive MR a positive MR or a positive VP testa positive VP test.

Several species are positive for both, such as Proteus mirabilis.Proteus mirabilis.

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PrinciplePrinciple This is to detect the ability of an organism

to produce and maintain stable acid end products from glucose fermentation.

Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system.

Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less.

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IMViC testMethyl Red-Voges Proskauer (MR-VP) Tests Glucose

Acidic pathway

Mixed acids pH less than 4.4

Methyl Redindicator

Red color

Principle

MR positiveE. coli

Or Neutral pathway

Acety methyl carbinol(ACETOIN)

Barrit’s ABarrit’s B

Pink colorVP positiveKlebsiella

Positive Control: E.Coli Negative Control: Klebsiella pneumoniae

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1. The tubes of MR-VP broth were inoculated using sterile technique with given bacterial culture.

2. One tube was served as control.3. Incubate at 37 0 C for 24-48

hours.4. Test the presence of mixed acids

by the addition of methyl red indicator.

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Principle VP test detects butylene glycol

producers. Acetyl-methyl carbinol (acetoin) is

an intermediate in the production of butylene glycol.

In this test two reagents, 40% KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen.

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If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl.

Diacetyl then reacts with guanidine

components of peptone, in the presence of alpha naphthol to produce red color.

Role of alpha-naphthol is that of a catalyst and a color intensifier.

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Positive Control: Klebsiella pneumoniae

Negative Control: E.Coli

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1. The tubes of MR-VP broth were inoculated using sterile technique withgiven bacterial culture.

2. One tube was served as control. 3. Incubate at 37 0 C for 24-48 hours. 4. Test the presence of acetyl-methyl

carbinol by the addition of Barrit’s reagent.

5. Shacked well and allowed for 15 minutes. The presence of rose colouration is a positive result

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IMVC testIndole Methyl Red-Voges Proskauer (MR-VP) and Citrate Tests.

This Four test used to differentiation between of Enterobacteriacea member.

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Name test

I M V C

E. Coli + + - -

Enterobacter

- - + +

GOOD LUCK GIRLS