Notes For Purpose Easy to Microbiology 3rd Sem B.Pharm

1. ❑ BIOCHEMICAL TEST (IMViC)
It is the series of biochemical tests including as fallow;
I = Production of indole from tryptophan.
M = Methyl red test production of acid from glucose.
Vi = Voges-Proskauer test production of acetoin from glucose.
C = usage of citrate as a carbon source.
Note: These all tests detect the motility of bacteria & disulfide production.
 Indole test:
Microorganisms can metabolize tryptophan by the tryptophanase. It based on the formation of
hydrolytic products from amino acid tryptophan.
Media: tryptophan used as substrate.
Reagent: kovac’s reagent.
Colour: Pink & red.
Tryptophan  Tryptophanase indole  Acid pyruvate  Kovac’s reagent
Example:
Proteus mirabilis can differentiate from Proteus vulgaris.
 METHYL RED TEST:
It is test for acid production. The bacteria are growth in MR-VP broth. Which organisms that are
maintained low pH, methyl red indicates for mixed acid fermentation.
Yellow colour indicating negative test for fermentation.
Media: MR-VP medium.
Note: this test is used to differentiate E. coli & E. aerogens.
Glucose + water Organic acids + CO2 + H2
Methyl red test
Red (+ve MR) Yellow (-ve MR)

2. Example:
E. aerogen
Glucose + H2O acetic acid 2,3 butanediol + methyl red + CO2 + H2O
Yellow color [negative test]
E. coli:
Glucose + H2O Lactic acid,
Acetic acid + methyl red CO2 + H2 + red colour [positive]
Formic acid
The test is based on the principle of conversion of glucose to
acetyl methyl carbinol.
After break down of glucose it reacts with alpha naphthol & potassium hydroxide to form red colour
known as acetoin.
Reagent: Barritt’s A & Barritt’s B are mainly alpha naphthol & potassium hydroxide respectively.
When these reagents react with acetyl methyl carbinol & change in pink colour.
Example:
E. coli does not produce red colour whereas Enterobacter & klebsiella give positive test.
Principle of MR-VP test:
Glucose
Acidic pathway Neutral pathway
Mixed acids pH less than 4 Acetyl methyl carbinol
Methyl red indicator Barritt’s A & Barritt’s B
Red color Ex: R. coli Pink color Ex: klebsiella
VOGES-PROSKAUER TEST:

3. This test used to determine the ability of an organism, using the citrate
enzyme to use citrate as its sole carbon source.
Principle=
Breakdown of ammonium salt gives ammonia & hydroxides. These hydroxyl ions raise the pH & that
changes the colour of bromophenol blue indicators from green to royal blue.
When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia which
increases alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to
blue above pH 7.6.
Reagent: bromothymol blue indicator.
Positive reaction: growth with colour change from green to intense blue along the slant.
Example: salmonella, edwardsiella, Citrobacter & serratia etc.
Negative reaction= no growth & no colour change, slant remains green.
Example: Escherichia, shigella & morganella etc.
CITRATE TEST: