This document describes biochemical tests used to identify and classify microorganisms, specifically the IMViC tests. It explains that IMViC is an acronym for Indole, Methyl Red, Voges-Proskauer, and Citrate tests. These tests examine the enzymes and byproducts of microbes like E. coli and Enterobacter to determine if they produce certain compounds or change the pH or color of growth media. The document provides detailed procedures and expected results for each of the IMViC tests.

1. Biochemical Test
• Living micro-organism are differentiated on the basis of various
enzyme based catalyzed metabolized reaction.
• Presence or absence of certain enzyme metabolites or end
products give valuable information in identifying and classifying
m.o.
• IMViC stands for “I” is for indole; “M” is for methyl red; “V” is for
Voges-Proskauer, and “C” is for citrate, lowercase “i” is added for
the ease of pronunciation. IMViC is an acronym that stands
for four different tests
• Indole test
• Methyl red test
• Voges-Proskauer test
• Citrate utilization test
• IMViC tests are employed in the identification/differentiation of
members of family enterobacteriaceae.

2. • General procedure for performing IMViC Tests
and their interpretations:
• Cultures of any members of enterobacteriaceae
have to grow for 24 to 48 hours at 37°C and the
respective tests can be performed:

3. Methyl Red Test
For this test we require
Micro-organism- E.coli, Enterobacteraerogenes
Indicator- Methyl red
Media- Glucose phosphate broth media
Principle-
Some micro-organism utilise glucose for energy production
During these process they release acid which changes pH of
broth.
These change in pH is indicated by change in colour of broth due
presence of methyl red.

4. • Procedure-
• Prepare Glucose phosphate broth & sterlize it.
• Cool media & inoculate test micro-organism in test tube
• Incubate these test tube to grow for 24 to 48 hours at 37°C .
• After growth add the Methyl red indicator into it mix well &
observe the result.
Result
• Positive test – development of
red color
• negative test – no color change
We can identify specific m.o. by using its biochemical
characteristics.

5. Citrate Utilization Test
For this test we require
Micro-organism- E.coli, Enterobacteraerogenes
Indicator- Bromothymol blue
Media- Citrate broth media
Principle-
• These test differentiate m.o. which utilise citric acid as source
of carbon for growth.
• But test is depend on amount of enzyme.
• During utilization of citrate it release turbid substance which
make solution alkaline.
• pH change determine by addition of BTB (Bromothymol blue)

6. Procedure-
• Prepare citrate broth & sterlize it.
• Cool media & inoculate test micro-organism in test tube
• Incubate these test tube to grow for 24 to 48 hours at 37°C .
• After growth add the Bromothymol blue indicator into it mix well &
observe the result.
Result
Positive test – development of
blue color
negative test – no color change
We can identify specific m.o. by using its biochemical characteristics.

7. Indole Production Test
For this test we require
Micro-organism- E.coli, Enterobacteraerogenes
Indicator- Kovac’s Reagent
Media- Peptone broth media
Principle-
• Some micro-organism consist of enzyme tryptophanase.
• This enzyme degrade amino acid tryptophan into indole,
pyruvic acid and ammonia.
• Indole production is detected by inoculating test m.o. in
peptone water.

8. Procedure-
• Prepare peptone broth & sterlize it.
• Cool media & inoculate test micro-organism in test tube
• Incubate these test tube to grow for 24 to 48 hours at 37°C .
• After growth add the Kovacs reagent into it mix well & observe the
result.
Result
Positive test – development of
red color in alcohol layer
(Idole Produced)
negative test – no color change
We can identify specific m.o. by using its biochemical characteristics.

9. Voges-Proskauer (VP) test:
For this test we require
Micro-organism- E.coli, Enterobacteraerogenes
Indicator- 1% NaOH + 5% ά- Napthol
Media- Glucose Phosphate broth media
Principle-
• Some micro-organism ferment m.o. carbohydrate with
production of acetyl methyl carbonyl (Acetoin).
• In presence of alkali and atmospheric oxygen , small amount of
acetoin present into the media oxidised to diacetyl .
• These diacetyl react with peptone of broth to give red colour.

10. Procedure-
• Prepare glucose phosphate broth & sterlize it.
• Cool media & inoculate test micro-organism in test tube
• Incubate these test tube to grow for 24 to 48 hours at 37°C .
• After growth add 1 ml 1% NaOH + 3 ml 5% ά- Napthol into it mix
well & observe the result.
Result
Positive test – development of
pink color
negative test – no color change
We can identify specific m.o. by using its biochemical characteristics.

11. • Thank You