IMViC Tests

Contents
• Introductuon of IMViC tests
• Purpose of IMViC tests
• Principle of IMViC tests
• Requirements
• Reagents and thir compostion
• Procedure of IMViC tests
• Interpretation
IMViC tests:
• IMViC tests, a set of four useful reactions
that are commonly designed for the
differentiation of enterics (members of family
Enterobacteriaceae). used in microbiology
lab testing to identify an organism in the
coliform group. A coliform is a gram negative
, aerobic or facultative anaerobic rod which
produces gas from lactose within 48 hours.
The presence of some coliforms indicates
fecal contamination.
The IMViC series includes
following four tests
• Indole test
• Methyl red test
• Voges-Proskauer test
• Citrate test
Indole Test:

• Indole test is the a biochemical test

performed on bacterial species to
detect the ability of an organism to
degrade the amino acid tryptophan to
produce three possible end products
– indole , pyruvate and ammonium.
Tryptophan is hydrolyzed by
tryptophanase enzyme.
Purpose:

• The indole test screens for the

ability of an organism to degrade
the amino acid tryptophan and
produce indole.
Principle of Indole Test:

• Tryptophanase catalyzes the

deamination reaction, during which the
amine (-NH2) group of the tryptophan
molecule is removed. Final products of the
reaction are indole, pyruvic acid,
ammonium (NH4+) and energy.
• When indole is combined with Kovac’s
Reagent (which contains hydrochloric
acid and p-dimethylaminobenzaldehyde in
amyl alcohol) the solution turns from
yellow to cherry red.
Figure: 1.1
Requirements:

• Peptone broth ( a nutrient enriched

with Amino acid tryptophan)
• Bacterial sample/culture
• Kovac’s reagent
Tryptophan broth composition:

• Tryptophan 1gram
• Sodium chloride 0.5gram
Kovac’s reagent:
• P-dimethylaminobenzaldehyde (DMAB)
• Hydrochloric Acid, 37%
• Amyl Alcohol
Procedure of Indole Test
• Take sterilized test tubes containing 4 ml
of tryptophan broth.
• Inoculate the tube aseptically by taking the
growth from 18 to 24 hrs culture.
• Incubate the tube at 37°C for 24-28 hours.
• Add 0.5 ml of Kovac’s reagent to the broth
culture.
• Observe for the presence or absence of
ring.
Interpretation:
• 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.
• Examples: Escherichia coli
• Negative: No color change even after the
addition of appropriate reagent.
• Examples: Actinobacillus spp Klebsiella
pneumonia
Methyl Red (MR) Test
• Methyl Red (MR) test is a
biochemical test performed on
bacterial species to detect the ability
of an organism to produce stable
acids end products (Mixed-acid
fermentation) from supplied
glucose
Principle of Methyl Red (MR) Test

• When some types of bacteria, such as

E-coli, come in contact with glucose,
the bacteria use the glucose as an
energy source. The process of
breaking down glucose for energy will
ferment the glucose and form acetic
acid, lactic acid, and succinic acid.
Continue
• The Methyl Red test involves adding the pH
indicator methyl red to an inoculated tube of
MR-VP broth. If the organism uses the mixed
acid fermentation pathway and produces
stable acidic end-products, the acids will
overcome the buffers in the medium and
produce an acidic environment in the
medium. When methyl red is added, if acidic
so produced decreases the pH to 4.5 or below,
end products are present, the methyl red will
stay red.
Figure: 1.2
Requirements:
• Glucose broth/Clark broth
• Peptone= 7.0gm
Glucose=5.0gm
Dipotassium phosphate= 5.0 gms
• Methyl red solution, 0.02%
a. Dissolve 0.1 g of methyl red in 300 ml of ethyl
alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
• The pH indicator methyl red (p-
dimethylaminoaeobenzene-O-carboxylic acid)
Procedure for Methyl Red (MR) Test
• Take sterilized test tubes containing 4 ml of
glucose broth.
• Inoculate the tube aseptically by taking the
growth from 18 to 24 hrs culture.
• Incubate the tube at 37°C for 24-28 hours.
• Add about 5 drops of the methyl red
indicator solution
• A positive reaction is indicated, if the colour
of the medium changes to red within a few
minutes.
Interpretation of Methyl Red (MR) test

• Positive Reaction: A distinct red color

Examples: E. coli, Yersinia sps, etc.
• Negative Reaction: A yellow color
Examples: Enterobacter aerogenes,
Klebsiella pneumoniae, etc.
Voges–Proskauer (VP) Test

• voges-Proskauer test is one of the tests used

for identification of Enterobacteriaceae. It is
usually performed alongside the methyl red
test since both tests are performed on
cultures grown in MR-VP broth.  Both tests
are based on the detection of end products
from the metabolism of glucose The test
depends on the digestion of glucose to
acetylmethylcarbinol.
Principle of Voges–Proskauer (VP) Test

• The principle of Voges–Proskauer Test is to check for

microorganism’s ability to produce acetylmethyl carbinol
from the fermentation of glucose. If present,
acetylmethyl carbinol is converted to diacetyl in the
presence of ∝- naphthol, strong alkali (40%
KOH), and atmospheric oxygen. The ∝-naphthol was
not part of the original procedure but was found to act as
a color intensifier by Barritt and must be added first. The
second reagent, potassium hydroxide, absorbs carbon
dioxide present in the medium and acts as an oxidizing
agent thereby hastening the critical reaction that
converts acetoin to diacetyl. The diacetyl and
quanidine-containing compounds found in the
peptones of the broth then condense to form a pinkish
red polymer.
Figure: 1.3
Requirements:
• Culture:
•  24-48 hour broth culture.
•  Media:
•  MR-VP medium/glucose/ Clark broth
• Peptone= 7.0gm
Glucose=5.0gm
Dipotassium phosphate= 5.0 gm
• Reagents:
•  Barrett’s reagents A and B.
• Preparation of Barritt's reagent:
•  It consists of two solutions;
▫ Alpha-naphthol, 5% color intensifier
 Alpha Naphthol-5g
 Absolute ethyl alcohol- 100 mL
▫ Potassium Hydrooxide, 40%, oxidizing agent
 Potassium hydroxide 40g
 Distilled water to: 100 mL
 Procedure of Voges–Proskauer (VP) Test

• Take sterilized test tubes containing 4 ml of glucose

broth.
• Inoculate the tube aseptically by taking the growth from
18 to 24 hrs culture.
• Incubate the tube at 37°C for 24-28 hours.
• Add 6 drops of 5% alpha-naphthol, and mix well to
aerate.
• Add 2 drops of 40% potassium hydroxide, and mix well
to aerate.
• Observe for a pink-red color at the surface within 30 min.
Shake the tube vigorously during the 30-min period.
Interpretation of Voges–Proskauer (VP)
Test
•  Glucose ------Glucose Metabolism-------> Pyruvic Acid.
• Pyruvic acid ---------------> Acetoin.
• Acetoin + added alpha-naphthol + added KOH = red color.
• Examples Listeria, Enterobacter, Klebsiella
•  Negative Result:
•  Glucose ------Glucose Metabolism-------> Pyruvic Acid.
• Pyruvic acid -----------------> No Acetoin.
• No acetoin + added alpha-naphthol + added KOH = copper
color.
• Examples: Streptococcus mitis, Citrobacter sp., Shigella,
Yersinia,
Citrate Utilization Test

• A citrate utilization test helps detect

the organism’s ability to produce
citrase enzyme. Such organisms can
use sodium citrate as a source of
carbon as well as inorganic
ammonium salt as the source of
nitrogen.
Principle of Citrate Utilization Test
• Bacteria that can grow on this medium produce an
enzyme, citrate-permease, capable of
converting citrate to pyruvate. Pyruvate can
then enter the organism’s metabolic cycle for
the production of energy. Growth is indicative of
utilization of citrate, an intermediate metabolite in
the Krebs cycle.
• When the bacteria metabolize citrate, the
ammonium salts are broken down to ammonia,
which increases alkalinity. The shift in pH turns
the bromthymol blue indicator in the medium
from green to blue above pH 7.6.
Figure: 1.4
Requirements

• Culture:
•  24-48 hour broth culture
• Composition of simmon citrate agar
• Sodium chloride and citrate
• Dipotassium phosphate
• Ammonium dihydrogen phosphate
• Agar
• Magnesium sulphate
• Bromothymol blue – It serves as a pH indicator. At
neutral pH, the color is green. It turns blue if the pH
becomes alkaline and yellow if the pH becomes acidic.
• Deionized water
Procedure of Citrate Utilization Test

• Streak the slant back and forth with a light

inoculum picked from the center of a well-
isolated colony.
• Incubate aerobically at 35 to 37C for up to
4-7 days.
• Observe a color change from green to blue
along the slant.
Interpretation
• Positive Reaction: Growth with color change
from green to intense blue along the slant.
Examples:Salmonella,, Citrobacter, Klebsiella,
Enterobacter,,, etc.
• Negative Reaction: No growth and No color
change; Slant remains green.
Examples: Escherichia, Shigella,, Yersinia etc.