The document discusses biochemical tests called IMViC tests that are used to identify bacteria, especially in the coliform group. The IMViC tests include Indole, Methyl Red, Voges-Proskauer, and Citrate tests. These tests help distinguish members of the Enterobacteriaceae family based on how they metabolize different substrates. The document provides details on how each test is performed and interpreted. It gives examples of reactions for common bacteria like E. coli, Salmonella, Klebsiella, and Proteus species. While most aerobic bacteria are citrate positive, E. coli is citrate negative despite being an aerobic bacterium.

1. Food Micro BioL9-10
Biochemical Tests IMViC

2. IMViC Test
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an
organism in the coliform group or different other food borne pathogens groups . 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 term "IMViC" is an acronym for each of these tests. "I" is for indole test; "M" is for methyl red
test; "V" is for Voges-Proskauer test, and "C" is for citrate test. The lower case "i" is merely for "in"
as the Citrate test requires coliform samples to be placed "in Citrate". Triple sugar iron test
(TSI), Urease Test and Malonate Test are also included in this test.
These tests are useful in distinguishing members of Enterobacteriaceae.

3. Peptone water broth rich in
tryptophan (Growth media for
microbes)
Microbes
Convert tryptophan into
indole by fermentation
Indole is extracted
by xylene (Solvent
extraction)
Kovac's reagent added : it
consist of amyl alcohol and para-
dimethylaminobenzaldehyde and
concentrated hydrochloric acid.
Cherry red coloured layer is
formed if indole is produced.
Because amyl alcohol is
not water soluble, the red
coloration will form in
an oily layer at the top of
the broth in xylene.
Inference – The microorganism
produces tryptophanase
Indole Test

4. MRVP Test
("MR" is
for methyl red
test; "V" is
for Voges-
Proskauer test)
Microbes grown in
Cultured in a
broth
containing
Pyruvate or
Glucose as
sole carbon
source
Microbes produces Mixed
acids like Lactic acid, fumaric
acid, Acetic acid, succinic acid
and CO2 if it follows krebs
cycle aerobic pathway.
After 24 hrs of growth
divide the broth into two
test tubes
Add methyl red to the first
half
Keep the other half broth in
incubation for another 24 hrs to
make it ready for VP test in
anaerobic condition ***
If methyl red positive no
need to perform VP test
with the rest half of the
broth
If methyl red
positive
immediately
gives red colour
Inference: Aerobic bacteria :
Produces enzymes of
aerobic pathways if positive

5. Principle of Voges–Proskauer (VP) Test (Barrit’s reagents)
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl carbinol or acetoin
(by 2,3-butanediol fermentation pathway) which is an intermediate of the pathway from glucose
pyruvate fermentation. If present, acetylmethyl carbinol is converted to diacetyl in the presence of ∝-
naphthol in ethanol, 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 diacetyl
and quanidine-containing compounds found in the peptones of the broth then condense to form a pinkish
red polymer.
2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+
Take the rest half
of the broth after
48 hrs incubation
(in anaerobic
condition)
Add VP reagent ∝-naphthol in alcohol
and strong alkali (40% KOH), mix well
and aerate
Observe for a pink-red color
at the surface within 30 min.
Shake the tube vigorously
during the 30-min period.
Anaerobic bacteria alcohol
fermenters / Facultatives

7. Citrate Utilzation test in Simmons Citrate agar
Citrate agar is used to test an organism’s ability to utilize citrate as a
source of energy. The medium contains citrate as the sole carbon
source and inorganic ammonium salts (NH4H2PO4) as the sole
source of nitrogen.
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.
Inference : Aerobes /
Facultative aerobes
and producers of
citrate permease

8. Species Indole Methyl Red Voges-Proskauer Citrate
Escherichia coli Positive Positive Negative Negative
Shigella spp. [2] Negative Positive Negative Negative
Salmonella spp. Negative Positive Negative Positive
Klebsiella spp. Negative Negative Positive Positive
Proteus vulgaris [3] Positive Positive Negative Negative
Proteus mirabilis Negative Positive Negative Positive
Citrobacter freundii Negative Positive Negative Positive
Enterobacter aerogenes Negative Negative Positive Positive

9. Why E. coli is citrate negative although it falls in the
group of aerobes / Facultative aerobes ?