IMViC
the organism to split indole from the amino acid tryptophan. This division is performed by a
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The IMViC tests are a group of individual tests used in microbiology lab testing to identify an
organism in the coliform group. A coliform is a gram negative, aerobic or facultative aerobic
rod which produces gas from lactose within 48 hours. The presence of some coliforms indicate
fecal contamination.
Except for the lowercase “i”, which is added for ease of pronunciation, each of the letters in
“IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for
Voges-Proskauer, and “C” is for citrate.
 Indole test
In this test, the organism under consideration is grown in peptone Water Broth. It contains
tryptophan, which under the action of enzyme tryptophanase is converted to an Indole
molecule, pyruvate and carbon dioxide.The indole is then extracted from the broth by means
of xylene. To test the broth for indole production, Kovac's reagent is added. A positive result
is indicated by a Pink/Red layer forming on top of the liquid.
 Methyl Red test
 Voges–Proskauer test
These tests both use the same broth for bacterial growth. The broth is called MRVP broth.
After growth, the broth is separated into two different tubes, one for the Methyl Red (MR) test
and one for the Voges-Proskauer (VP) test. The pH indicator Methyl Red is added to one tube
and a red color appears at pH's lower than 4.2, and indicated positive test. The VP test uses
alpha-naphthol and potassium hydroxide to indicate a positive or negative test.
 Citrate Test
This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate
as its sole carbon source. The citrate agar is green before inoculation, and turns blue as a
positive test indicator.
These IMViC tests are useful for differentiating the family Enterobacteriaceae, especially
when used alongside the Urease test.
Indole test
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The indole test is a biochemical test performed on bacterial species to determine the ability of
chain of a number of different intracellular enzymes, a system generally referred to as
"tryptophanase."
Performing a Test
Like many biochemical tests on bacteria, results of an indole test are indicated by a change in
color following a reaction with an added reagent.
Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours
before performing the test. Following incubation, add 5 drops of Kovac's reagent (isoamyl
alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture broth.
A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl
alcohol, developed by Paul Ehrlich) is used when performing the test on nonfermenters and
anaerobes.
A positive result is shown by the presence of a red or red-violet color in the surface alcohol
layer of the broth. A negative result appears yellow. A variable result can also occur, showing
an orange color as a result. This is due to the presence of skatole, also known as methyl indole
or methylated indole, another possible product of tryptophan degradation.
Indole-Positive Bacteria
Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas
hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter sp., Edwardsiella sp.,
Escherichia coli, Flavobacterium sp., Haemophilus influenzae, most Proteus sp. (not P.
mirabilis), Plesiomonas shigelloides, Pasturella multocida, Pasturella pneumotropica,
Streptococcus faecalis, and Vibrio sp.
Indole-Negative Bacteria
Bacteria which give negative results for the indole test include: Actinobacillus spp.,
Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordtella sp., Enterobacter sp.,
Lactobasillus spp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasturella
haemolytica, Pasturella ureae, Proteus mirabilis, Pseudomonas sp., Salmonella sp., Serratia
sp., Yersinia sp.
Methyl red, also called C.I. Acid Red 2, is an indicator dye that turns red in acidic solutions.
It is an azo dye, and is a dark red crystalline powder.
Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2, and orange in
between, with a pKa of 5.1 [1].
Murexide and methyl red are investigated as promising enhancers of sonochemical destruction
of chlorinated hydrocarbon pollutants.[2]
Methyl red is classed by the IARC in group 3 - unclassified as to carcinogenic potential in
humans.
Also known as benzenediazonium 2-carboxylate.
Methyl red test
In microbiology, methyl red is used in the Methyl Red (MR) Test, used to identify bacteria
producing stable acids by mechanisms of mixed acid fermentation of glucose (cf. Voges–
Proskauer (VP) test).
The methyl red test is the "M" portion of the four IMViC tests used to characterize enteric
bacteria. The methyl red test is used to identify enteric bacteria based on their pattern of
glucose metabolism. All enterics initially produce pyruvic acid from glucose metabolism.
Some enteric subsequently use the mixed acid pathway to metabolize pyruvic acid to other
acids, such as lactic, acetic, and formic acids. These bacteria are called methyl-red positive
and include Escherichia coli and Proteus vulgaris. Other enterics subsequently use the
buytylene glycol pathway to metabolize pyruvic acid to neutral end-products. These bacteria
are called methyl-red-negative and include Serratia marcescens and Enterobacter aerogenes.
[2]
Process
An isolate is inoculated into a tube with a sterile transfer loop. The tube is incubated at 35°C
for 2-5 days. After incubation, 2.5ml of the medium is transferred to another tube. Five drops
of the pH indicator methyl red is added to this tube. The tube is gently rolled between the
palms of the hands to disperse the methyl red.[2]
Expected results
Enterics that subsequently metabolize pyruvic acid to other acids lower the pH of the medium
to 4.2. At this pH, methyl red turns red. A red color represents a positive test. Enterics that
subsequently metabolize pyruvic acid to neutral end-products lower the pH of the medium to
only 6.0. At this pH, methyl red is yellow. A yellow color represents a negative test
Voges–Proskauer test
Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. By adding
a concentrated solution of sodium hydroxide a red color appears.[1] [2]
History
The reaction was developed by Daniel Wilhelm Otto Voges and Bernhard Proskauer —
German bacteriologists in 1898 at the Institute for Infectious Diseases.
The citrate test detects the ability of an organism to use citrate as the sole source of carbon
and energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator
such as bromothymol blue. The medium also contains inorganic ammonium salts, which are
utilized as sole source of nitrogen. Use of citrate involves the enzyme citritase, which breaks
down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and
carbon dioxide (CO2). Production of sodium bicarbonate (Na2CO3) as well as ammonia (NH3)
from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a
change of the medium’s color from green to blue.
Bacterial colonies are picked up from a straight wire and inoculated into slope of Simmon’s
citrate agar and incubated overnight at 37 °C. If the organism has the ability to use citrate, the
medium changes its color from green to blue.
Examples:
 Escherichia coli: Negative
 Klebsiella pneumoniae: Positive
 Frateuria Aurantia: Positive
IMViC 
Enterobacteriaeae (enterics) are Gram-negative bacteria that grow in the intestinal tract of
humans and other animals. The IMViC tests are frequently employed for identification of this
group of microbes which includes such organisms as Klebsiella, Enterobacter, and
Escherichia coli. The presence of E. coli is used by public health officials as an indicator of
fecal contamination of food and water supplies. While Enterobacter and Klebsiella resemble
E.coli in being lactose fermenters, their presence does not necessarily indicate fecal
contamination because they are widespread in soil and grass. The IMViC tests can be used to
differentiate these three organisms.
IMViC is an acronym that stands for indole, methyl red, Voges-Proskauer, and citrate. To
obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole
test), methyl red - Voges Proskauer broth (MR-VP broth), and citrate.
The Indole Test
The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan.
Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that
cleaves tryptophan, producing indole and other products. When Kovac's reagent (p-
dimethylaminobenzaldehyde) is added to a broth with indole in it, a dark pink color develops.
The indole test must be read by 48 hours of incubation because the indole can be further
degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits
its growth.
The Methyl Red and Voges-Proskauer Tests
The methyl red (MR) and Voges-Proskauer (VP) tests are read from a single inoculated tube
of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes.
One tube is used for the MR test; the other is used for the VP test.
MR-VP media contains glucose and peptone. All enterics oxidize glucose for energy; however
the end products vary depending on bacterial enzymes. Both the MR and VP tests are used to
determine what end products result when the test organism degrades glucose. E. coli is one of
the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator
methyl red is added to this acidic broth it will be cherry red (a positive MR test).  

Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl alcohol,
acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The
bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. At this
pH, methyl red indicator is a yellow color (a negative MR test).

The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium
hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is
present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30
minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and
Klebsiella do.

The Citrate Test

The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing
citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH
indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and
gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate
agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads
to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are
citrate positive while E.coli is negative.

Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and Klebsiella give the
reverse: --++