IMViC Test- Principle, Result Chart, Examples,

Uses

IMViC test is a series of four different biochemical tests used in identifying

and differentiating bacteria, especially the members of Enterobacteriaceae.
Though it can be (and is) used for the identification of any type of bacteria,
it is mainly used for identifying Gram-negative bacteria. It is the key to
identifying and differentiating members of the Enterobacteriaceae family.

IMViC is an acronym for four different biochemical tests; each letter except
“I” represents an individual test making this series of biochemical tests.
IMViC series contains the following biochemical tests:
1. “I” = Indole Test
2. “M” = Methyl Red (MR) Test
3. “V” = Voges – Proskauer (VP) Test
4. “C” = Citrate Utilization Test (simply Citrate Test)
The letter “i” after ‘V’ is only for the rhyming purpose, it does not indicate
any test.
On making a minor modification like the use of Sulfide-Indole-Motility
(SIM) Agar medium, instead of 4, we can test 6 biochemical properties. The
use of SIM medium gives information on the ability to test bacteria’s
‘motility’ and ‘sulfide production’ (H2S production) ability. Hence, it is a
widely accepted series of biochemical tests.
IMViC is the most widely used primary biochemical test series. Though
considered key to selectively differentiating Enterobacterales, it is also used
for characterization and identification of several Gram-positive bacteria. It is
routinely used in clinical laboratories for teaching and research purposes.
All the tests in the series are easy to perform and give results within 24 – 48
hours. Hence, used for the primary screening purpose.

Objectives of IMViC Test

1. To study some biochemical properties – indole production, acid
production, acetylmethylcarbinol (acetoin) production, and citrate
utilization – of isolated unknown bacteria in order to characterize and
identify them.
2. To selectively differentiate and identify members of
the Enterobacteriaceae family.

Principle of IMViC Test

The IMViC test is based on the variations in the metabolic requirements and
properties of different genera and species of bacteria. The ‘indole test’ and
‘citrate utilization test’ in the series detect the ability of bacteria to produce
specific enzymes and utilize specific nutrients. On the other hand, the ‘MR
test’ and ‘VP test’ in the series detect the final metabolic products produced
by the bacteria utilizing specific nutrients. For this purpose, the test bacteria
is cultured in specific culture media; different media for the different tests
(expect MR and VP tests which need the same culture media; MR-VP
media).

Requirements for IMViC Test

The IMViC test is a series of different biochemical tests requiring different
culture media and reagents. Traditionally, broths were only used but now
different solid media are widely recommended for ease and ability to test
multiple properties.

Requirements for Indole Test:

Culture media:
 Tryptophan broth was traditionally used.
 Recently, Sulfide – Indole – Motility (SIM) medium is widely
recommended (because it gives the result of H 2S production and motility
also).
 Motility – Indole – Urea (MIU) medium is also preferred (because it gives
the result of urease production and motility also).
Reagents:
 Kovac’s Indole Reagent (a solution of 4- (dimethylamino)benzaldehyde
and hydrochloric acid in n-butanol or amyl alcohol) is mostly used. It is
preferred for aerobic organisms.
 Ehrlich’s Reagent (a solution of p-dimethylaminobenzaldehydre and
hydrochloric acid in ethyl alcohol) is preferred for anaerobic and weak
indole producing organisms.
 5% p-dimethylaminobenzaldehyde or
1% pdimethylaminocinnamaldehyde in 10% (v/v) concentrated HCl for
the spot indole test.

Requirements for MR Test:

Culture media:
 MR-VP broth
Reagents:
 Methyl red indicator

Requirements for VP Test:

Culture media:
 MR-VP broth
Reagents:
 Barritt’s A solution or VP reagent I (5% – α-naphthol solution)
 Barritt’s B solution or VP reagent II (40% KOH solution)
 Requirements for Citrate Utilization Test:
Culture media:
 Simmon’s Citrate Agar
Reagent:
 Bromothymol blue indicator (it is already incorporated in the Simmon’s
citrate medium)
 The simplified IMViC agar plate containing modified media containing all
four IMVic test media is also prepared, but it is not applied widely.

What is Indole Test?

The indole test is a biochemical test in the IMViC test series which detects
the ability of organisms (bacteria) to produce indole as a metabolic product
utilizing tryptophan. It is indicated by the letter “I” of the IMViC.
Principle of Indole Test
Some bacteria can produce an enzyme called ‘tryptophanase’ which helps
them to metabolize the amino acid ‘tryptophan’ into ‘indole, pyruvic acid,
and ammonia’.

When the indole reagent is added to a medium with a bacterial culture that
has produced indole, the indole combines with the aldehyde present in the
reagent to give a distinctive color.
If benzaldehyde is present in the reagent, a pink to a violet-red quinoidal
compound is formed, hence; a pink to red color ring is formed.
If cinnamaldehyde is present in the reagent, a blue to the green color
compound is formed, hence; a green to blue color ring is formed.
(Kovac’s indole reagent uses amyl alcohol and benzaldehyde. The amyl
alcohol is water-insoluble and forms an oily layer, thus giving a cherry-red
or pink – red ring on the top.)”
Test bacteria are cultured in the medium containing tryptophan for 24 – 48
hours and an indole reagent is added following the incubation to read the
result. A positive result is indicated by the formation of a pink to violet-red
or green to blue color ring according to the type of reagent used. A
negative color is indicated by the formation of a lack of color change or a
slight yellowish color ring at the top.

Indole Test
Indole Positive Bacteria: Escherichia coli, Klebsiella oxytoca, V. cholerae,
Proteus vulgaris, Porphyromonas asaccharolytica, Vibrio spp., Flavobacterium
spp., Providencia spp., Enterococcus faecalis, Haemophilus influenzae,
Morganella morganii, Aeromonas spp., Citrobacter koseri,
Indole Negative Bacteria: Klebsiella pneumoniae, Proteus mirabilis,
Salmonella spp., Shigella spp., Citrobacter freundii, Pseudomonas aeruginosa,
Bacteroides fragilis, Staphylococcus aureus,

What is Methyl Red (MR) Test?

Methyl Red (MR) Test is a biochemical test that detects the ability of
organisms (bacteria) to produce stable mixed acids as metabolic end
products of glucose metabolism. It is indicated by the letter “M” of the
IMViC.
Principle of MR Test
Some species of bacteria use the mixed acid fermentation pathway as their
glucose metabolism process. Following this metabolic pathway, they
convert pyruvate into stable mixed acids.
When such acid fermenters bacteria are grown in a medium containing
glucose (or carbohydrate), they will release the acids, hence; decreasing the
pH of the medium to 4.4 or lower. When methyl red indicator is added in a
medium containing such acid fermenters, it will turn the medium red.”
Following the 24-hour incubation on the MR-VP broth, a methyl red
indicator is added to the broth. A positive result is indicated by the
development of red color while a negative result is indicated by the
development of yellowish color.

MR Test.

MR Positive Bacteria: Escherichia coli, Salmonella spp., Shigella spp.,

Citrobacter spp., Proteus spp., Yersinia spp., Edwardsiella spp., Staphylococcus
aureus,
MR Negative Bacteria: Klebsiella pneumoniae, Enterobacter spp., Hafnia
spp., Serratia marcescens.
What is Voges-Proskauer (VP) Test?
Voges-Proskauer (VP) Test is a biochemical test in the IMViC test series
which detects the ability of organisms (bacteria) to metabolize the pyruvate
into a neutral intermediate product called ‘acetylmethylcarbinol’ or
‘acetoin’. It is indicated by the letter “V” of the IMViC.
Principle of VP Test
Pyruvate can be metabolized into a neutral intermediate product called
‘acetyl methyl carbinol’, commonly called the ‘acetoin’ during the
butanediol pathway of 2,3-butanediol production.

If acetoin is present in the media, it is oxidized readily to diacetyl in

presence of air and KOH. Thus produced diacetyl, in the presence of ∝ –
naphthol, will react with the guanidine component of peptone forming a
pink to a red colored product.
Following the 48-hour aerobic incubation on MR-VP broth, VP reagents I
and II are added and the color change is observed within 30 minutes. A
positive result is indicated by the development of pink – red color at the
top of the broth immediately or within 30 minutes but not more than 1
hour. No change in color represents a negative VP test.
 VP Test.

VP Positive Bacteria: Klebsiella spp., Enterobacter spp., Viridans Streptococci

(except S. mitis, and S. vestibularis), Proteus mirabilis, Hafnia spp., Serratia
spp., Staphylococcus aureus,
VP Negative Bacteria: Escherichia spp., Proteus vulgaris, Citrobacter
freundii,

What is Citrate Utilization Test?

Citrate Utilization Test is a biochemical test in the IMViC test series which
detects the ability of organisms (bacteria) to utilize citrate as a sole source
of energy. It is indicated by the letter “C” of the IMViC.
Principle of Citrate Utilization Test
Some bacteria can utilize ‘citrate’ as their sole source of carbon. Such
bacteria produce citrase enzymes which will break the citrate into
oxaloacetic acid and acetic acid. The oxaloacetic acid will then be
decarboxylated to produce pyruvate and CO 2.
Released CO2 will combine with H2O and excess sodium from sodium citrate
to produce alkaline ‘sodium carbonate’. The sodium carbonate will increase
the pH of the medium.
CO2 + H2O + excess sodium from sodium citrate → Na2CO3 (alkaline)
Additionally, the released CO2 will trigger the metabolism of ammonium
salts. Utilization of the ammonium salts as a source of nitrogen will cause
the production of ammonia (or ammonium hydroxide).
Ammonium salt → Ammonium hydroxide (alkaline)
The combined effect of ammonium hydroxide and sodium carbonate will
increase the pH of the media above 7.6. This increase in pH will turn the pH
indicator bromothymol blue in the medium from deep forest green (at
neutral pH) to Prussian blue.

Following the incubation of 24 – 48 hours (up to 4 days for some), bacterial

growth and color change in the slant portion is observed. A positive result
is indicated by growth and change in color of slant from green to intense
blue. A negative result is indicated by no change in the color of the slant.

Citrate Utilization Test

Citrate Positive Bacteria: Klebsiella spp., Citrobacter spp., Serratia
marcescens, Proteus mirabilis, Enterobacter spp., Salmonella spp. (except
Salmonella Typhi and Paratyphi A), Edwardsiella spp., Providencia spp.,
Citrate Variable Bacteria: Proteus vulgaris, V. cholera, V. parahaemolyticus.
Citrate Negative Bacteria: Escherichia coli, Shigella spp., Salmonella Typhi
and Paratyphi A, Yersinia spp., Morganella morganii, Staphylococcus aureus,
etc.

IMViC test result chart of some common bacteria

Name of Bacteria Indole Test MR Test VP Test Citrate Test

E. coli + ve + ve – ve – ve

Klebsiella pneumoniae – ve – ve + ve + ve

Klebsiella oxytoca + ve – ve + ve + ve

Pseudomonas aeruginosa – ve – ve – ve + ve

Salmonella Typhi – ve + ve – ve – ve

Salmonella Paratyphi A – ve + ve – ve – ve

Salmonella Typhimurium – ve + ve – ve + ve

Citrobacter freundii – ve + ve – ve + ve

Citrobacter koseri + ve + ve – ve + ve

Enterobacter cloacae – ve – ve + ve + ve

Enterobacter aerogenes – ve – ve + ve + ve

Shigella flexneri Variable + ve – ve – ve

Shigella dysenteriae Variable + ve – ve – ve

Proteus vulgaris + ve + ve – ve (11 – 25% +ve) rest – ve

Proteus mirabilis – ve + ve – ve + ve

Serratia marcescens – ve – ve + ve + ve

Morganella morganii + ve + ve – ve – ve

Yersinia enterocolitica (26 – 75% + ve) + ve – ve – ve

Yersinia pestis – ve + ve – ve – ve

Vibrio cholerae + ve – ve Variable Variable

Vibrio parahaemolyticus + ve – ve – ve – ve

Staphylococcus aureus – ve + ve + ve + ve

Staphylococcus epidermidis – ve – ve + ve – ve

Staphylococcus saprophyticus – ve – ve + ve – ve

Uses of IMViC Test

1. It is routinely used in clinical, research, and teaching laboratory to
characterize and identify unknown isolated bacteria up to the level of
genus.
2. Alongside the Urease test and TSI (Triple Sugar Iron) test, the test can
differentiate and identify the members of the Enterobacteriaceae family.
3. Some tests are used to differentiate species of a genus. E.g., the indole
test is used to differentiate K. oxytoca and K. pneumoniae, C.
koseri and C. freundii, P. vulgaris and P. mirabilis, etc.

Limitations of IMViC Test

1. It is not enough to completely identify the bacteria up to the level of
species. Additional tests are required, even for differentiating
Enterobacterales.
2. Different genera give the same results, hence results become
ambiguous.
3. It is culture-based test series and hence requires a longer period. Some
bacteria require more than 2 days of incubation to show VP reaction,
and for the citrate test, we may have to incubate for more than 4 days.
4. Only the culturable bacteria can be characterized.
5. The whole process is complex and requires good culture skills and
various resources to do the procedure.
6. False positive results and false negative results are encountered if the
incubation period is not appropriate and the reagents are old or not
used appropriately.