26/03/2024, 22:25 IMViC Test- Principle, Result Chart, Examples, Uses

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IMViC Test- Principle, Result Chart, Examples, Uses

July 10, 2022 by Prashant Dahal

Edited By: Sagar Aryal

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

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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.

Table of Contents
Objectives of IMViC Test
Principle of IMViC Test
Requirements for IMViC Test
Requirements for Indole Test:
Requirements for MR Test:
Requirements for VP Test:
Requirements for Citrate Utilization Test:
What is Indole Test?
Principle of Indole Test
What is Methyl Red (MR) Test?
Principle of MR Test
What is Voges-Proskauer (VP) Test?
Principle of VP Test
What is Citrate Utilization Test?
Principle of Citrate Utilization Test
IMViC test result chart of some common bacteria
Uses of IMViC Test
Limitations of IMViC Test
References

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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

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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 H2S 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)

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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.)”
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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,

Read More: Indole Test- Principle, Media, Procedure, Types, Results,

Uses

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

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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,

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MR Negative Bacteria: Klebsiella pneumoniae, Enterobacter spp., Hafnia spp., Serratia

marcescens.

Read More: Methyl Red (MR) Test- Principle, Procedure, Results, Uses

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.

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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,

Read More: Voges Proskauer (VP) Test- Principle, Procedure, Results,

Uses

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 CO2.

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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.

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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.

Read More: Citrate Utilization Test- Principle, Procedure, Results,

Uses

IMViC test result chart of some common bacteria

MR
Name of Bacteria Indole Test VP Test Citrate Test
Test

E. coli + ve + ve – ve – ve

Klebsiella pneumoniae – ve – ve + ve + ve

Klebsiella oxytoca + ve – ve + ve + ve

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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

(11 – 25% +ve) rest –

Proteus vulgaris + ve + ve – ve
ve

Proteus mirabilis – ve + ve – ve + ve

Serratia marcescens – ve – ve + ve + ve

Morganella morganii + ve + ve – ve – ve

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

Yersinia pestis – ve + ve – ve – ve

Vibrio cholerae + ve – ve Variable Variable

Vibrio parahaemolyticus + ve – ve – ve – ve

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Staphylococcus aureus – ve + ve + ve + ve

Staphylococcus epidermidis – ve – ve + ve – ve

Staphylococcus
– ve – ve + ve – ve
saprophyticus

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.

References

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1. Powers, E. M., & Latt, T. G. (1977). Simplified 48-hour IMVic test: an agar plate method.
Applied and environmental microbiology, 34(3), 274–279.
https://doi.org/10.1128/aem.34.3.274-279.1977
2. Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology
Procedures Handbook, 3.17.1.1–3.17.48.3. DOI:10.1128/9781555818814.ch3.17.1
3. MacWilliams, M. P. (2009, December 8). Indole Test Protocol.
https://www.asmscience.org/content/education/protocol/protocol.3202
4. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
5. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice
S. Weissfeld, 12th ed 2007, Publisher Elsevier.
6. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D.,
Dowell V.R. Jr, and Sommers H.M.
7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P.
Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans
Franscisco 1996.
8. Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2nd Edition
9. Hansen DS, Aucken HM, Abiola T, Podschun R. Recommended test panel for differentiation
of Klebsiella species on the basis of a trilateral interlaboratory evaluation of 18 biochemical
tests. J Clin Microbiol. 2004 Aug;42(8):3665-9. doi: 10.1128/JCM.42.8.3665-3669.2004.
PMID: 15297514; PMCID: PMC497635.
10. Jones JL, Lüdeke CH, Bowers JC, Garrett N, Fischer M, Parsons MB, Bopp CA, DePaola A.
Biochemical, serological, and virulence characterization of clinical and oyster Vibrio
parahaemolyticus isolates. J Clin Microbiol. 2012 Jul;50(7):2343-52. doi:
10.1128/JCM.00196-12. Epub 2012 Apr 25. PMID: 22535979; PMCID: PMC3405591.
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2130244/pdf/jhyg00067-0137.pdf
12. IMVIC Test | Microbiology | Biotechnology Methods | Botany Laboratory Experiments |
Biocyclopedia.com
13. IMViC Tests: Principle, Procedure, Results • Microbe Online
14. Biochemical Test (IMViC) In Microbiology – Solution Pharmacy
15. IMViC Tests : Principle, Procedure and results – Lab Tests Guide
16. 37: IMViC tests – Biology LibreTexts

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17. IMViC test: Introduction, Principle, Test Procedure and Result Interpretation
(universe84a.com)
18. IMViC tests: Principle, Procedure and Results, Notes & MCQ For Microbiology Exams –
Gpatindia: Pharmacy Jobs, Admissions, Scholarships, Conference,Grants, Exam Alerts
19. https://bioinfo.bisr.res.in/cgi-bin/project/docter/get_details.cgi?
query=all&&organism=proteus&&species=Proteus%20vulgaris

About Author

Prashant Dahal

Prashant Dahal completed his bachelor’s degree (B.Sc.) Microbiology from Sunsari Technical
College, affiliated with Tribhuvan University. He is interested in topics related to
Antimicrobial resistance, the mechanism of resistance development, Infectious diseases
(Pneumonia, tuberculosis, HIV, malaria, dengue), Host-pathogen interaction,
Actinomycetes, fungal metabolites, and phytochemicals as novel sources of antimicrobials
and Vaccines.

5 thoughts on “IMViC Test- Principle, Result Chart, Examples, Uses”

Akhilesh
December 12, 2023 at 7:40 PM

Thank you for this valuable effort, greatly benefiting students and ensuring
accessibility for all by making it 🆓 . Again thank you by heart

Reply

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Emília Sêneca
April 2, 2023 at 9:06 PM

Very useful! How do I reference it?

Reply

Sagar Aryal
April 3, 2023 at 8:01 AM

Thank you.
You can cite as below:
Dahal, P. (2022) IMVIC test- principle, result chart, examples, uses, Microbe Notes.
Available at: https://microbenotes.com/imvic-tests/ (Accessed: April 3, 2023).

Reply

Selestine
July 25, 2023 at 10:48 AM

The article is very oky ….

Reply

Gomathi
June 19, 2022 at 7:48 AM

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It’s so use full .. thank you

Reply

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