IMViC Test- Principle, Result Chart, Examples, Uses

IMViC test is an acronym for four different biochemical tests that are used to identify and differentiate members of the Enterobacteriaceae family and other related bacteria. The four tests are:

• Indole test: This test detects the ability of bacteria to produce indole from tryptophan, an amino acid. Indole is a chemical compound that has a characteristic smell and can be detected by adding a reagent such as Kovac`s or Ehrlich`s.
• Methyl red (MR) test: This test detects the ability of bacteria to produce stable mixed acids from glucose fermentation. The pH of the medium is measured by adding methyl red indicator, which turns red at acidic pH and yellow at neutral or alkaline pH.
• Voges-Proskauer (VP) test: This test detects the ability of bacteria to produce acetylmethylcarbinol (acetoin) from glucose fermentation. Acetoin is a neutral intermediate product that can be oxidized to diacetyl, which reacts with alpha-naphthol and potassium hydroxide to form a pink-red complex.
• Citrate utilization test: This test detects the ability of bacteria to use citrate as a sole source of carbon and energy. The medium contains sodium citrate and bromothymol blue indicator, which changes color from green to blue when the pH increases due to citrate utilization.

The IMViC tests are performed on isolated pure cultures of bacteria that are grown on specific media. The results are interpreted by observing the color changes or the formation of rings or precipitates in the media or tubes. The results are usually reported as positive (+) or negative (-) for each test. For example, Escherichia coli is IMViC + + - -, meaning that it is positive for indole and methyl red tests, and negative for Voges-Proskauer and citrate utilization tests.

The IMViC tests are useful for characterizing and identifying bacteria up to the level of genus, and sometimes species. They are also helpful for distinguishing between closely related bacteria that have similar morphology and gram staining characteristics. For example, the indole test can differentiate between Klebsiella pneumoniae (-) and Klebsiella oxytoca (+), while the citrate utilization test can differentiate between Salmonella typhi (-) and Salmonella paratyphi A (+).

The IMViC tests are simple, inexpensive, and reliable methods for bacterial identification. However, they have some limitations, such as requiring pure cultures, long incubation periods, and additional tests for confirmation. Therefore, they are often used in combination with other biochemical, serological, or molecular methods for more accurate and comprehensive identification of bacteria.

The IMViC test is a set of four biochemical tests that are commonly used to identify members of the Enterobacteriaceae family. The name IMViC is an acronym for the four tests: Indole, Methyl Red (MR), Voges-Proskauer (VP), and Citrate. Each test detects a different metabolic property or product of the bacteria.

The main objectives of the IMViC test are:

• 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.
• To selectively differentiate and identify members of the Enterobacteriaceae family, which includes many medically important genera such as Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter, Proteus, Citrobacter, and Yersinia.
• To differentiate species within a genus based on their IMViC test results. For example, the indole test can distinguish between Klebsiella pneumoniae (indole negative) and Klebsiella oxytoca (indole positive), or between Proteus vulgaris (indole positive) and Proteus mirabilis (indole negative).

The IMViC test is a useful tool for bacterial identification because it is simple, inexpensive, and reliable. However, it has some limitations such as requiring pure cultures, long incubation periods, and additional tests for confirmation. Therefore, it is usually combined with other tests such as Urease test and Triple Sugar Iron (TSI) test for more accurate identification of Enterobacteriaceae.

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 .

• The indole test detects the ability of bacteria to produce indole from tryptophan by the enzyme tryptophanase. Indole reacts with a reagent (such as Kovac`s or Ehrlich`s) to form a colored compound that indicates a positive result .
• The MR test detects the ability of bacteria to produce stable mixed acids from glucose fermentation. The pH indicator methyl red turns red when the pH of the medium drops below 4.4 due to acid production .
• The VP test detects the ability of bacteria to produce acetylmethylcarbinol (acetoin) from glucose fermentation by the butanediol pathway. Acetoin is oxidized to diacetyl by KOH and reacts with alpha-naphthol to form a pink-red compound that indicates a positive result .
• The citrate utilization test detects the ability of bacteria to use citrate as a sole carbon source. Bacteria that can use citrate produce alkaline products that raise the pH of the medium and change the color of the pH indicator bromothymol blue from green to blue .

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 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) 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.

The indole test is a biochemical test in the IMViC test series that detects the ability of bacteria to produce indole as a metabolic product from tryptophan. It is indicated by the letter "I" of the IMViC.

Principle of Indole Test

Some bacteria can produce an enzyme called tryptophanase that helps them to break down the amino acid tryptophan into indole, pyruvic acid, and ammonia. When an indole reagent is added to a medium with a bacterial culture that has produced indole, the indole reacts with the aldehyde present in the reagent to form a colored compound. Depending on the type of reagent used, the color can be pink to violet-red or green to blue.

Procedure of Indole Test

There are different types of media and reagents that can be used for the indole test. Some of the commonly used ones are:

• Tryptophan broth: This is a simple broth containing tryptophan as the only source of nitrogen. After inoculating the bacteria and incubating for 24-48 hours, Kovac`s or Ehrlich`s reagent is added to detect indole production.
• SIM (Sulfide-Indole-Motility) medium: This is a semi-solid agar medium that can also test for hydrogen sulfide production and motility of bacteria. After inoculating the bacteria with a needle and incubating for 24-48 hours, Kovac`s or Ehrlich`s reagent is added to the surface of the medium to detect indole production.
• MIU (Motility-Indole-Urea) medium: This is another semi-solid agar medium that can also test for urease production and motility of bacteria. After inoculating the bacteria with a needle and incubating for 24-48 hours, Kovac`s or Ehrlich`s reagent is added to the surface of the medium to detect indole production.

Interpretation of Indole Test

The result of the indole test is indicated by the color change of the reagent after adding it to the medium. A positive result means that the bacteria can produce indole from tryptophan, while a negative result means that they cannot.

• Kovac`s reagent: A positive result is indicated by a pink to violet-red ring at the top of the medium or broth. A negative result is indicated by no color change or a slight yellowish ring.
• Ehrlich`s reagent: A positive result is indicated by a green to blue ring at the top of the medium or broth. A negative result is indicated by no color change or a slight yellowish ring.

Examples of Indole Test

Some examples of bacteria that are indole positive are Escherichia coli, Klebsiella oxytoca, Proteus vulgaris, Vibrio cholerae, and Enterococcus faecalis. Some examples of bacteria that are indole negative are Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhi, Shigella dysenteriae, and Pseudomonas aeruginosa.

Uses of Indole Test

The indole test is useful for differentiating and identifying bacteria based on their ability to produce indole from tryptophan. It can help to distinguish between different species within a genus, such as Klebsiella, Proteus, Citrobacter, and Vibrio. It can also help to identify some members of the Enterobacteriaceae family, which are mostly gram-negative rods that ferment glucose and are facultative anaerobes.

Limitations of Indole Test

The indole test has some limitations that should be considered when interpreting the results. Some of them are:

• The test may give false-negative results if the incubation time is too short or if there is insufficient tryptophan in the medium.
• The test may give false-positive results if there is contamination by other indole-producing bacteria or if there is degradation of tryptophan by other enzymes.
• The test may not be reliable for some anaerobic or weakly indole-producing bacteria, which may require Ehrlich`s reagent instead of Kovac`s reagent.
• The test may not be specific enough to identify some bacteria that have similar indole reactions, such as Escherichia coli and Enterobacter aerogenes.

The methyl red (MR) test is a biochemical test that detects the ability of bacteria to produce stable mixed acids as metabolic end products of glucose fermentation. It is indicated by the letter "M" of the IMViC.

Principle of MR Test

Some bacteria use the mixed acid fermentation pathway to metabolize glucose. In this pathway, they convert pyruvate into various acids, such as lactic, acetic, and succinic acids, lowering the pH of the medium below 4.4. The pH change can be detected by adding a pH indicator, methyl red, which is red at pH ≤ 4.4 and yellow at pH ≥ 5.8 .

Requirements for MR Test

The medium used for the MR test is the same as the one used for the VP test: MR-VP broth. It contains peptone, glucose, and dipotassium phosphate . The reagent required for the MR test is methyl red indicator, which is a solution of methyl red in ethanol and water.

Procedure for MR Test

• Inoculate two tubes of MR-VP broth with a pure culture of the test organism.
• Incubate at 35°C for at least 48 hours .
• Transfer 1 ml of broth from one tube to a clean test tube and add 2-3 drops of methyl red indicator .
• Observe the color change immediately or within 15 minutes.

Results and Interpretation of MR Test

• A positive result is indicated by the development of a red color in the broth, which means that the organism produces stable mixed acids from glucose fermentation and lowers the pH below 4.4 .
• A negative result is indicated by the development of a yellow color in the broth, which means that the organism does not produce stable mixed acids from glucose fermentation or produces less acid that does not lower the pH below 4.4 .
• A weak positive result is indicated by the development of a red-orange color in the broth.

Examples of MR Test

Some examples of bacteria that give positive MR test are Escherichia coli, Salmonella spp., Shigella spp., Proteus spp., Yersinia spp., and Staphylococcus aureus .

Some examples of bacteria that give negative MR test are Klebsiella pneumoniae, Enterobacter spp., Serratia marcescens, Hafnia spp., and Viridans streptococci .

Uses and Limitations of MR Test

The MR test is used to differentiate and identify members of the Enterobacteriaceae family based on their pattern of glucose metabolism. It can also help to distinguish some species within a genus, such as Proteus vulgaris (MR positive) and Proteus mirabilis (MR negative).

The MR test has some limitations, such as:

• It may not be enough to identify bacteria up to the species level and may require additional tests.
• It may give ambiguous results when different genera give the same reaction.
• It may give false positive or false negative results if the incubation period or the reagent quality is not appropriate.

The 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 .

Media and Reagents used in VP Test

The media and reagents used in VP test are:

• MR-VP broth (pH 6.9): It contains buffered peptone, glucose, and dipotassium phosphate.
• Voges-Proskauer Reagent A: It is also known as Barritt’s reagent A or VP reagent I. It is a solution of 5% alpha-naphthol in absolute ethanol.
• Voges-Proskauer Reagent B: It is also known as Barritt’s reagent B or VP reagent II. It is a solution of 40% potassium hydroxide .

Procedure of VP Test

The procedure of VP test is as follows:

• Prior to inoculation, allow medium to equilibrate to room temperature.
• Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
• Incubate aerobically at 37 degrees C. for 24 hours.
• Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube. Re-incubate the remaining broth for an additional 24 hours.
• Add 6 drops of Voges-Proskauer Reagent A, and mix well to aerate.
• Add 2 drops of Voges-Proskauer Reagent B, 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 .

Result Interpretation of VP Test

The result interpretation of VP test is based on the color change at the surface of the broth:

• Positive Reaction: A pink-red color at the surface Examples: Viridans group streptococci (except Streptococcus mitis and Streptococcus vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio cholerae biotype El Tor, and Vibrio alginolyticus .
• Negative Reaction: A lack of a pink-red color or a yellowish color at the surface Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus .

Uses of VP Test

The uses of VP test are:

• It is used to differentiate and identify some members of Enterobacteriaceae family based on their glucose metabolism pathway.
• It is used to differentiate some species within a genus based on their acetoin production ability. For example, it can differentiate Proteus mirabilis (VP positive) from Proteus vulgaris (VP negative).

The citrate utilization test is a biochemical test that detects the ability of bacteria to use citrate as their sole source of carbon and energy. It is indicated by the letter "C" of the IMViC test series. The test is commonly used to differentiate members of the Enterobacteriaceae family, such as Klebsiella, Enterobacter, Citrobacter, Salmonella and Escherichia.

Principle of Citrate Utilization Test

The principle of the citrate utilization test is based on the production of alkaline compounds by bacteria that can metabolize citrate. The test medium contains sodium citrate as the only carbon source and ammonium dihydrogen phosphate as the only nitrogen source. Bacteria that can grow on this medium produce an enzyme called citrate permease, which transports citrate into the cell and converts it to pyruvate and carbon dioxide. Pyruvate can then enter the bacterial metabolic cycle for energy production.

The carbon dioxide released by the bacteria reacts with water and sodium ions in the medium to form sodium carbonate, which increases the pH of the medium. In addition, the bacteria use ammonium salts as a nitrogen source, producing ammonia or ammonium hydroxide, which also raises the pH of the medium. The increase in pH changes the color of the pH indicator bromothymol blue from green to blue.

Requirements for Citrate Utilization Test

The citrate utilization test requires a specific culture medium and a pH indicator. The most commonly used medium is Simmon`s citrate agar, which contains sodium citrate, ammonium dihydrogen phosphate, sodium chloride, magnesium sulfate, agar and bromothymol blue. The medium is prepared by dissolving the salts in deionized water, adjusting the pH to 6.9, adding agar and bromothymol blue, heating to boiling until agar dissolves, dispensing into tubes and autoclaving at 121°C for 15 minutes. The medium is then cooled in a slanted position to form a long slant and a shallow butt.

Procedure of Citrate Utilization Test

The procedure of the citrate utilization test is simple and involves streaking a light inoculum of bacteria on the slant of Simmon`s citrate agar and incubating aerobically at 35-37°C for up to 4-7 days. The result is observed by looking for growth and color change on the slant.

Interpretation of Citrate Utilization Test

The interpretation of the citrate utilization test is based on the presence or absence of growth and color change on the slant. A positive result is indicated by growth and color change from green to intense blue along the slant, showing that the bacteria can utilize citrate and produce alkaline compounds. A negative result is indicated by no growth and no color change; slant remains green, showing that the bacteria cannot utilize citrate.

Some examples of citrate positive bacteria are Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Salmonella spp. (except S. Typhi and S. Paratyphi A), Edwardsiella spp., Providencia spp., etc. Some examples of citrate negative bacteria are Escherichia coli, Shigella spp., Salmonella Typhi and Paratyphi A, Yersinia spp., Morganella morganii, Staphylococcus aureus, etc.

The IMViC test results can be used to differentiate and identify some common bacteria, especially the members of the Enterobacteriaceae family. The table below shows the IMViC test result chart of some common bacteria :

*Variable results may occur depending on the strain and incubation time.

A positive result is indicated by a "+" sign and a negative result is indicated by a "-" sign. The indole test is positive if a pink-red ring forms after adding Kovac`s reagent. The methyl red test is positive if a red color develops after adding methyl red indicator. The Voges-Proskauer test is positive if a pink-red color develops after adding Barritt`s reagents A and B. The citrate test is positive if the medium turns blue after incubation.

• The IMViC test is a useful tool for identifying and differentiating bacteria, especially the members of the Enterobacteriaceae family . The Enterobacteriaceae are a large and diverse group of gram-negative bacteria that include many pathogens such as Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, and Yersinia.
• The IMViC test can help to narrow down the possible genera and species of bacteria based on their metabolic properties and enzyme production . For example, the indole test can differentiate Escherichia coli (indole positive) from Klebsiella pneumoniae (indole negative), the methyl red test can differentiate Escherichia coli (methyl red positive) from Enterobacter aerogenes (methyl red negative), the Voges-Proskauer test can differentiate Klebsiella pneumoniae (Voges-Proskauer positive) from Salmonella typhimurium (Voges-Proskauer negative), and the citrate test can differentiate Citrobacter freundii (citrate positive) from Shigella dysenteriae (citrate negative) .
• The IMViC test is also used for the characterization and identification of some gram-positive bacteria, such as Streptococcus, Staphylococcus, Bacillus, and Listeria. For example, the Voges-Proskauer test can differentiate Streptococcus pyogenes (Voges-Proskauer positive) from Streptococcus pneumoniae (Voges-Proskauer negative), and the citrate test can differentiate Bacillus subtilis (citrate positive) from Staphylococcus aureus (citrate negative).
• The IMViC test is routinely used in clinical, research, and teaching laboratories for the primary screening and identification of unknown isolated bacteria . It is a simple, inexpensive, and reliable method that can provide results within 24 to 48 hours. It can also be combined with other biochemical tests, such as urease test and triple sugar iron test, to further confirm the identity of bacteria .

The IMViC test is a useful tool for characterizing and identifying some bacteria, especially the members of the Enterobacteriaceae family. However, it also has some limitations that should be considered when interpreting the results. Some of the limitations are:

• The IMViC test cannot provide a definitive identification of bacteria at the species level. Additional tests, such as biochemical tests, serological tests, or molecular tests, are often required to confirm the identity of the bacteria.
• The IMViC test may give ambiguous results when different genera or species have the same IMViC pattern. For example, both Escherichia coli and Salmonella spp. are IMViC ++-- (positive for indole and methyl red, negative for Voges-Proskauer and citrate), but they are clearly different bacteria with different pathogenicity and epidemiology. Therefore, other tests are needed to differentiate them.
• The IMViC test is a culture-based test that requires a pure culture of bacteria and an appropriate incubation period. This may take several days or even weeks depending on the growth rate and metabolic characteristics of the bacteria. Some bacteria may not grow well or at all on the culture media used for the IMViC test, which may lead to false negative results or no results at all.
• The IMViC test is a complex and labor-intensive test that requires good culture skills and various resources, such as culture media, reagents, indicators, and equipment. The test may be prone to errors or inaccuracies if the procedure is not followed correctly or if the reagents are old or contaminated. For example, false positive results may occur if the indole reagent is oxidized or if the VP reagents are not added in the correct order or amount. False negative results may occur if the culture media are not properly prepared or sterilized or if the incubation temperature is not optimal.

These limitations suggest that the IMViC test should be used with caution and in conjunction with other tests to obtain reliable and accurate identification of bacteria. The IMViC test is a valuable diagnostic tool, but it is not a definitive one.

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