IMViC Tests- Principle, Procedure, Results, Uses, Limitations

IMViC is an acronym for four separate tests which are used to isolate and identify bacterial, especially mainly coliforms. The IMViC stands for:

  1. Indole test
  2. Methyl red test
  3. Voges-Proskauer test
  4. Citrate utilization test

This test is used to identify and selectively differentiate the members of the family Enterobacteriaceae. There are different broths for each test for inoculation, such as tryptone broth for indole test, methyl red-Voges Proskauer broth (MR-VP broth) for Voges-Proskauer test and methyl red test, and citrate broth for citrate test.

IMViC Tests
IMViC Tests

Objective of IMViC Tests

  1. To differentiate members of the family Enterobacteriaceae, especially E. coli from Enterobacter and Klebsiella.
  2. To perform indole, methyl red, Voges-Proskauer, and citrate utilization tests.

Indole Test

Indole Test Principle

The indole test selectively differentiates the type of bacteria that decomposes amino acid tryptophan into indole which accumulates in the medium and changes the pH of the medium. The enzyme involved in the process is known as tryptophanase. When this indole reacts with Kovac’s reagent, it changes the broth color from yellow to cherry red. 

An oily layer is observed on the top surface of the broth because of the insolubility of amyl alcohol present in the red coloration.

Sulfide-indole-motility (SIM) medium, tryptophan broth, or motility urease indole (MIU) medium is generally used to perform this experiment. Observation is done after the addition of Kovac’s reagent.

It is an important test to identify the enterobacteria family including E. coli, P. Vulgaris, P. rettgeri, M. morgani, and Providencia species.

Indole Test Material Required

  • Kovac’s reagent containing p-Dimethylaminobenzaldehyde
  • SIM medium
  • 37% Hydrochloric acid
  • Amyl alcohol
  • Glasswares
  • Distilled water

Indole Test Procedure

  1. Take sterilized test tubes containing 4 ml of tryptophan broth.
  2. Inoculate the tube aseptically by taking the growth from 18 to 24 hrs culture.
  3. Incubate the tube at 37°C for 24-28 hours.
  4. Add 0.5 ml of Kovac’s reagent to the broth culture.
  5. Observe for the presence or absence of a ring.

Indole Test Expected outcomes

  • Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent.
    Examples: Escherichia coli, Flavobacterium sp., Haemophilus influenzaeKlebsiella oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Pasteurella pneumotropicaEnterococcus faecalis, and Vibrio sp. 
  • Negative: No color change even after the addition of an appropriate reagent.
    Examples: Actinobacillus spp.,  Alcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasteurella haemolyticaPasteurellaureae.

Methyl Red (MR) Test

MR Test Principle

  • Using a mixed acid pathway, some bacteria can convert glucose into acids such as lactic acid, acetic acid, or formic acid, having pyruvic acid as an intermediate. Thus, this produced acid decreases the pH of the medium to 4.5 or below which can be detected using pH indicators such as methyl red which converts the medium color from yellow to red. 
  • This test utilizes this property of bacteria by first culturing it on a glucose-containing medium and later adding methyl red to the broth culture containing those bacteria.

MR Test Media and reagents

  • MRVP broth (pH 6.9), deionized water, buffered peptone, glucose, and dipotassium phosphate.
  • Methyl red solution

MR Test Procedure

  1. Before inoculation, allow the medium to equilibrate to room temperature.
  2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
  3. Incubate aerobically at 37 degrees C. for 24 hours.
  4. Following 24 hours of incubation, aliquot 1ml of the broth into a clean test tube.
  5. Reincubate the remaining broth for an additional 24 hours.
  6. Add 2 to 3 drops of methyl red indicator to the aliquot.

MR Test Expected outcomes

  • Positive Reaction: A distinct red color.
    Examples: E. coli, Yersinia sps, etc.
  • Negative Reaction: A yellow color.
    Examples: Enterobacter aerogenes, Klebsiellapneumoniae, etc.

Voges-Proskauer (VP) Test

VP Test Principle

The Voges-Proskauer (VP) test is used to determine if an organism produces acetyl methyl carbinol from glucose fermentation. If present, acetylmethylcarbinol is converted to diacetyl in the presence of ∝- naphthol, strong alkali (40% KOH), and atmospheric oxygen.

2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+

VP Test Material required

  • Voges-Proskauer Reagent A: Barritt’s reagent A , Alpha-Naphthol, 5%, Absolute Ethanol
  • Voges-Proskauer Reagent B: Barritt’s reagent B, Potassium Hydroxide, Deionized Water, Test tube, distilled water.

VP Test Procedure

  1. Before inoculation, allow the medium to equilibrate to room temperature.
  2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
  3. Incubate aerobically at 37 degrees C. for 24 hours.
  4. Following 24 hours of incubation, aliquot 2 ml of the broth into a clean test tube. Re-incubate the remaining broth for an additional 24 hours.
  5. Add 6 drops of 5% alpha-naphthol, and mix well to aerate.
  6. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
  7. Observe for a pink-red color on the surface within 30 min. Shake the tube vigorously during the 30-min period.

VP Test Expected outcomes

  • Positive Reaction: A pink-red color at the surface.
    Examples: Viridans group streptococci (except Streptococcus vestibular), Listeria, Enterobacter, Klebsiella, Serratiamarcescens, Hafniaalvei, Vibrio eltor, Vibrio alginolyticus, etc.
  • Negative Reaction: A lack of a pink-red color.
    Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus, etc.

Citrate Utilization Test

Citrate Utilization Test Principle

  • Some bacteria utilize citrate as their energy source with the help of an enzyme, citrate-permease, that converts citrate to pyruvate which creates energy through the metabolic cycle, and later in the process, the inorganic ammonium salt breaks down to ammonia to increase the alkanility of medium thus making medium basic and thus are capable of showing growth on citrate agar containing inorganic ammonium salts as nitrogen source. 
  • Bromthymol blue is used as a pH indicator that changes the medium color from green to blue.

Citrate Utilization Test Media and requirements

  • Simon’s citrate agar containing sodium chloride, sodium citrate, ammonium dihydrogen phosphate, dipotassium phosphate, magnesium sulfate
  • Bromothymol blue
  • Agar

Citrate Utilization Test Procedure

  1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony.
  2. Incubate aerobically at 35 to 37C for up to 4-7 days.
  3. Observe a color change from green to blue along the slant.

Citrate Utilization Test Expected outcomes

  • Positive Reaction: Growth with color change from green to intense blue along the slant.
    Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia, etc.
  • Negative Reaction: No growth and No color change; Slant remains green.
    Examples: Escherichia, Shigella, Morganella, Yersinia, etc.

Uses of IMViC test

  1. IMViC is sufficient to differentiate the Enterobacteriaceae family when used with the urease test.
  2. Especially used for E. coli, Enterobacter aerogens, Enterobacter cloacae, and Klebsiella pneumonia.
  3. It can be used to determine the motility of bacteria and disulfide production.

Limitation of IMViC test

  1. Indole test cannot be performed with the medium that already contains dye, such as MacConkey agar.
  2. Some bacteria do not show color change despite showing growth on citrate agar, but still, the bacteria is considered positive for citrate utilization.
  3. Tests with equivocal results should be repeated.
  4. The reaction of citrate agar is not alone sufficient to categorize the species as positive for citrate utilization.



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