Biochemical Test

IMViC test procedure, Purpose, Result, and Organism List.

Table of Contents show 1 What is IMViC test? 2 Purpose of IMViC Test 3 IMViC test Procedure 4 IMViC Test Result...

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MN Editors
This article writter by MN Editors on June 22, 2020

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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IMViC test procedure
IMViC test procedure

Hey, there in this article I have discussed imvic test procedure and it’s purpose in detail. If you face any problem related to imvic test procedure than feel free and comment below.

Before we know about imvic test procedure, first of all, we have to know what is imvic test and IMViC test Purpose

What is IMViC test?

    • IMVIC is a group of individual biochemical tests, used for the detection of coliform group.
    • The full form of IMViC test is “Indole Methyl red Voges-Proskauer Citrate” test, whereas “I” indicating “Indol”, “M” indicating “Methyl red”, “Vi” indicating a scientist name “Voges-Proskauer”, and “C” indicating “Citrate”.
    • IMVIC test is the combination of four tests, such as Indol test, Methyl red test, Voges-Proskauer test, and Citrate utilization test.
    • The indole test detects the production of indole from the amino acid tryptophan.
    • Methyl red is a pH indicator which determines whether the bacterium carries out mixed acid fermentation.
    • Voges-Proskauer (VP) detects the production of acetoin.
    • The citrate test determines whether or not the bacterium can use sodium citrate as a sole source of carbon.
    • Used to distinguish Escherichia (MR-, VP-, indole+) from Enterobacter (MR-, VP +, indole-) and Klebsiella pneumoniae, (MR- , VP I , indole- ); also used to characterize members of the genus Bacillus

Purpose of IMViC Test

To find out the procedure of treatment or prevention of a specific foodborne disease or waterborne disease it needs to identify the enteric bacteria or intestinal bacteria that are responsible for these infections.

All the bacteria present in intestine of a human or other mammals belong to the family of Enterobacteriaceae. These enterobacteria are short, gram-negative, and nonsporing bacilli.

Some of them have the ability to ferment lactose, for example, occasional pathogens Klebsiella and Escherichia, lactose fermenters, and some of them lack this activity for example Salmonella and Shigella, lactose non fermenters.

The identification of these fermentative and non fermentative bacteria can be accomplished by IMViC test. In medical research, it il help to identify the pathogen responsible for the disease.

IMViC test Procedure

IMViC test performs in four test; each step of 

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

1. Indole test

IMVIC test procedure starts with, it’s first test called the Indole test. Procedure, result, and example of the indole test I have already discussed in my previous article, you can check them out by clicking here.

    • In this test, at first, the test organism is grown in peptone water broth, which is contain tryptophan.
    • The enzyme tryptophanase will convert tryptophan to an Indole molecule, pyruvate, and ammonium. 
    • Then, indole is extracted from the broth by means of xylene. 
    • To confirm indole production, Kovac’s reagent is added to the broth, a positive result will be indicated by forming a pink/red layer on top of the medium.
    • Kovac’s reagent is composed of amyl alcohol and para-dimethylaminobenzaldehyde and concentrated hydrochloric acid. Kovac’s reagent is mainly used for the detection of the ability of an organism to separate indole from amino acid tryptophan and it is added after incubation.

Methyl red and Voges–Proskauer test

Methyl red and Voges–Proskauer is the second step of IMVIC test procedure. Methyl red and Voges–Proskauer test is composed of two different tests, but it performed simultaneously because both of them use same broth during the experiment, which is called MRVP broth. Methyl red and Voges–Proskauer test is completed in this following steps;

Check here full procedure, results, requirements of Methyl red, and Voges–Proskauer test.

    • First of all, test organism is grown in an MRVP broth.
    • After growth, the organism containing broth is divided into two separate tubes, one for the MR test and one for the VP test.
    • The MR test helps in the detection of acid-forming bacteria during mixed acid fermentation pathway. They use pyruvic acid as a substrate for mixed acid fermentation pathway during their metabolic activity. If a red color appears after the addition of pH indicator Methyl Red to one tube (at pH’s lower than 4.2). indicating a positive test (mixed acid fermentation is used). The solution remaining yellow (pH = 6.2 or above) indicates a negative test, meaning the butanediol fermentation is used.
    • The VP test uses alpha-naphthol and potassium hydroxide for the detection of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-butanediol fermentation pathway. After the addition of both reagents, the tube is shaken vigorously and then kept in undisturbed condition for 5-10 minutes. The positive test will indicate by forming A pinkish-red color, meaning the 2,3-butanediol fermentation pathway is used.

Citrate utilization test

    • The third and important step of imvic test procedure is Citrate utilization test.
    • Citrate utilization test uses Simmon’s citrate agar to detect the ability of a microorganism to use citrate as its sole carbon source.
    • The Simmon’s citrate agar is composed of citrate as a main carbon source and ammonium ions as the main nitrogen source, and bromothymol blue as an indicator.
    • If the citrate agar is turned into blue after inoculation, it indicating positive results, meaning citrate is utilized.

Read Also

  1. Simmons citrate agar test – Procedure, Principle, Result, Example.
  2. Methyl Red and Voges Proskauer Test – Principle, Result
  3. Indole test – Principle, Procedure, Uses.

IMViC Test Result

S. No Test Name Positive result Negative Result
1. Indole Test color changes pink to red (“cherry-red ring”) no color change occurs 
2. Methyl Red test  a stable red color develops  a yellow color develops
3. Voges Proskauer Test a pink-red color develops a yellow color develops 
4. Citrate Test develops blue color from green No color change
IMViC test procedure
Figur: IMViC test procedure | Image Source:

IMViC Positive and Negative Organisms

List of Positive and Negative Organism in Indole Test

Microorganism Name Indole Positive Indole Negative
Actinobacillus spp. No Yes
Aeromonas hydrophila Yes No
Aeromonas punctata Yes No
Aeromonas salmonicida No Yes
Alcaligenes sp. No Yes
Bacillus alvei Yes No
Bacillus sp. No Yes
Bordetella sp. No Yes
Edwardsiella sp Yes No
Enterobacter sp. No Yes
Enterococcus faecalis Yes No
Escherichia coli Yes No
Flavobacterium sp. Yes No
Haemophilus influenzae Yes No
Haemophilus sp. No Yes
Klebsiella oxytoca Yes No
Klebsiella sp. No Yes
Lactobacillus reuteri Yes No
Mannheimia haemolytica No Yes
Neisseria sp. No Yes
P. penneri No Yes
Pasteurella multocida Yes No
Pasteurella pneumotropica Yes No
Pasteurella ureae No Yes
Plesiomonas shigelloides Yes No
Proteus mirabilis No Yes
Proteus sp. (not P. mirabilis and P. penneri) Yes No
Pseudomonas sp. No Yes
Rhizobium sp. No Yes
Salmonella sp. No Yes
Serratia sp. No Yes
Vibrio sp Yes No
Yersinia sp. No Yes

List of Positive and Negative Organism in Simmons citrate test

Microorganism Result
Klebsiella pneumoniae Positive (+)
Salmonella Typhi Negative (-)
Enterobacter species  Positive (+)
Citrobacter freundii Positive (+)
Escherichia coli Negative (-)
Shigella spp Negative (-)
Serratia marcescens Positive (+)
Proteus mirabilis Positive (+)
Salmonella Paratyphi A Negative (-)
Providencia Positive (+)
Morganella morganii Negative (-)
Yersinia enterocolitica Negative (-)
Salmonella other than Typhi and Paratyphi A Positive (+)

List of Positive and Negative Organism in Methyl Red test

Organism Name Result
Escherichia coli Positive (MR+)
Klebsiella pneumoniae Negative (MR-)
Shigella species Positive (MR+)
Enterobacter species Negative (MR-)
Salmonella species Positive (MR+)
Hafnia species Negative (MR-)
Proteus species Positive (MR+)
Citrobacter species Positive (MR+)
Serratia marcescens Negative (MR-)
Yersinia species Positive (MR+)

List of Positive and Negative Organism in Voges-Proskauer test

Organism Name Result
Enterobacter Positive (VP+)
 Citrobacter sp. Negative (VP-)
Klebsiella Positive (VP+)
Shigella Negative (VP-)
Serratia marcescens Positive (VP+)
Yersinia Negative (VP-)
Hafnia alvei Positive (VP+)
Vibrio cholera biotype eltor Positive (VP+)
Edwardsiella Negative (VP-)
Salmonella Negative (VP-)
Vibrio furnissii Negative (VP-)
Vibrio fluvialis Negative (VP-)
 Vibrio vulnificus Negative (VP-)
 Vibrio parahaemolyticus. Negative (VP-)
Vibrio alginolyticus Positive (VP+)

Read Also

  1. Simmons citrate agar test – Procedure, Principle, Result, Example.
  2. Methyl Red and Voges Proskauer Test – Principle, Result
  3. Indole test – Principle, Procedure, Uses.
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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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