BIOchemical Test - IMViC reactions - Indole/Methyl Red/Voges-Proskauer/Citrate tests

Sep 27, 2026

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Lecture notes on IMViC reactions used to identify members of Enterobacteriaceae, especially Escherichia and Enterobacter. The document explains the four constituent tests—Indole, Methyl Red, Voges Proskauer, and Citrate utilization—along with their key principles based on bacterial metabolism and relevant pH/color changes. It provides concise experimental procedures using reagents such as Kovac’s/Ehrlich’s, MR indicator, KOH with alpha-naphthol, and Simmons citrate agar, plus interpretation criteria and example outcomes with E. coli and Klebsiella pneumoniae.

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IMViC reactions

Overview and purpose

Indole test

Methyl Red (MR) test

Voges Proskauer (VP) test

Citrate utilization test

Result table

BIOchemical Test IMViC REACTIONS IMViC reactions are a set of four useful reactions that are commonly employed in the identification of members of family enterobacteriaceae. A series of tests used to study the physiological characteristics of bacteria from the family Enterobacteriaceae, especially Escherichia and Enterobacter. They are designed to differenciate Gram – negative intestinal bacilli of family Enterobacteriaceae Which contains a large number of genera that are biochemically and genetically related to one another. IMViC tests consist of four different tests each of the letters in “IMViC” stands for one of these tests. I- Indole M – methyl red V- Voges-proskauer C- citrate The letter “i” is only for rhyming purpose. The four reactions are: Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test. The letter “i” is only for rhyming purpose. INDOLE TEST: Principle: Some bacteria can produce indole from amino acid tryptophan using the enzyme typtophanase. Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent. Indole reacts with the aldehyde in the reagent to give a red color. An alcoholic layer concentrates the red color as a ring at the top. Procedure: 1. Bacterium to be tested is inoculated in peptone water, which contains amino acid tryptophan and incubated overnight at 37oC.Prepare 1% tryptophan broset thbate one . 2. Incubate one set of test tube withtest organism and maintain one set as negative control without inoculation . inoculate one set of test tube with E.coli use as positive control. 3. Following incubation few drops of Kovac’s reagent are added. Shake gently. 4. Kovac’s reagent consists of para-dimethyl aminobenzaldehyde 10 gm, isoamyl alcohol 150gm and con. HCl 50 ml 5. Allow the tubes to [stand for 2 min. so](stand for 2 min. so) that the reagent comes to the top and then compare test culture with the control tubes. 6. Ehrlich’s reagent is more sensitive in detecting indole production inanerobes and non-fermenters. Observation:-Formation of a red or pink coloured ring at the top is taken as positive. Example: Escherichia coli: Positive; Klebsiella pneumoniae: Negative METHYL RED (MR) TEST: Principle: This is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less. Procedure: 1. the bacterium to be tested in inoculated into glucose phosphate broth, which contains glucose and a phosphate buffer and incubated at 37oC for 48 hours. 2. Over the 48 hours the mixed-acid producing organism must produce sufficient acid to overcome the phosphate buffer and remain acid. 3. The pH of the medium is tested by the addition of 5 drops of MR reagent. 4. Development of red color is taken as positive. MR negative organism produce yellow color. Example: Eschericihia coli: Positive; Klebsiella pneumoniae: Negative VOGES PROSKAUER (VP) TEST: Principle: While MR test is useful in detecting mixed acid producers, VP test detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene glycol. In this test two reagents, 40% KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of peptone, in the presence of alpha- naphthol to produce red color. Role of alpha-naphthol is that ofa catalyst and a color intensifier Procedure: 1. Bacterium to be tested is inoculated into glucose phosphate broth and incubated for at least 48 hours. 2. 0.6 ml of alpha-naphthol is added to the test broth and shaken. 0.2 ml of 40% KOH is added to the

FAQs

What do IMViC reactions consist of?

IMViC reactions consist of four different tests: Indole test, Methyl Red test, Voges Proskauer test, and Citrate utilization test.

How is the Indole test interpreted?

A red or pink coloured ring at the top indicates a positive Indole test; examples given show E. coli positive and Klebsiella pneumoniae negative.

What color change indicates a positive Citrate utilization test?

When the medium changes from green to blue, the test is citrate positive; the document states E. coli as citrate positive.

Sep 27, 2026

Uploaded by Kurz