Biochemical tests: IMViC Series

• Biochemical tests are crucial in identifying bacteria by assessing their metabolic and enzymatic activities.

• The IMViC series—comprising the Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization tests—is particularly valuable for differentiating members of the Enterobacteriaceae family.

• Each test examines specific metabolic pathways or enzyme activities, contributing to a comprehensive biochemical profile for bacterial identification.

  

  Biochemical tests: IMViC Series

(image may contain incorrect data, it is just for illustration purpose)

1) Indole Test

Purpose:

• Determines the ability of bacteria to produce indole from the amino acid tryptophan using the enzyme tryptophanase.

Procedure:

1. Inoculation: Inoculate an indole reagent tube (e.g., Kovac’s or Ehrlich’s reagent) with a bacterial culture grown on a tryptophan-containing medium such as tryptone broth or SIM agar.

2. Incubation: Incubate the inoculated tube at 37°C for 24 hours.

3. Reagent Addition: After incubation, add a few drops of Kovac’s or Ehrlich’s reagent to the culture.

4. Observation:

   1. A red or pink layer at the top indicates a positive result.

   2. No color change or a yellow layer indicates a negative result.

Interpretation:

1) Positive Indole Test:

• Bacteria produce indole.

• Example: Escherichia coli

2) Negative Indole Test:

• Bacteria do not produce indole.

• Examples: Enterobacter aerogenes, Klebsiella pneumoniae

Significance:

• The test is useful for distinguishing species within the Enterobacteriaceae family.

• For instance, E. coli is typically indole positive, while closely related species may be indole negative.

2) Methyl Red (MR) Test

Purpose:

• Assesses the ability of bacteria to perform mixed acid fermentation of glucose, leading to the production of stable acid end-products.

Procedure:

1. Inoculation: Inoculate a Methyl Red test tube with a fresh bacterial culture grown in MR-VP broth.

2. Incubation: Incubate at 37°C for 24-48 hours.

3. Indicator Addition: Add a few drops of Methyl Red indicator solution directly to the culture after incubation.

4. Observation:

   1. A red or pink color indicates a positive result.

   2. Yellow or no color change indicates a negative result.

Interpretation:

1) Positive Methyl Red Test:

• Production of stable acid end-products, resulting in a lower pH.

• Example: Escherichia coli

2) Negative Methyl Red Test:

• Production of neutral end-products, maintaining a higher pH.

• Examples: Enterobacter aerogenes, Klebsiella pneumoniae

Significance:

• This test differentiates species based on their glucose fermentation pathways.

• A positive MR result is typically associated with organisms that fully ferment glucose to acids.

3) Voges-Proskauer (VP) Test of IMViC Series

Purpose:

• Detects the ability of bacteria to produce acetoin (a precursor to 2,3-butanediol) from glucose fermentation, indicating a fermentation pathway distinct from that assessed by the MR test.

Procedure:

1. Inoculation: Inoculate a VP test tube with a fresh culture grown in MR-VP broth.

2. Incubation: Incubate at 37°C for 24-48 hours.

3. Reagent Addition: Add equal volumes of alpha-naphthol and potassium hydroxide (KOH) solution to the culture.

4. Reflux: Gently heat the mixture in a boiling water bath for a few minutes to develop color.

5. Observation:

   1. A red or pink color indicates a positive result.

   2. No color change or a yellow color indicates a negative result.

Interpretation:

1) Positive VP Test:

• Acetoin production.

• Examples: Enterobacter aerogenes, Enterobacter cloacae

2) Negative VP Test:

• No acetoin production.

• Example: Escherichia coli

Significance:

• The VP test complements the MR test by identifying bacteria that utilize different glucose fermentation pathways, helping to differentiate closely related species within the Enterobacteriaceae family.

4.Citrate Utilization Test

Purpose:

• Evaluates whether bacteria can use citrate as the sole carbon source and ammonium ions as the sole nitrogen source, indicating specific metabolic capabilities.

Procedure:

1. Inoculation: Inoculate a citrate agar slant (e.g., Simmons citrate agar) with the bacterial culture using an inoculating needle.

2. Incubation: Incubate at 37°C for 24-48 hours.

3. Observation:

   1. A blue color change in the medium indicates a positive result.

   2. No color change (medium remains green) indicates a negative result.

Interpretation:

1) Positive Citrate Utilization:

• The bacteria can utilize citrate as a sole carbon source.

• Examples: Enterobacter aerogenes, Klebsiella pneumoniae

2) Negative Citrate Utilization:

• The bacteria cannot utilize citrate and rely on other carbon sources.

• Examples: Escherichia coli, Proteus mirabilis

Significance:

• The citrate utilization test helps differentiate bacterial species based on their ability to metabolize citrate, which is characteristic of certain genera within the Enterobacteriaceae family.

• This series of tests, when used together, provides a powerful tool for identifying and distinguishing bacterial species based on their metabolic characteristics.

Summary of IMViC Tests

Indole	Red/pink layer	E. coli	Enterobacter, Klebsiella
Test	Positive Indicator	Positive Organisms	Negative Organisms
Methyl Red	Red/pink color	E. coli	Enterobacter, Klebsiella
VP	Red/pink color	Enterobacter, Klebsiella	E. coli
Citrate	Blue medium	Enterobacter, Klebsiella	E. coli, Proteus