IMViC Battery of Tests

  • Definition and Purpose

    • The IMViC battery of tests includes:
    • Tryptone broth (indole test)
    • MRVP broths (Methyl Red and Voges Proskauer tests)
    • Citrate slant (citrate test)
    • Purpose: To differentiate among members of the Enterobacteriaceae family, particularly among Gram-negative short rod enteric lactose fermenters.

  • Enterobacteriaceae Family

    • General Characteristics:
    • Composed of Gram-negative short rods.
    • Example members: E. coli, Enterobacter, Serratia, Proteus.
    • Ferment glucose but not all family members ferment lactose.
    • Facultative anaerobes:
      • Can reduce nitrate.
      • Produce catalase.

Indole Production (Tryptophan Hydrolysis)

  • Biochemical Reaction

    • Process:
    • Tryptophan + Water
    • Enzyme: Tryptophanase
    • Products: Pyruvic acid and Indole.

  • Procedure

    • Method:
    • Add Kovac’s reagent to the stab culture of SIM (Sulfide Indole Motility) medium.

  • Results Interpretation

    • Observation:
    • A red layer develops on top of the SIM stab.
    • Presence of E. coli or Proteus may indicate test results.
    • Conclusion:
    • A red layer signifies that Kovac’s reagent is binding to indole, indicating the presence of tryptophanase.
    • Alternate method: Use tryptone broth instead by shaking it, adding 8-10 drops of Kovac’s reagent, and do not shake after.
    • E. coli and Proteus are both positive for indole production.

MRVP Tests (Methyl Red vs. Voges Proskauer)

  • Purpose and Convenience

    • Two broths are used to allow inoculation into one broth, then splitting into two for separate testing.

  • Composition of the Broth

    • Buffered peptone and glucose are present.
    • Characteristics:
    • Glucose broth, minus phenol red, but includes a phosphate buffer and protein.
    • Investigation focuses on the microbe's ability to ferment glucose through two pathways:
    • Mixed acid fermentation pathway
    • 2,3-butanediol fermentation pathway
    • A microbe will pick either pathway but not both simultaneously.

Methyl Red Test

  • Purpose

    • Tests the ability of the microbe to produce mixed acids via the mixed acid fermentation pathway.

  • Procedure

    • Grow MRVP broth for at least 72 hours.
    • Shake the broth thoroughly and subsequently add 15 drops of methyl red reagent.

  • Indicator Characteristics

    • Methyl red acts as a pH indicator:
    • Red: pH of 4 or less (positive for mixed acids)
    • Yellow: pH higher than 6
    • Orange: pH between 4 and 6
    • Positive results for mixed acids: E. coli and Proteus.

Voges Proskauer Test

  • Function

    • Opposite of the methyl red test.
    • Tests for neutral end products produced by bacteria via fermentation.

  • Target Compound

    • The key compound sought is acetoin (not 2,3-butanediol, which follows acetoin in the pathway).

  • Procedure

    • Add Alpha naphthol and KOH in a 3:1 ratio into the tubes.

  • Results Interpretation

    • Red coloration indicates a positive reaction for acetoin.
    • Expected outcomes: Enterobacter and Serratia are positive for Voges Proskauer since they were negative for the Methyl Red test.

Citrate Utilization Test

  • Purpose

    • Tests the ability of the microbe to utilize citrate as its sole carbon source.

  • Results Interpretation

    • Blue color indicates a positive result for using citrate; green indicates a negative result.
    • Mechanism
    • If a microbe can utilize citrate, it must also utilize nitrogen in the form of ammonium salts, resulting in ammonia production through cleavage of these salts.

  • Indicator Used

    • Bromthymol blue serves as the pH indicator:
    • Green: pH below 7 (negative result)
    • Prussian blue: pH above 7 (positive result).