IMVIC Tests and Microbial Biochemistry

IMViC Testing Overview

• IMViC tests comprise four specific biochemical tests: Indole, Methyl Red (MR), Voges-Proskauer (VP), and Citrate.
• Primarily used to differentiate members of the Enterobacteriaceae family from other Gram-negative rods.
• Enterics characteristics:
  • Gram-negative
  • Bacilli
  • Facultative anaerobes
  • Glucose fermenters
• Includes:
  • Normal flora (e.g., Escherichia coli, Proteus, Klebsiella, Enterobacter)
  • Pathogens (e.g., Salmonella, Shigella)
  • Coliforms (e.g., Escherichia coli, Enterobacter aerogenes) are lactose fermenters.

1. SIM Test: Sulfur, Indole, Motility

Sulfur Reduction

• Medium: SIM containing thiosulfate and iron.
• Two enzyme systems:
  • Cysteine desulfurase:
  • Hydrolyzes cysteine to produce pyruvate, ammonia, and H2S.
  • H2S reacts with iron forming black precipitate (ferric sulfide).
  • Thiosulfate reductase:
  • Reduces sulfate to H2S in anaerobic respiration.
• Both systems generate H2S gas leading to black precipitate.

Indole Production

• Tryptophan hydrolyzation by tryptophanase in SIM medium produces pyruvate, ammonia, and indole.
• Post-incubation: add 3 drops of Kovac’s reagent:
  • Positive: Red ring signifies indole presence.

Motility

• Lower agar concentration (0.4% vs. 1.5%) in SIM allows for bacterial movement.
• Inoculation method: stabbing with a needle.
• Observations:
  • Motile: Fuzzy growth diffusing from stab line.
  • Non-motile: No diffusion, clear tube except along the stab line.

2. TTC Motility Agar

• Semisolid medium with 0.4% agar containing triphenyltetrazolium chloride (TTC).
• TTC as an electron acceptor:
  • Oxidized (colorless, soluble) vs. Reduced (red precipitate).
• Inoculation:
  • Motile: Red growth radiating from stab line.
  • Non-motile: Red growth not diffusing from stab line.

3. Methyl Red (MR) Test

• Medium: MR-VP which contains glucose and potassium phosphate.
• Pacifies between acidic fermentation products and neutral products:
  • Acidic: Mixed acid fermentation (e.g., lactic acid, acetic acid).
  • Test organisms: MR positive include Escherichia coli, Shigella sp., Salmonella sp.

Procedure for MR Test

• Add 5 drops of Methyl Red pH indicator:
  • Red: pH < 5 (Positive: Acid produced).
  • Yellow: pH > 6 (Negative: No acid).

4. Voges-Proskauer (VP) Test

• Introduces means to identify organisms with neutral products from glucose fermentation (e.g., 2,3-butanediol, acetoin).
• Distinguishes VP positive organisms (e.g., Enterobacter sp., Serratia sp., Klebsiella aerogenes) from negative ones (e.g., Escherichia coli).

Procedure for VP Test

• Add reagents A & B; reactions lead to:
  • Red color: Indicates presence of acetoin (Positive).
  • Yellow color: Indicates absence (Negative).

5. Citrate Utilization Test

• Media is specifically designed to differentiate organisms based on their ability to use citrate as the sole carbon source.
• Example media: Simmons Citrate.
• The presence of citrate permease allows for citrate transport into the cell, facilitating citrate fermentation.
• Ammonium ion (NH4+) as the nitrogen source enables growth that increases pH.

Growth Indication

• Bromothymol blue pH indicator used:
  • Green: pH 7 (Neutral, no growth).
  • Blue: pH > 7.6 (Alkaline, positive growth indicating citrate metabolism).

Summary of IMViC Tests

• Importance of distinguishing Enterobacteriaceae members.
• Application in clinical diagnostics for identifying pathogens and commensals.