Protein Catabolism II

Theory: Amino Acid Breakdown in Microbial Metabolism

Microorganisms utilize proteins as nutrient sources, breaking down amino acids through specific biochemical reactions. Two primary mechanisms are involved:

• Deamination: The removal of an amino group (NH2) from an amino acid. This process produces ammonia and organic acids, which can be further metabolized by the cell.

• Decarboxylation: The removal of a carboxyl group (COOH) from an amino acid, resulting in the release of carbon dioxide (CO2) gas. This reaction often produces amines, which can affect the pH of the environment.

These reactions are essential for microbial metabolism and are used diagnostically to identify bacterial species based on their enzymatic capabilities.

Diagnostic Tests for Amino Acid Catabolism

Three laboratory tests are commonly used to detect deamination, decarboxylation, and related metabolic activities in bacteria:

Phenylalanine Slant

• Purpose: Detects the presence of phenylalanine deaminase enzyme.

• Procedure: After incubation, add ferric chloride (FeCl3) reagent to the slant.

• Positive Result: The slant turns dark green, indicating phenylalanine deamination.

• Negative Result: No color change.

• Example: Proteus mirabilis is known to produce phenylalanine deaminase.

Peptone Iron Deep

• Purpose: Detects hydrogen sulfide (H2S) production from amino acid breakdown.

• Procedure: Stab inoculation along the center of the tube.

• Positive Result: Formation of a black precipitate along the stab line, indicating H2S reacts with iron salts to form iron sulfide.

• Negative Result: No blackening.

• Example: Proteus mirabilis and Escherichia coli may produce H2S under certain conditions.

MIO Tube (Motility, Indole, Ornithine Decarboxylase)

The MIO tube is a multipurpose test that assesses three characteristics in a single medium:

• Motility:

  • Positive: Diffuse, cloudy growth spreading from the stab line.

  • Negative: Growth confined to the stab line.

• Indole Production:

  • Positive: Cherry-red layer forms on top after adding Kovac's reagent, indicating tryptophanase activity.

  • Negative: No color change after Kovac's reagent.

• Ornithine Decarboxylase:

  • Positive: Medium turns purple throughout, indicating decarboxylation of ornithine.

  • Negative: Medium remains yellow.

Example: Enterobacter aerogenes is motile and can decarboxylate ornithine.

Laboratory Procedure: Inoculation and Organisms

Students are instructed to inoculate three different media with one of four bacterial species:

• Escherichia coli

• Bacillus subtilis

• Proteus mirabilis

• Enterobacter aerogenes

Each student should:

1. Label all tubes with name, date, and class time.

2. Phenylalanine slant: streak surface in a zigzag pattern.

3. MIO tube: stab straight down the center to the bottom and withdraw along the same line.

4. Peptone iron deep: stab straight down the center to the bottom and withdraw along the same line.

5. Place tubes in the rack for collective incubation and result reading.

Summary Table: Diagnostic Tests and Their Purposes

Key Equations

• Deamination Reaction:

• Decarboxylation Reaction:

Additional info: These tests are part of microbial identification protocols, helping differentiate bacteria based on their metabolic properties. The ability to deaminate or decarboxylate amino acids is often species-specific and can be used in clinical and environmental microbiology.