Biochemical Tests for Bacteria Identification

Overview

• Biochemical profiles are built to determine which biochemical reactions bacteria can perform.
• Indicators are used to determine if certain reactions are occurring.
• Focus is on nitrogen metabolism, specifically breaking down amino acids and proteins for energy when carbohydrates are lacking.

Hydrolysis of Gelatin

• Tests for the presence of gelatinase, an enzyme that breaks down the protein gelatin into amino acids.
• Negative result: Gelatin remains solid at room temperature.
• Positive result: Gelatin liquefies at room temperature.

Indole Production

• Tests for the presence of tryptophanase, an enzyme that breaks down tryptophan (an amino acid) into pyruvic acid and indole.
• Indicator: Kovac's reagent.
• Positive result: Kovac's reagent turns pink in the presence of indole.
• Negative result: Kovac's reagent remains orange or green.

Hydrogen Sulfide (H2S) Production

• Tests for the presence of cysteine desulfurase, an enzyme that breaks down cysteine (an amino acid) releasing hydrogen sulfide gas.
• Hydrogen sulfide gas interacts with iron in the agar to produce a black precipitate.
• Positive result: Black precipitate forms.
• Negative result: No black precipitate.

Urea Hydrolysis

• Tests for the presence of urease, an enzyme that breaks down urea into ammonia and carbon dioxide.
• Ammonia raises the pH, which is detected by the indicator phenol red.
• Indicator: Phenol red.
• Positive result: Media turns pink (basic pH due to ammonia production).
• Negative result: Media remains orange.

Nitrate Reduction

• Determines if nitrate (NO3) is reduced to nitrite (NO2) by nitrate reductase, and/or if nitrite is further reduced to ammonia or nitrogen gas by nitrite reductase.
• Reagents used: Nitrate reagents A and B (detect nitrite) and zinc dust (catalyzes the conversion of nitrate to nitrite).
• Procedure:
  • Add reagents A and B.
  • If red color develops, nitrite is present (positive for nitrate reduction to nitrite).
  • If no color change, add zinc dust.
  • If red color develops after adding zinc, nitrate was present (negative for both reactions).
  • If no color change after adding zinc, nitrite was further reduced (positive for both nitrate and nitrite reduction).