Title: Assay of Urease Enzyme
Aim: To detect and quantify the activity of the urease enzyme using the urea hydrolysis method.
Introduction:
Urease is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. It is found in bacteria, fungi, and plants, playing a crucial role in nitrogen metabolism. Urease activity is significant in various biotechnological applications, including agriculture, medicine, and environmental science. In pathogenic bacteria like Helicobacter pylori, urease production aids in colonization and survival in acidic environments.
Theory:
Urease hydrolyzes urea into ammonia and carbon dioxide. The release of ammonia increases the pH of the medium, which can be detected using pH indicators such as phenol red. The change in pH results in a color shift from yellow to pink, indicating urease activity. This assay helps in the identification of urease-producing organisms and their enzymatic activity.
Principle:
Urease catalyzes the hydrolysis of urea:
Urea + H₂O → 2NH₃ + CO₂
The released ammonia increases the pH of the medium. Phenol red, a pH indicator, changes color from yellow (acidic) to pink (alkaline) in response to ammonia production, confirming urease activity.
Requirements:
Urea agar slants or broth
Proteus vulgaris (positive control) and test organisms
Sterile inoculating loop
pH indicator (Phenol red)
Sterile distilled water
Incubator (35–37°C)
Test tubes and culture media
Pipettes and sterile swabs
Procedure:
- Prepare urea agar or urea broth containing urea and phenol red as a pH indicator.
- Dispense into sterile test tubes and sterilize by filtration.
- Using a sterile inoculating loop, pick a colony of the test organism.
- Streak the slant surface (for agar) or inoculate the broth with the test organism.
- Include Proteus vulgaris as a positive control and an uninoculated tube as a negative control.
- Incubate the tubes at 35–37°C for 24 hours.
- Observe the color change in the medium.
- A pink color indicates a positive urease test, while a yellow color suggests a negative result.
Result: A positive urease test is indicated by a pink color change due to ammonia production.
Conclusion:
The urease test is a simple and effective method to detect urease enzyme activity in bacteria and fungi. It is widely used in clinical microbiology for identifying urease-positive pathogens such as Helicobacter pylori and Proteus species. The ability to hydrolyze urea has significant applications in medical diagnostics, agriculture, and biotechnology.
