A urease test is a common diagnostic test used to detect the presence of the urease enzyme in microorganisms. It is often used to identify certain types of bacteria that can cause urinary tract infections, such as Proteus species. The basis of the test relies on the fact that some microbes produce the enzyme urease, which catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The production of ammonia leads to an increase in pH that is detected by a color change of a pH indicator in the test medium. Understanding what causes this color change is important for interpreting urease test results.
The Urease Test Procedure
The typical urease test procedure involves the following steps:
- A urea-containing medium is inoculated with a bacterial culture.
- The medium also contains a pH indicator such as phenol red which is yellow at acidic pH and bright pink at alkaline pH.
- The inoculated medium is incubated for a period of time, usually 24 hours.
- If the bacterium produces urease, the urea in the medium is hydrolyzed to produce ammonia and carbon dioxide.
- The ammonia increases the pH of the medium causing the pH indicator to change color.
- A positive urease test is indicated by a color change of the medium from yellow to bright pink.
The Role of Urease
The key component of the urease test is the bacterial enzyme urease. Urease catalyzes the following hydrolysis reaction:
(NH2)2CO + H2O → CO2 + 2NH3
The enzyme breaks down urea into two products – ammonia (NH3) and carbon dioxide (CO2). The critical product that leads to the observed color change is ammonia.
Ammonia Causes Alkalinization
Ammonia is a base that increases the pH of the medium making it more alkaline. The chemical structure of ammonia allows it to accept protons leading to the increased pH:
NH3 + H+ → NH4+
As ammonia removes protons, the hydronium ion (H3O+) concentration decreases, and the pH rises. The more ammonia produced, the more alkaline the medium becomes.
Color Change of pH Indicator
The urease test medium contains a pH indicator such as phenol red which undergoes a color change depending on the pH. At acidic pH, phenol red is yellow with a maximal absorbance at 430 nm. At alkaline pH, the molecule loses a proton turning into a bright pink/fuchsia color with maximal absorbance at 560 nm. Therefore, as the pH rises due to the production of ammonia, phenol red changes from yellow to bright pink indicating a positive urease test.
| pH | Phenol Red Color |
|---|---|
| Acidic (pH 6.8 or lower) | Yellow |
| Alkaline (pH 8.2 or higher) | Bright pink |
Other pH Indicators
While phenol red is a commonly used pH indicator for the urease test, other pH-sensitive dyes can also be used such as:
- Cresol red – yellow (acidic), fuchsia (alkaline)
- Bromthymol blue – yellow (acidic), blue (alkaline)
- Neutral red – yellow/orange (acidic), red (alkaline)
The key is the pH indicator has a color transition range between pH 6-8 which spans the change induced by ammonia production.
Role of Urea Concentration
For the urease test to work properly, an optimal concentration of urea must be present in the medium. Too little urea may not produce enough ammonia for an obvious color change. Too much urea can create a strong buffering capacity preventing pH change. Typically urea concentrations between 0.1 – 1% are used. The amount of ammonia produced is proportional to the urea concentration as long as saturation kinetics are avoided.
Time Considerations
Sufficient time must be allowed for urease-positive organisms to produce ammonia and induce a color change. This requires viable, actively metabolizing bacteria. Optimal incubation times are typically 24 hours for urease production. However, weaker or slower urease producers may require 48 hours for a detectable color change.
Role of Bacterial Concentration
A certain threshold number of viable bacteria must be present in the urease test inoculum to produce enough urease enzyme to generate sufficient ammonia for color change. Too few bacteria may appear urease-negative. Recommended inoculum concentrations are 106-108 CFU/ml for reliable urease testing.
Interfering Substances
Certain substances in bacterial cultures can interfere with the urease test and cause false results:
- Acids: Bacterial acids lower pH causing false negative results.
- Alkalis: Bacterial alkalis increase pH causing false positive results.
- Urease inhibitors: Substances that inhibit urease activity prevent color change.
To maximize accuracy, pure cultures should be prepared in neutral pH buffer before inoculating urease test medium.
Conclusion
In summary, a positive urease test producing a color change from yellow to pink results from the following steps:
- Urease produced by bacteria hydrolyzes urea to generate ammonia.
- Ammonia increases pH by consuming protons.
- Increased pH changes pH indicator color from acidic (yellow) to alkaline (pink).
- Adequate urea, incubation time, and inoculum density are required.
Understanding this mechanism allows proper interpretation of results and troubleshooting of unexpected outcomes when performing the urease testing procedure.