The urease test is a common biochemical procedure utilized in microbiology to differentiate bacteria based on their ability to produce the enzyme urease. This test is a diagnostic tool, particularly in clinical settings where the rapid identification of pathogens is important for patient care. It works by detecting the presence of this enzyme in a bacterial sample, helping scientists classify the organism and diagnose various infections.
The Biochemical Reaction Driving the Test
The enzyme urease acts as a catalyst, specifically targeting the compound urea, a common nitrogen-containing waste product. Urease hydrolyzes urea in the presence of water to generate two products: ammonia and carbon dioxide.
The production of ammonia is the event that dictates a positive outcome, as ammonia is a highly basic compound. As ammonia accumulates in the test medium, it reacts with water and carbon dioxide to form ammonium carbonate, which significantly raises the medium’s pH. This shift to an alkaline environment indicates a positive result.
To make this chemical change visible, the test medium contains a pH indicator dye, most commonly phenol red. Phenol red is sensitive to pH fluctuations, starting at a yellowish-orange color when the medium is neutral or slightly acidic. When the pH rises above approximately 8.1 due to ammonia production, the indicator changes color, providing a clear visual signal of urease activity.
Performing the Urease Test: Different Formats
The urease test uses several formats, each tailored to the speed and purpose required in the laboratory. A common method involves using a urea broth or urea agar slant, such as Christensen’s urea agar, which is inoculated with the bacterial sample. This format is typically incubated for a period ranging from a few hours up to 24 hours or more, allowing time for the organism to grow and produce the enzyme.
The medium in these standard tests often contains a low concentration of buffering agents, making it easier for small amounts of ammonia to cause a noticeable pH change. While effective for general identification, these methods may take a full day to confirm a positive result for organisms with moderate urease activity. Strong urease producers, like certain Proteus species, often show a positive result much faster, sometimes within an hour.
Rapid Urease Test (RUT)
A specialized application is the Rapid Urease Test (RUT), also known as the CLO test, designed specifically for diagnosing Helicobacter pylori infection. For the RUT, a small biopsy sample of the stomach lining, taken during an endoscopy, is placed directly into a medium containing urea and a pH indicator. This test is rapid because H. pylori has exceptionally high urease activity, leading to a quick color change, often within minutes to a few hours. The RUT provides a diagnosis at the time of the procedure, aiding in treatment planning.
Interpreting Results and Key Applications
A positive result is represented by the medium changing color from its initial yellow or yellowish-orange to a bright pink or magenta. This color shift confirms the presence of the urease enzyme, as the pink hue is caused by the rise in pH above 8.1 due to the accumulation of alkaline ammonia.
A negative result is indicated when the medium remains yellow, orange, or shows no color change after the incubation period. This signifies that the organism either does not produce urease or produces it in quantities too small to overcome the medium’s buffering capacity.
The most recognized application of this test is the identification of Helicobacter pylori, the bacterium responsible for stomach ulcers and chronic gastritis. The bacterium’s ability to produce large amounts of urease allows it to survive in the highly acidic environment of the stomach by creating a localized alkaline cloud around itself. Detecting this activity via the RUT is a standard method for diagnosing the infection.
Beyond H. pylori, the urease test is routinely used in clinical laboratories to identify members of the Enterobacteriaceae family. Specifically, it is a differentiating test for Proteus species, which are known for their strong urease production and are a frequent cause of urinary tract infections. The test helps distinguish Proteus from other urease-negative Enterobacteriaceae like Escherichia coli or Salmonella.