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10.11: Urease Test

HGTC BIO 225: Microbiology Lab Manual · LibreTexts

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    Learning Objectives
    • Understand the role of the urease enzyme in microbial metabolism and its importance in nitrogen metabolism.
    • Explain the principles and methods of urease detection.
    • Interpret experimental results and analyze data obtained from urease activity assays.
    • Explore factors affecting urease activity, such as pH and temperature.
    • Understand the significance of urease activity in various microbial processes, such as its relevance in medical diagnostics.

    Urea Hydrolysis  

    The urease test is used to identify bacteria capable of producing the enzyme urease. The urease enzyme catalyzes the hydrolysis of urea into ammonia and carbon dioxide. When added to urea broth, a urease-positive bacterial species will hydrolyze urea. This results in the production of ammonia, which raises the pH of the medium and turns it pink due to the phenol red pH indicator present in the medium. This color change indicates a positive result for urease activity. 

    Urease activity differentiates urease-producing Proteus bacteria (e.g., Proteus vulgaris) from Enterobacteriaceae (e.g., Escherichia coli). In urea broth, Proteus can use urea as a sole source of nitrogen. In the process, ammonia is released, resulting in a color change from orange to bright pink (Fig.1).

     

    urea.jpg

    Figure 1: Urea broth test results: (a) Proteus vulgaris (urease positive) and (b) Escherichia coli (urease negative).

    Note

    The urease test is commonly used in clinical microbiology to aid in the identification of bacterial pathogens, particularly those associated with urinary tract infections (UTIs) and gastrointestinal disorders

     
    Real-World Connections

    Peptic Ulcers

    Peptic ulcers (e.g. stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori. This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005.

    The ability of H. pylori to survive the low pH of the stomach suggests that it is an extreme acidophile. As it turns out, this is not the case. H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment.

    This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a solution of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the urea, producing radioactive CO2 that can be detected in the patient’s breath. Because peptic ulcers may lead to gastric cancer, patients who are determined to have H. pylori infections are treated with antibiotics.

     

    Laboratory Instructions

    1. Obtain two (2) urea broth test tubes.
    2. Using labeling tape, label the tube with your name/group name, date, course section, species initials, and test type (e.g., urea).
    3. Using a sterile inoculating loop, aseptically obtain a generous sample of bacteria and mix it into the urea broth. Repeat for both species.
    4. Place the inoculated broths into a test tube rack and incubate for 24 hours at 37 °C.
    5. Observe, record, and interpret results.
    Note

    Rapidly urease-positive Proteeae (e.g., Proteus mirabilis) for which this medium is differential, will produce a strong positive reaction within 24 hours of incubation. Delayed-positive organisms (e.g., Klebsiella pneumonia) will not produce a positive reaction due to the high buffering capacity of this medium.

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