Urease test is a procedure used to find out the organism’s ability to split urea by producing an enzyme urease.
Image 1: Two test tubes set for urease test; the pink test tube is urease positive while the orange test tube is negative.
Picture Source: microbeonline.com
Image 2: Four test tubes of urease test with varying degree of results.
Picture Source: biologypractical.com
Decarboxylation of amino acids leads to the production of urea. Once urea undergoes hydrolysis, it produces ammonia and carbon dioxide. Urea is acidic but the formation of ammonia turns the medium into alkaline.
The change in pH is indicated by the changes in color from light orange to magenta. Hence, an organism that tests positive for urease makes the medium pink within 24 hours. (1, 2, 3, and 4)
- Agar or broth medium
- Phenol red as an indicator
- Sodium chloride
- Monopotassium phosphate (4, 5)
What is urease test used for?
- It is used to detect the presence of H. pylori in tissue biopsy material.
- It is used to differentiate between Candida albicans and Cryptococcus neoformans. The former is urease negative while the latter is urease positive.
- It is performed to identify some Enterobacteriaceae species such as Klesiella and Proteus.
- Urease test is done to identify brucella and Cryptococcus species. (4, 5, and 6)
Preparation and Procedures
- The dry ingredients are dissolved in 100 m distilled water.
- Filter and sterile the dissolved ingredients.
- The agar is suspended in 900 ml distilled water.
- Bring to boil to make sure that the agar is completely dissolved.
- Autoclave for 15 minutes. The temperature is set at 121 degree Celsius.
- Allow the agar to cool down (50 to 55 degree Celsius).
- Add at least 100 ml of urea base to the previously prepared agar.
- Put at least 5 ml of the preparation into the test tube and slant the tube. Allow it to cool until such time it reaches the solid state. (5, 6, 7, and 8)
Once it solidifies, you can proceed with the next procedure:
- The surface of the slant containing urea agar is streaked with an isolated colony with at least two drops from brain-heart infusion broth culture.
- Leave the cap but make sure it is loose.
- Incubate the test tube for 48 hours. You can incubate it for up to seven days. The ideal temperature is between 35 and 37 degree Celsius.
- After the incubation period, check for any changes in color. Pink is an affirmation result. (6, 8, 9, and 10)
The color of the slant changes from light orange to pink if the organism being tested is positive for urease. On the other hand, the color of the slant remains the same (light orange) if the organism being tested didn’t produce urease enzyme. (2, 4)
Organisms that have the ability to produce urease include:
- Helicobacter pylori
- Brucella (5, 6)
Image 3:A rapid urease test can be done using a test kit.
Picture Source: researchgate.net
Rapid Urease Test
It is one of the commonly used procedures to check for the presence of Helicobacter pylori. It is widely used because the process is straightforward and cheap.
Rapid urease test checks the gastric mucosa for the presence of urease. Before performing a rapid urease test, a procedure like gastric endoscopy and biopsy of stomach lining cells should be performed first. (6, 7)
- Microorganisms like H. pylori and Brucella rapidly splits urea which may alter the test result.
- For complete identification, a biochemical and/or serological test should be done.
- Avoid using inoculum from a broth suspension to enable the growth and hydrolysis of urea.
- A false-positive alkaline reaction is observed if the culture is incubated for a long period of time.
- Do not put the urea agar slant in direct heat as urea is heat sensitive. Exposure to heat causes decomposition of urea.
- If you are testing Proteus species, make sure you check the inoculation for any reaction within six hours.
- Make sure you incubate the medium with a loose cap. Failure to do so can alter the result.
- Store the test tube in a dark place as urea undergoes auto-hydrolysis when exposed to light.
- Urea agar should not only be the basis for identifying the quantitative rate of urease activity because various organisms have different capability and hydrolysis rate. (1, 6, 9, and 10)
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