Urease Test- Principle, Media, Procedure, Result, Uses
The urease test is a biochemical test that detects the presence of the enzyme urease in microorganisms. Urease is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The production of ammonia raises the pH of the medium, which can be detected by a color change of a pH indicator.
The urease test is useful for identifying and differentiating various bacteria, especially those that cause urinary tract infections, such as Proteus, Klebsiella, and some Citrobacter and Yersinia species. It can also be used to identify other bacteria that produce urease, such as Brucella, Helicobacter pylori, Cryptococcus, and some Corynebacterium species.
The urease test can be performed using different media, such as agar slants, broth tubes, or disks. The most commonly used medium is the urea agar base (Christensen agar), which contains urea and phenol red as a pH indicator. Phenol red turns from orange-yellow to bright pink when the pH rises above 8.4. The test can also be performed using a rapid urease test kit, which involves placing a gastric biopsy sample on a urea broth with a red phenol indicator and observing for a color change within minutes.
The urease test is simple, inexpensive, and reliable. However, it has some limitations, such as the variability in the speed of urease production among different organisms, the possibility of false-positive results due to peptone hydrolysis or autohydrolysis of urea, and the need for proper storage and buffering of the medium.
In this article, we will discuss the objectives, principle, procedure, result interpretation, control organisms, uses, and limitations of the urease test in detail.
The urease test is a biochemical test that detects the ability of microorganisms to produce the enzyme urease, which hydrolyzes urea into ammonia and carbon dioxide. The test has two main objectives:
- To test the ability of an organism to use urea as a sole source of nitrogen and produce alkaline fermentation.
- To differentiate urease-positive organisms, such as Proteus, from other members of the Enterobacteriaceae family and other bacteria that do not produce urease.
The test is useful for the identification of several genera and species of bacteria that are associated with human infections, such as urinary tract infections, gastric ulcers, brucellosis, and cryptococcosis. The test can also be performed as a rapid test on gastric biopsy samples to detect the presence of Helicobacter pylori, which is a major cause of peptic ulcers and gastric cancer.
The urease test is used to detect microorganisms that are capable of hydrolyzing urea with the enzyme urease. Urea is a product of the decarboxylation of amino acids and is found in urine and other body fluids. Hydrolysis of urea produces ammonia and carbon dioxide, which raise the pH of the medium and change the color of the indicator phenol red from orange-yellow to bright pink.
The urease test is especially useful for the identification of Proteus species, which are highly urease-positive and can cause urinary tract infections and wound infections. Proteus species can rapidly turn the entire medium pink within 24 hours or less. The test can also differentiate Proteus from other non-lactose-fermenting bacteria, such as Salmonella and Shigella, which are urease-negative.
Other members of the Enterobacteriaceae family that can be tested using the urease test include Klebsiella, Enterobacter, Citrobacter, Yersinia, and some Providencia and Morganella species. These organisms may produce weak positive reactions in 6 hours or more or delayed positive reactions after 24 hours or longer.
Besides Enterobacteriaceae, the urease test can also identify some fastidious Gram-negative rods, such as Brucella, Helicobacter pylori, and Pasteurella. Brucella and H. pylori are rapid urease-positive organisms that can cause serious infections such as brucellosis and gastritis. Pasteurella is a weakly positive organism that can cause animal bite infections.
Some Gram-positive rods, such as Corynebacterium and Rhodococcus, can also be tested using the urease test. Corynebacterium species are usually positive for urease, except for C. diphtheriae, which is negative. Rhodococcus species are variable for urease production.
Additionally, some yeasts, such as Cryptococcus, can also be detected by the urease test. Cryptococcus species are positive for urease and can cause cryptococcosis, a fungal infection of the central nervous system.
Therefore, the urease test is a valuable tool for the identification of various microorganisms that produce the urease enzyme and can cause different types of infections in humans and animals.
The urease test is based on the ability of some microorganisms to produce an enzyme called urease that can hydrolyze urea into ammonia and carbon dioxide. Urea is a nitrogenous compound that is produced by the breakdown of proteins in the body and excreted in urine.
The urease test uses a medium that contains urea and a pH indicator, such as phenol red. Phenol red is an acid-base indicator that changes color depending on the pH of the solution. It is yellow at acidic pH, orange at neutral pH, and pink at alkaline pH.
When a microorganism that produces urease is inoculated into the medium, it will break down the urea and release ammonia and carbon dioxide. The ammonia will raise the pH of the medium, making it more alkaline. This will cause the red phenol indicator to turn from orange-yellow to bright pink, indicating a positive urease test.
On the other hand, if a microorganism that does not produce urease is inoculated into the medium, it will not affect the urea or the pH of the medium. The red phenol indicator will remain orange-yellow, indicating a negative urease test.
The urease test can be performed on different types of media, such as agar slants, broth tubes, or disks. The agar slants and broth tubes are incubated for up to 7 days at 35°C or 30°C, depending on the organism tested. The disks are used for rapid tests that can give results within minutes or hours.
The urease test is useful for identifying and differentiating various microorganisms that produce urease, such as Proteus, Klebsiella, Yersinia, Brucella, Helicobacter pylori, Cryptococcus, and some Corynebacterium species. The test can also help diagnose infections caused by these microorganisms, such as urinary tract infections, gastric ulcers, brucellosis, and cryptococcosis.
There are different types of media that can be used to perform the Urease test, depending on the purpose and the organism to be tested. Some of the common media are:
- Urea agar base: This is also known as Christensen agar, and it contains urea, yeast extract, peptone, glucose, and phenol red as a pH indicator. The medium is prepared by dissolving the dehydrated powder in distilled water, boiling it to dissolve completely, autoclaving it at 121°C for 15 minutes, and adding sterile 40% urea solution after cooling it to 50°C. The medium is then dispensed into tubes and set as slants. The medium turns from orange-yellow to bright pink when urea is hydrolyzed by the urease enzyme.
- Urea broth: This is a liquid medium that contains urea, yeast extract, peptone, and phenol red as a pH indicator. The medium is prepared by dissolving the dehydrated powder in distilled water, boiling it to dissolve completely, autoclaving it at 121°C for 15 minutes, and adding sterile 40% urea solution after cooling it to 50°C. The medium is then dispensed into tubes or bottles. The medium turns from orange-yellow to bright pink when urea is hydrolyzed by the urease enzyme.
- Rapid urease test kit: This is a commercially available kit that contains a urea broth with a red phenol indicator and a plastic device with a well to hold a gastric biopsy sample. The kit is used for the rapid detection of Helicobacter pylori in gastric mucosa. The biopsy sample is placed in the well and covered with the broth. The device is then observed for the change in color from yellow to pink within 24 hours.
Some of the reagents and supplies that are used in the Urease test are:
- Sterile wooden sticks or loops: These are used to inoculate the media with the test organisms.
- Saline or water in a small plastic tube: This is used for the disk test that combines urea and phenylalanine deaminase (PDA) to identify Proteus, Providencia, and Morganella species.
- Incubator at 35°C and 30°C: This is used to incubate the media with the inoculated organisms for different periods of time depending on their growth rate and urease activity.
There are two main methods for performing the urease test: the agar slant method and the rapid urease test. The agar slant method is more commonly used for identifying urease-positive bacteria from the Enterobacteriaceae family and other genera. The rapid urease test is mainly used for detecting Helicobacter pylori from gastric biopsy samples.
Agar Slant Method
The agar slant method involves the following steps:
- Prepare the urea agar base by dissolving 24.52 grams of the dehydrated medium in 950 ml of distilled water in a beaker. Heat the solution to boiling to dissolve the medium completely. Sterilize the solution by autoclaving at 15 lbs pressure, 121°C for 15 minutes. Cool the solution to 50°C and add 50 ml of sterile 40% urea solution. Mix well and dispense into tubes. Set the tubes in a position to obtain agar slants.
- Inoculate a loopful of a well-isolated colony on the agar slant using a sterile wooden stick or loop. Do not stab the butt of the tube. Incubate the tubes with loosened caps at 35 to 37°C for up to 7 days. For non-fermenters, incubate at 30°C.
- Observe the tubes for color change from orange-yellow to bright pink, indicating an alkaline reaction due to urease activity. If no growth is seen on the slant, inoculate again with a heavy inoculum.
Rapid Urease Test
The rapid urease test, also known as the CLO test (Campylobacter-like organism), is used for the quick identification of Helicobacter pylori from gastric biopsy samples. The test involves the following steps:
- Take a biopsy of the mucosal layer of the antrum of the stomach using an endoscope and place it on a urea broth with a phenol red indicator in a small plastic tube.
- Observe the tube for color change from yellow to pink within 24 hours, indicating a positive reaction due to urease activity.
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The result of the urease test is based on the color change of the pH indicator in the medium due to the production of ammonia and carbon dioxide from the hydrolysis of urea. The color change indicates whether the organism is urease-positive or urease-negative.
- Urease-positive: The organism produces urease and splits urea into ammonia and carbon dioxide. This raises the pH of the medium and turns it alkaline. The pH indicator changes from its original orange-yellow color to bright pink. A positive test is demonstrated by an intense magenta to bright pink color in 15 min to 24 hours. Examples of urease-positive organisms are Proteus, Klebsiella, Yersinia, Cryptococcus, Brucella, and Helicobacter pylori.
- Urease-negative: The organism does not produce urease and does not split urea. The pH of the medium remains unchanged or slightly acidic. The pH indicator does not change color and remains orange-yellow. A negative test shows no color change. Examples of urease-negative organisms are Escherichia coli, Salmonella, Shigella, and Staphylococcus.
The result of the rapid urease test for Helicobacter pylori is also based on the color change of the pH indicator in the urea broth. The biopsy sample from the gastric mucosa is placed in the broth and incubated for a short time.
- Positive test: The presence of H. pylori in the biopsy sample indicates that the organism produces urease and splits urea in the broth. This raises the pH of the broth and turns it alkaline. The pH indicator changes from yellow to pink. A positive test is demonstrated by a pink color within 1 hour.
- Negative test: The absence of H. pylori in the biopsy sample indicates that there is no urease production and no urea hydrolysis in the broth. The pH of the broth remains unchanged or slightly acidic. The pH indicator does not change color and remains yellow. A negative test shows no color change within 1 hour.
Control organisms are used to verify the quality and performance of the urease test media and reagents. They are also used to compare the results of the test with known positive and negative reactions. Control organisms should be inoculated and incubated under the same conditions as the test organisms.
The following control organisms are commonly used for the urease test:
- Positive control: Proteus mirabilis (Urease positive bacteria)
- Negative control: Escherichia coli (Urease negative bacteria)
A positive control should produce a bright pink color change in the urea agar or broth within 24 hours, indicating a strong urease activity. A negative control should produce no color change or a yellow color change in the urea agar or broth, indicating a lack of urease activity.
If the control organisms do not show the expected results, the test should be repeated with fresh media and reagents. The test results of the unknown organisms should not be reported until the quality of the test is assured.
The urease test is used for various purposes in microbiology, such as:
- To identify organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide. This is a useful biochemical characteristic that helps to differentiate some bacteria from others. For example, the urease test can help to distinguish urease-positive Klebsiella from urease-negative Escherichia coli.
- To presumptively identify Proteus species from other members of the Enterobacteriaceae family. Proteus species are known to produce large amounts of urease, which results in a rapid and strong alkaline reaction in the urea medium. This can be observed as a bright pink color change within 15 minutes to 24 hours. Other Enterobacteriaceae may also produce urease, but at a slower rate and with a weaker reaction.
- To differentiate Proteus from other non-lactose-fermenting bacteria. Proteus species are non-lactose-fermenting bacteria that can be confused with other similar bacteria, such as Salmonella, Shigella, and Yersinia. However, Proteus species are usually urease-positive, while the others are usually urease-negative. Therefore, the urease test can help to rule out Proteus from the differential diagnosis of non-lactose-fermenting bacteria.
- To perform a rapid test on gastric biopsy samples to detect the presence of Helicobacter pylori. Helicobacter pylori is a gram-negative bacterium that causes gastritis, peptic ulcers, and gastric cancer. It is able to colonize the human stomach by producing large amounts of urease, which neutralizes the acidic environment and protects the bacterium from the host`s immune system. The rapid urease test, also known as the CLO test (Campylobacter-like organism), is a simple and inexpensive method to diagnose H. pylori infection. It involves placing a gastric biopsy sample on a urea broth with a red phenol indicator and observing for a color change from yellow to pink within 24 hours. A positive result indicates the presence of H. pylori in the stomach.
These are some of the main uses of the urease test in microbiology. The test is easy to perform, inexpensive, and reliable for detecting urease-producing organisms. However, it also has some limitations that should be considered when interpreting the results.
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The urease test is a simple and useful method to identify urease-producing microorganisms, but it also has some limitations that should be considered. Some of these limitations are:
- The urease test may not be reliable for some slow-growing or fastidious organisms, such as Brucella and Helicobacter pylori, which may require longer incubation times or special media to show a positive reaction. Therefore, the test should be performed in conjunction with other biochemical and serological tests for confirmation.
- The urease test may give false-positive results if the medium contains peptone or other protein sources that can be hydrolyzed by some bacteria to produce ammonia. This can raise the pH of the medium and change the color of the indicator. To avoid this, the medium should be free of peptone or other nitrogenous compounds, or a control tube without inoculum should be included to compare the results.
- The urease test may give false-negative results if the medium is not properly buffered or if the inoculum is too small or too old. A buffered medium is necessary to maintain a stable pH and prevent the neutralization of the alkaline reaction by the acidic products of bacterial metabolism. A sufficient amount of fresh inoculum is also important to ensure adequate enzyme activity and visible color change.
- The urease test may be affected by light exposure, which can cause the degradation of urea and the formation of ammonia. This can lead to a spontaneous color change in the medium without inoculation. To prevent this, the medium should be stored in a dark place at low temperatures and used within its expiry date.
- The urease test may not be specific for some organisms that produce other enzymes that can hydrolyze urea, such as amidases or arginases. These enzymes can also generate ammonia from urea or other substrates and cause a similar alkaline reaction. To differentiate these organisms, additional tests such as the arginine dihydrolase test or indole test may be required.
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