Urease Test – Principle, Media, Procedure, And Result

Before we get to testing, let’s take a second to talk about urease. You may be wondering what urease is and why we test for it. Well, ureases belong to a superfamily of amidohydrolases and phosphotriesterases.

They are found in lots of common things such as numerous bacteria, fungi, algae, and plants. 

So, what is the role of urease? What does it actually do? Well, urease is essential in the colonization of a host organism and it’s also incredibly important in the maintenance of bacterial cells in tissues. 

Urease Test - Principle, Media, Procedure, And Result

You might be wondering what any of this has to do with you. If it’s found in bacteria and fungi, why do we test for it in humans? The answer is pretty simple.

Due to its enzymatic activity, urease has quite a toxic effect on humans and so you’re going to want it in your system, ideally not at all, but if it is there, you’re going to want it out as quickly as possible.

This is why we have tests. The sooner you can test and know that urease is present, the sooner you can get the treatment needed to sort it. 

Principle Of The Urease Test. 

Urea’s a product of a chemical process of amino acids also known as decarboxylation. When urea is hydrolyzed it makes ammonia and carbon dioxide.

Ammonia in turn then alkalinizes the medium so the shift in pH can be detected by a change of color.

When you use Phenol red when it reaches 6.8 on the pH scale it turns orange in color, when it hits 8.1 on the pH scale it turns to a magenta color which is a shade of pink.

If the bacteria is present the medium should turn completely pink within one day (24 hours.)

Test One:  Rapid Urease Test 

This test is a really popular test for diagnosing Helicobacter Pylori.

Helicobacter Pylori is a type of bacteria that can infect your stomach and be quite difficult to cure as it is capable of becoming resistant to antibiotics. 

It’s a quick, cheap, and easy test that discovers urease’s presence in the mucus membrane layer of your stomach which is called your gastric mucosa.

The procedure is known as gastric endoscopy or gastric biopsy. During this procedure, they will collect cells in your stomach’s lining.

The test will usually take place at the same time as the gastroscopy. By taking a biopsy of the mucosa from the lower portion of your stomach (antrum.)

It will be then moved into a medium that contains the urea as well as an indicator like phenol red.

If H. pylori are present it will then hydrolyze the urea into ammonia, this will then raise the medium’s pH and change its color to red if it’s positive. The color will be yellow if H. pylori aren’t present. 

Test One:  Rapid Urease Test 

Test Two: Urea Breath Test

This test is also another option. It also tests for Helicobacter Pylori by monitoring urease activity. It’s a really popular option because it is both non-invasive and also highly specific and sensitive. 

If you go for this test you will ingest radioactively labeled urea. The two common types are carbon-14 (radioactive) and carbon-13 (nonradioactive) Now before you hear the word radioactive and get anxious, don’t panic.

Your body receives a very low and safe amount, there’s nothing to worry about. 

If there is an infection, the urease that is created by H. Pylori will hydrolyze the urea and form ammonia, much like in the aforementioned test, but this time will also produce labeled bicarbonate which is breathed out as carbon dioxide.

This labeled carbon dioxide can then be detected by an instrument that can detect and measure ionizing radiation (scintillation counter) for carbon-14 or by a mass correlation spectrometry for carbon-13. 

Test Two: Urea Breath Test

The Uses Of The Urease Test

There are lots of uses for a urease test, we’ve gone over one or two already but below you’ll find a more comprehensive list of reasons we may use one of the tests we’ve just learned about. 

  • This test can differentiate which, if any, organisms are present by how well urea is hydrolyzed by urease.
  • You can use this test to identify several genera as well as several species of Enterobacteriaceae (a type of bacteria) such as Proteus (responsible for gastroenteritis and UTI infections) and Klebsiella (responsible for pneumonia and meningitis.) 
  • It can also be helpful in identifying Cryptococcus spp (an infection associated with immunosuppressive individuals) Helicobacter Pylori (stomach infection) and plenty of other types of bacteria that create urease enzymes that can make you sick. 


Here I’m going to list a step-by-step guide as to how it’s prepared. 

  1. First, dissolve all of the aforementioned ingredients in 100ml of water (distilled) with 0.45 mm of filter sterilizer.
  2. Then you’ll want to hang the agar in about 900 ml of water (distilled) and then boil this so that it completely dissolves. 
  3. Next, autoclave or steam sterilize at 121 degrees C/249.8 degrees F for around 15 minutes. 
  4. Now leave the agar to cool until it comes down to around 50/55 degrees c or 122/131 degrees F.
  5.  Aseptically add 100ml of urea base to the agar solution, making sure that it has cooled, and then mix well. Your urea base should always be filter-sterilized.
  6. Distribute 4 or 5 ml per sterile tube then slant the tubes while they’re cooling until it has solidified. 

Urease Test – Result Interpretation

 The procedure of a urease test isn’t very complicated. It can be explained through 3 simple steps

  1. First, you’ll need to cover the exterior of the slant with a small amount of a well-isolated colony with about one or two drops from a 24-hour long brain-heart infused broth culture. 
  2. Keep the cap on but do so loosely. Then you can incubate the tube. It needs to be between 35-37 degrees C / 95-98.6 degrees F in room-temperature air for anywhere between two to seven days. 
  3. Finally, wait to see if it develops into a magenta (pink) color.  

If a deep magenta to vivid pink color develops in 15 minutes to 24 hours it is a strong indication of harmful bacteria being present. 

If there is no change in color it indicates that you do not have an extremely harmful bacteria present although it can still mean examples such as Salmonella may be present. 

The Limitations Of A Urease Test

Nothing is completely perfect, and so of course there are some limitations to using this kind of test. These are listed below. 

  • Organisms can differ – You have to be patient with this test, just because you do not rapidly turn the medium pink, doesn’t mean that it won’t. Organisms will split urea at different rates, some quick and some slow, so it’s important to keep checking for up to seven days. 
  • Complete identification – It is usually recommended that the types of tests be done on colonies from pure cultures for thorough identification which is not always possible with these tests. 
  • Don’t use inoculum if it comes from broth suspension – If you do this you will stunt growth as well as the hydrolysis reaction
  • Possible False Positives – After a prolonged incubation time you can sometimes get a false positive. 
  • Heated Urea Agar Slants – If the slants are heated the urea will decompose really quickly. 

Final Thoughts 

Although we have just gone through some limitations of these tests, ultimately they’re still really helpful tests to identify something toxic in your body and can potentially save lives.

If you are ever worried about any of the illnesses mentioned throughout this article and feel like you may need one of these tests, speak to your doctor as soon as possible.   

Jennifer Dawkins