What is Urease Test?

Urease test is one of the biochemical tests which helps in detecting the fermentation of alkaline in the urine which results in the production of ammonia by the microorganisms.

The alkaline fermentation results in the formation of urea. Usually, the fermentation process of urea happens in the presence of an enzyme “Urease”, which results in the formation of two molecules of ammonia and carbon dioxide.

This test was first developed by Christensen in the year 1946 to differentiate a pathogen enteric bacilli.

Christensen used Urea Agar base initially to test the urease, so it was later named after him as Christensen Urea Agar.

Urease activity is considered as an important characteristic for identifying the species Proteus.

Urease activity helps to identify the proteus from the non-lasting fermentation members of the Enterobacteriaceae.

During Urease test, the organisms tend to utilize urea as the source of nitrogen which helps in producing enough amount of ammonia to overcome the buffering capacity of the culture medium. When the color of the medium changes due to pH, it conveys the test result.

Objective of Urease Test

To test the ability of an organism to synthesize the enzyme urease, which hydrolysis the enzyme urease into urea.

This test also enables us to differentiate Urease-positive proteus from other Enterobacteriaceae

Microorganisms Types and Urease Test

Urease test helps in identifying the following microorganisms.

1. Gram negative enteric pathogens including those of Yersinia spp

2. Fastidious Gram-negative rods-Brucells, Pasteurella, Helicobacter pylori.

3. Gram-positive rods-Corynebacterium and Rhodococcus spp.

4. Yeasts-Cryptococcus spp. This test is performed directly as a rapid test to detect the presence of H. pylori in the gastric biopsy samples.

Urease Test Principle

Medium of the urea culture is made up of either broth or agar, which contains urea and phenol red acts as pH indicator. Many of the organism especially which causes urinary tract infections, produces an enzyme urease, and catalyzes the splitting of urea in the presence of water, and releases tow molecules of ammonia and the carbon dioxide the ammonia then combines with the carbondioide and turns the medium into alkaline thud turning the indicator from its natural orange yellow to bright pink color.

This test is performed as performed to identify the several genera and species of the Enterobacteriaceae family including some species like Klebsiella, Proteus, and some Citrobacter and Yersinia and also some of the species of Corynebacterium.

This test also helps in identifying the pathogens such as Cryptococcus, Brucella, Helicobacter pylori and other bacteria’s which produces the enzyme ureases.

Disks are available to combine Urea and the phenylalanine deaminase, which allows one-disk test is used to identify the species proteus, Morganells and Providencia. it also helps in separating them from Klensiella and Yersinia enterocolitica.

The disk reactions are rapid and sensitive and allows the rapid detection of disease-causing agents such as Brucella and cryptococcus.

Reagents, Media and Supplies Required for Urease Test

Both agar slant and broth media are used for detecting urease production. Agar media includes the Urea Agar Base namely Christensen Agar and the broth includes Urea broth. Besides the usage of rapid test kits are available for detecting the activity of urease.

The composition of the urea and agar base is listed below.

Ingredients	Gram/liter
Dextrose	1.0
Sodium chloride	5.0
Peptic digest of animal tissue	1.5
Monopotassium Phosphate	2.0
Agar	15.00
Phenol red	0.012

Supplies:

Loops or sterile wooden sticks are used. Saline or water in a small plastic tube for the disk test, Incubator at 35ºC and 30ºC.

Urease Test Procedure

1. Preparation of media

 Initially 24.52 grams of dehydrated medium is dissolved in the 950ml of distilled water in a beaker.

 The solution is heated to bring into boil in order to dissolve it in the medium completely.

 Then the prepared suspension is sterilized by autoclaving at 15 lbs. pressure for 121º for 15 minutes.

 after sterilizing the beaker is taken out and cooled to 50ºC.

 Then 50ml of sterilized 40 percentage of urea solution is added to the beaker and mixed well.

 The medium is then dispensed into the test tubes and they are set in a position to obtain an agar slant.

2. Urease Activity

 To test the urease activity a loop full of isolated colony is taking with the help of an inoculating loop and it is inoculated further on the agar slant that were kept prepared.

 The inoculation is done on just the slant it must be checked often that it should not be stabbed.

 Then the tubes are incubated by loosening their caps at 35 to 37ºC. When using non-fermenters, it is incubated at 30ºC.

 Now the tubes are observed for the appearance of pink color for as long as 7 days.

 If there is no change or growth is seen on the agar slant, further inoculation using heavy inoculum can be done.

3. Rapid Urease Test

 This test is often called as CLO test which is Campylobacter-like Organisms, and it is commonly used for rapid identification of Helicobacter pylori.

 For performing this test biopsy is taken from the mucosal layer of the antrum of the stomach and the sample taken is placed in the urea broth and phenol red indicator is added.

 The tube is then noted for the change from yellow to pink color.