- 1 Urease test principle
- 2 Composition of Christensen’s Urea Agar
- 3 Preparation of Christensen’s Urea Agar
- 4 Composition and preparation of Stuart’s Urea Broth
- 5 Procedure for urease test
- 6 Result and Interpretation
- 7 Diagnostic utility of Urease test
- 8 Principle of Urea Breath Test:
Urease test principle
Composition of Christensen’s Urea Agar
|Sodium chloride||5 g|
|Potassium phosphate, monobasic||2 g|
|Phenol red||0.012 g|
|Agar||15 to 20 g|
Preparation of Christensen’s Urea Agar
- Dissolve the first six ingredients in 100 ml of distilled water and filter sterilize (0.45 mm pore size).
- Suspend the agar in 900 ml of distilled water, boil to dissolve completely, and autoclave at 121°C and 15 psi for 15 minutes
- Cool the agar to 50-55 °C
- Aseptically add 100 ml of filter-sterilized urea based to the cooled agar solution and mix thoroughly
- Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling until solidified (It is desirable to have a long slant and short butt).
Composition and preparation of Stuart’s Urea Broth
|Yeast extract||0.1 g|
|Potassium phosphate, monobasic||9.1 g|
|Potassium phosphate, dibasic||9.5 g|
|Phenol red||0.01 g|
- Dissolve all ingredients in 1 liter of distilled water and filter sterilize (0.45-mm pore size).
- Distribute 3 ml of prepared broth per sterile tube (13X100 mm).
Note: Prepared media (both Christensen’s Urea Agar and Stuart’s Urea Broth) will have a yellow-orange color. Once prepared, do not reheat the medium as the urea will decompose. Prepared media can be stored in the refrigerator at 4 to 8 °C until needed.
Procedure for urease test
For Christensen’s Urea Agar
- Streak the entire slant surface with a heavy inoculum from an 18-24 hour pure culture (Do not stab the butt as it will serve as a color control).
- Incubate tubes with loosened caps at 35°C.
- Observe the slant for a color change at 6 hours and 24 hours unless specified for longer incubation.
For Stuart’s Urea Broth
- Inoculate the broth with a heavy inoculum from an 18-24 hour pure culture
- Shake the tube gently to suspend the bacteria
- Incubate the tubes with loosened caps at 35°C.
- Observe the broth for a color change at 8, 12, 24 hours.
Result and Interpretation
Organisms that hydrolyze urea rapidly (Proteus spp., Morganella morganii, and some Providencia stuartii strains) will produce strong positive reactions within 1 or 6 hours of incubation; delayed positive organisms (e.g. Klebsiella spp and Enterobacter species ) will produce weak positive reactions in the slant in 6 hours of incubation which will be intense during further incubation. The culture medium will remain a yellowish color if the organism is urease negative e.g. Escherichia coli.
In routine diagnostic laboratories the urease test result is read within 24 hours.
- If organism produces urease enzyme, the color of the slant changes from light orange to magenta.
- If organism do not produce urease the agar slant and butt remain light orange (medium retains original color).
If Stuart’s Urea Broth is used; rapidly urease positive organisms (Proteus spp., Morganella morganii) will produce a strong positive reactions within 8-24 hours of incubation but delayed positive organisms (e.g., Enterobacter) will not produce a positive reaction due to high buffering capacity of this medium.
Diagnostic utility of Urease test
- Urease test helps for the identification of Proteus species (urease positive) and to differenitate it from other non-lactose fermenting members of the Enterobacteriaceae family.
- Urease test is used for the presumptive evidence of the presence of Helicobacter pylori in tissue biopsy material. This is done by placing a portion of crushed tissue biopsy material directly into urease broth. A positive urease test is considered presence of Helicobacter pylori. Commercially available urease agar kits are also available.
- Rapid Urease test is can be used to differentiate between the yeasts, Candida albicans and Cryptococcus neoformans. A presumptive identification of C. neoformans may be based on rapid urease production, whereas Candida albicans do not.
- Urea breath test: A common noninvasive test to detect Helicobacter pylori also based on urease activity. This is highly sensitive and specific test.
Principle of Urea Breath Test:
Patient ingests radioactively labeled (13C or 14C) Urea. If infection is present, the urease produced by Helicobacter pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO2. The labeled CO2 is detected either by a scintillation counter or a special spectrometer.
- Proteeae (Proteus spp., Morganella morganii, and some Providencia stuartii strains)
- Cryptococcus spp
- Corynebacterium spp
- Helicobacter pylori
- Brucella spp
Note: Mneomonics to remember urease positive organisms: PUNCH (Similar mneomonic for oxidase positive organism is PVNCH)
- P: Proteus
- N: Nocardia
- C: Cryptococcus neoformans/Corynebacterium spp
- H: Helicobacter pylori
References and further reading
- Benita Brink. 2010. Urease test protocol. American Society for Microbiology