Urease Test

Urease Test
The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease.

Principle 

Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours.

Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production.

Uses 

1.    This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.

2.    This test can be used as part of the identification of several genera and species of Enterobacteriaceae, including Proteus, Klebsiella, and some Yersinia and Citrobacter species, as well as some Corynebacterium species.

3.    It is also useful to identify Cryptococcus spp., Brucella, Helicobacter pylori, and many other bacteria that produce the urease enzyme.

4.    Directly, this test is performed on gastric biopsy samples to detect the presence of H. pylori.

Media 

Composition

Urea                                       20.0 gm

Sodium Chloride                   5.0 gm

Monopotassium Phosphate   2.0 gm

Peptone                                 1.0 gm

Dextrose                                1.0 gm

Phenol Red                            0.012 gm

Agar                                      15.0 gm

Final pH 6.7 +/- 0.2 at 25 degrees C.

Preparation

1.    Dissolve the ingredients in 100 ml of distilled water and filter sterilize (0.45-mm pore size).

2.    Suspend the agar in 900 ml of distilled water, boil to dissolve completely.

3.    Autoclave at 121 degree C and 15 psi for 15 minutes.

4.    Cool the agar to 50 to 55 degree C.

5.    Aseptically add 100 ml of filter-sterilized urea base to the cooled agar solution and mix thoroughly.

6.    Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling until solidified.

Procedure 

1.    Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with 1 to 2 drops from an overnight brain-heart infusion broth culture.

2.    Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 48 hours to 7 days.

3.    Examine for the development of a pink color for as long as 7 days.

Result 

Positive Reaction: Development of an intense magenta to bright pink color in 15 min to 24 h.
Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter pylori, Yersinia spp, Brucella spp, etc.

Negative Reaction:  No color change.
Examples: Escherichia, Shigella, Salmonella, etc.

Quality Control 

Positive: Proteus vulgaris (ATCC13315)
Weak positive: Klebsiella pneumoniae (ATCC13883)
Negative: Escherichia coli (ATCC25922)

References

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