3BMT MICR 231 LEC Bacteriology

Biochemical Tests for Bacterial Identification

Urease Test (Christensen's Method)

Purpose:
Determination of an organism's ability to
produce urease, an enzyme which hydrolyzes
urea. Presumptive identification of Proteus sp.
may be possible through its ability to rapidly
hydrolyze urea.

Priniciple:
Urea is the product of decarboxylation of amino
acids. Hydrolysis of urea produces ammonia
and CO
2
. 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.

Media:
Enzymatic digest of gelatin (1 g), dextrose (1 g),
NaCl (5 g), KH
2
PO
4
(2 g), urea (20 g), phenol red
(0.012 g), per 1000 mL, pH 6.8.

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



Expected Results:
Positive: Change in color of slant from light
orange to magenta.
Negative: No color change (agar slant and butt
remain light orange)

Limitations:
Alkaline reactions may appear after prolonged
incubation and may be the result of peptone or
other protein utilization raising the pH. To
eliminate false-positive reactions, perform a
control test with the base medium without
urea.

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