UREASE TEST

UREASE TEST

AIM

            To determine the ability of an organism to produce an exoenzyme Urease and degrade Urea.

PRINCIPLE     

    The alkaline fermentation of urine (urea) with the resultant production of ammonia was hypothesized to occur due to the action of microorganisms by Reoch in 1875. Christensen later formulated a medium that would allow growth of other members of the Enterobacteriaceae that cannot utilize the by-product of urea hydrolysis (ammonia) as a sole nitrogen source for growth.

       Urea is a nitrogen containing compound that is produced during Decarboxylation of the amino acid Arginine in the Urea cycle. Urea is a diamide of Carbonic acid. Many organisms (Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae and Helicobacter pylori) especially those that infect the Urinary tract have Urease enzyme which is able to split Urea in the presence of Water to release Ammonia and Carbon-di-oxide. The Ammonia combines with Carbon-di-oxide and Water to form Ammonium carbonate which turns the Christensen’s urea agar medium Alkaline (from pH 6.8 to pH 8.1), turning the indicator Phenol red from its original Orange yellow (pH 6.8) color to Bright pink (pH 8.1). Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production.

      Christensen’s urea agar is widely used for the Urease test but sometimes Stuart’s Urea broth is also used. In Stuart’s urea broth, Urease production is indicated by a Pink color throughout the broth.

Figure – 1: Splitting of Urea into Ammonia by Urease

MATERIALS REQUIRED

1. Test bacteria
2. Christensen urea agar slant
3. Inoculation loop
4. Incubator

PROCEDURE

a)   Streak the surface of a Christensen urea agar slant with a portion of a well-isolated bacterial colony.

b)      Incubate the inoculated Christensen urea agar slant at 37 °C  for 24 hours in an incubator.

c)     Examine for the development of Pink colour.

d)     In case of unknown result incubate the tube for 7 days to check for slow Urease production.

OBSERVATION AND RESULTS

·   Positive Urease Test – The color of the slant changes from light Orange to Pink. Organisms that hydrolyze Urea rapidly (e.g. Proteus sp.) may produce positive reactions within 1 or 2 hours. Less active species (e.g. Klebsiella sp.) may require 3 or more days. In routine diagnostic laboratories, the Urease test result is read within 24 hours.

·       Negative Urease Test - The agar slant and butt remain light orange (medium retains original color).

(Left – Urease negative; Right – Urease positive)

Figure – 2: Urease test