Urease Test

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Urease test is an analytical method which is practised to identify the urease positive and negative organisms based on the production of cytosolic urease enzyme. It distinguishes the Proteus species from the non-lactose fermenting members belonging to the Enterobacteriaceae family. Urease test makes the use of urea base. Conventionally, Stuart formulated urea broth was used to identify the rapid urease positive Proteus species.

Later on, Christensen formulated urea agar was used to identify slow urease positive organisms belong to the Enterobacteriaceae family. Urease test comes into use to check whether the microorganism is capable of producing “Urease” or not. It involves splitting of urea into end products like alkaline ammonia and carbon dioxide, by the addition of water.

Content: Urease Test

  1. Definition
  2. Overview
  3. Principle
  4. Test Media
  5. Procedure
  6. Test Results

Definition

Urease test is a biochemical method that demarcates the difference between rapid positive, slow positive and negative urease test organisms, based on the existence of urease enzyme. Urease catalyses the hydrolysis of urea into ammonia and carbon dioxide. The test results of the urease test interpret by any colour change in the pH indicator (phenol red). The pH at 6.8 and between 7.5-8.1, the colour of the phenol red indicator appears yellow-orange and red, respectively. The pH above 8.2, the colour of the phenol red turns fluorescent pink.

Urea is an acidic compound, which on hydrolysis produces alkaline by-product, namely ammonia. Therefore, the colour change of the pH indicator is due to the formation of the ammonia end product.

Overview

Before discussing the theory of the urease test, let us study a few general terms that we must have an idea of.

Urease

It also refers as “Urease aminohydrolase”. Urease is a cytosolic enzyme that breaks down urea into by-products, namely carbon dioxide and ammonia. It functions as a nitrogen metabolizer, as it degrades the chief nitrogenous product “Urea”. Urease is a nickel-containing metalloenzyme.

It exists in archaea, bacteria, unicellular eukaryotes etc. and a member of the Aminohydrolases and phosphodiesterases superfamily. Urease is dependent on the substrate like urea and hydroxyurea. Its reactivity can alter from species to species.

Urea

It can define as a chief nitrogenous end product, which forms by the decarboxylation of protein subunits (amino acids). Urea is a diamide of carbonic acid. It is a chemical compound that comprises two –NH2 group connected to the other via the carbonyl (C=O) functional group.

Urea Broth

A scientist named Stuart formulated urea broth, and that’s why also refers by the name of Stuart’s Urea broth media. It was commonly used to get rapid results of urease test, and the bacteria giving quick positive results are Proteus sp, Providencia sp etc. This media has a high buffering capacity and limited nutrients, due to which the organisms degrading urea slowly, cannot be identified.

Urea Agar Media

A scientist named Christensen formulated urea agar in the year 1946, and that’s why the media is named after the name of the scientist. It contains urea base and a pH indicator (phenol red). The peptone contained in the media is to encourage faster growth and reaction time of the organisms. Dextrose contained in the media helps in stimulation of urease activity in the microorganisms to make them efficient to hydrolyze urea.

Principle of Urease Test

The mechanism of urease test depends on the hydrolysis reaction catalyzed by the production of urease enzyme. The nitrogenous compound “Urea” splits into the by-products like ammonia and carbon dioxide by the aid of urease. The organisms capable of hydrolyzing acidic urea (Proteus sp, Providencia sp etc.) can produce a sufficient amount of basic ammonia and a gaseous end-product carbon-dioxide.

The ammonia further protonates into ammonium and hydroxyl ion that causes alkalinity. Thus, the formation of base results in changing the pH value of the pH indicator that in turn, changes the colour of the media. The pH shift of the media from acidic (6.8) to alkaline (> 8.2), changes the colour of the media from yellow-orange to pink-red.

Principle of Urease Test
Few organisms like Klebsiella sp and Enterobacter sp can change the pH as well as the media’s colour, but give delayed result by slowing metabolizing the urea. Some microorganisms like Escherichia coli lack urease enzyme, as a result of which, they can neither split urea nor change the colour of media.

Test Media

Urease test makes the use of either Christensen’s urea agar medium or Stuart’s urea broth medium.

Stuart’s Urea Broth

The composition and preparation of Stuart’s urea broth include:

Composition of Stuart’s urea broth medium:
Yeast extract: 0.1 g
Potassium phosphate (monobasic): 9.1 g
Potassium phosphate (dibasic): 9.5 g
Urea: 20 g
Distilled water: 1L
Final pH: 6.8 0.2 at 25 degrees Celsius

Preparation: To prepare urea broth, accurately weigh the above ingredients of Stuart’s media and add 1000 ml of distilled water. Filter sterilize the solution with the filter paper having a pore size of 0.45mm. After that, autoclave the broth medium at 121 degrees Celsius for 15-20 minutes.

Christensen’s Urea Agar

The composition and preparation of Christensen’s urea agar include:

Composition of Christensen’s urea agar medium:
Urea: 20 g
Monopotassium phosphate: 2 g
Peptone: 1 g
Dextrose: 1 g
Phenol red: 0.012 g
Agar: 15 g
Distilled water: 1L
Final pH: 6.7 0.2 at 25 degrees Celsius

Preparation: To prepare urea agar slant, accurately weigh the above six contents of the Christensen’s media and add 100 ml of distilled water. Filter sterilize the solution containing urea base and 100ml of distilled water through the filter paper having 0.45mm porosity. Take 20grams of agar media into the sterile flask and autoclave it at 121 degrees Celsius for 15-20 minutes.

Allow to stand the agar media until it reaches the temperature of 50-55 degrees Celsius, and aseptically transfer the 100ml of a solution containing urea base into it. Thoroughly mix the contents by shaking the flask back and forth. Then, add 3-4ml of the media into sterile tubes and keep them in an inverted position until it solidifies. After solidification, store the test tubes at 4-8 degrees Celsius under refrigeration.

Procedure

The protocol of the urease test includes the following steps:

Experiment using Stuart’s urea broth media:
Urease test in Stuart's media

  1. After refrigerating the media, transfer the tubes into the laminar airflow.
  2. Dip the sterilized inoculating loop into Stuart’s urea broth medium.
  3. Incubate the culture tubes for 16-24 hours at 35 degrees Celsius.
  4. Observe the culture tubes for the appearance of a pink-red colour.

Experiment using Christensen’s urea agar media:
Protocol of urease test in Christensen's media

  1. After refrigerating the media, transfer the tubes into the laminar airflow.
  2. Sterilize the inoculating loop on the flame and streak the test organism from the isolated colony over the Christensen’s urea agar media.
  3. Incubate the culture tubes for at least 16 hours at 35 degrees Celsius.
  4. Regularly observe the culture tubes for any colour change, as few bacteria take time to split urea.

Test Results

The test results of the urease test in Christensen’s urea agar and Stuart’s urea broth media can be concluded simultaneously, by the given result interpretations.

Test Result Of Urease Test

Positive result: The whole media turns fluorescent pink-red colour within 16-24 hours, indicates a positive urease test.
Delayed positive result: The media slightly turns pink in colour and requires prolonged incubation of 6 days.
Negative result: The colour and the pH of the media will remain the same.

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