Principle
Some bacteria have the ability to hydrolyse (breakdown in presence of water) urea to NH3 and CO2, as they can produce the enzyme ‘urease’. NH3 is alkaline (basic), due to which, it increases the pH changing the color of phenol red from yellow to pink.
In the urease test, the test bacteria are grown on agar slants containing urea and phenol red. If the bacteria have the ability to hydrolyse urea, the color of the medium changes from yellow to pink. Some bacteria may produce NH3 by breaking down the peptones in the medium. In such cases false positive result is obtained.
Experiment
Reagent And Material Used
- Test tubes
- Conical flask
- Cotton plugs
- Inoculating needle
- Autoclave
- Bunsen burner
- Laminar flow chamber
- Dispose jar
- Incubator
- Membrane filtration apparatus
- Urea agar
- Isolated colonies or pure cultures of bacteria
Procedure
- Five test tubes are cotton-plugged, covered with craft paper and tied with thread or rubber band.
- The ingredients of urea agar medium (containing urea as the main component) or its ready-made powder (except urea) required for 100 ml of the medium is weighed and dissolved in 90 ml of distilled water in a 250 ml conical flask by shaking and swirling.
- Its pH is determined using a pH paper or pH meter and adjusted to 6.9 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
- The flask is heated to dissolve the agar in the medium completely.
- The flask is cotton-plugged, covered with craft paper and tied with thread or rubber band.
- The five test tubes and the conical flask containing urea agar (except urea) are sterilized at 121°C (15 psi pressure) for 15 minutes in an autoclave.
- After sterilization, they are removed from the autoclave and allowed to cool for sometime without allowing the medium to solidify. Cooling of the medium prevents condensation and accumulation of water droplets on the slants. If the medium has already been prepared and solidified during storage, it has to be liquefied by heating carefully till it melts completely.
- 20% urea solution is prepared by dissolving 20 grams of urea in 100 ml of distilled water and is sterilized by membrane filtration apparatus, because urea cannot be sterilized by heat, as it decomposes upon heating.
- 10 ml of the sterilized urea solution is aseptically mixed thoroughly with 90 ml of the sterilized cooled liquefied medium (liquefied by heating).
- Before it solidifies, the medium, in warm molten condition, is distributed aseptically into the 5 cotton-plugged sterilized test tubes (approximately 20 ml each).
- The test tubes are kept in a slanting position to cool and solidify the medium, so as to get urea agar slants.
- The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the slants by stabbing into the butt and streaking on the surface of the slants with the help of a flame- sterilized needle. The needle is sterilized after each inoculation.
- The inoculated slants are incubated at 37°C for 24 hours in an incubator.
Observations
1. Pink color produced: Urease positive.
Examples:Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter pylori, Yersinia spp, Brucella spp, etc.
2. Pink color not produced: Urease negative.
Examples:Escherichia, Shigella, Salmonella, etc.
Uses of Urease Test
- This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.
- 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.
- It is also useful to identify Cryptococcus spp., Brucella, Helicobacter pylori, and many other bacteria that produce the urease enzyme.
- Directly, this test is performed on gastric biopsy samples to detect the presence of H. pylori.
Limitations of Urease Test
- Some organisms rapidly split urea (Brucella and H. pylori), while others react slowly.
- It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.
- To facilitate growth and the urea hydrolysis reaction, do not use inoculum from a broth suspension.
- After prolonged incubation times a false-positive alkaline reaction may be seen. To rule out this occurrence, check the test with a control (an uninoculated tube of Urea Agar) along with the inoculated tube during prolonged incubation.
- Do not heat the Urea Agar Slants, as urea decomposes very readily when heated.
- To detect Proteus species, the Urea Agar, Slants must be examined within 6 hours of inoculation for a reaction.
- Urea Agar should not be used to determine the quantitative rate of urease activity, as organisms vary in their capability and rate of hydrolysis.
- Failure to incubate this medium with loose caps may cause erroneous results to occur.
- Urea is light sensitive and can undergo autohydrolysis. Store at 2 to 8C in the dark.