Urease Test for Mycobacteria – Principle, Procedure, Media and Results

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Principle

Urease is an enzyme possessed by many Mycobacterium species that can hydrolyze urea to form ammonia and carbon dioxide. The ammonia reacts in solution to form ammonium carbonate, resulting in alkalinisation and an increase in the pH of the medium.

Specimen

Type of Specimen

Mature colony of the unknown Mycobacterium species recovered from clinical material, grown on a Lowenstein–Jensen slant.

Materials

Equipment

  1. Biologic safety hood
  2.  37°C incubator

Medium

Urea Broth Preparation

  1. Add 10 g of Difco (Detroit, MI) Bacto urea agar base concentrate to 90 mL of sterile deionized water. Mix well to dissolve.
  2. Aseptically aliquot 3.0 mL portions into sterile screw-capped test tubes.
  3. Store at 2°C–8°C; shelf life is 1 month.

 Quality Control

Positive control: M. kansasii American Type Culture Collection (ATCC) 12478

Negative control: M. avium/M. intracellulare ATCC 13950

Procedure

  1. Inoculate each substrate tube with a loopful of growth from an actively growing culture. Thoroughly emulsify the culture in the broth.
  2. Incubate at 35°C for 5 days.
  3. Visually read the tubes at 1, 3, and 7 days.

Results

Color change from yellow to dark pink or red indicates a positive reaction. No color change is a negative reaction.


Procedure Notes

Repeat tests that appear slightly pink; if the repeat test result is still ±, report as negative.

Limitation of Procedure

  1. False-positive tests are usually due to incorrect interpretation of results, incubation beyond 5 days, or contaminated test or control cultures.
  2. False-negative results are due to the use of an incorrect medium (i.e., one used for testing bacteria other than acid-fast bacilli), insufficient inoculum, or cultures that are too old.

 Reference

  1. Kent PT, Kubica GP. Public Health Mycobacteriology: A Guide for the Level III Laboratory. Atlanta,
  2. GA: Centers for Disease Control, 1985. Department of Health and Human Services publication no.
  3. 86-21654-6.
  4. Murphy DB, Hawkins JE. Use of urease disks in the identification of mycobacteria. J Clin Microbiol
  5. 1975;1:465–468.



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