Cat. no. L65 Urea Agar, 16x100mm Tube, 5.5ml Slant 20 or 100 tubes/box
Cat. no. R42 Urea Agar, 13x100mm Tube, 3ml Slant 20 or 100 tubes/box


Hardy Diagnostics Urea Agar is recommended for use in the detection of urea hydrolysis in gram-negative organisms.


Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli, as an improvement over the broth method used at the time.(3) Urea Agar is used to differentiate between rapidly positive Proteus species and other slower urea positive members of the Enterobacteriaceae. This medium may also be used in the detection of urease activity in other gram-negative organisms, such as Pseudomonas, Pasteurella, and Brucella.(5) Webb, et al. also reported that Urea Agar is useful in differentiating Cryptococcus from other yeast species.(9)

Urea Agar contains urea and phenol red as the pH indicator. Organisms capable of hydrolyzing urea form ammonia as a by product, thus turning the medium alkaline. The pH indicator turns from pale yellow to pink-red in color in these conditions. The reduced buffer content and peptone in this medium promote more rapid growth and reaction time for many members of the Enterobacteriaceae. Dextrose is included in the formulation to stimulate urease activity in organisms that hydrolyze urea slowly, and to exclude false-negative reactions. Proteus species rapidly hydrolyze urea, and a positive reaction is usually seen within one to six hours. Other organisms perform this reaction slower, and may require a 24 to 48 hour, or longer, incubation time.


Ingredients per liter of deionized water:*

Urea 20.0gm
Sodium Chloride 5.0gm
Monopotassium Phosphate 2.0gm
Peptone 1.0gm
Dextrose 1.0gm
Phenol Red 0.012gm
Agar 15.0gm

Final pH 6.7 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of Use: Allow Urea Agar to warm to room temperature before use. Using a sterile loop, pick up three to four isolated colonies from a pure culture. Streak back and forth over the surface of the slant. Do not stab the butt, as it serves as a color control. Incubate aerobically at 35ºC. with a loosened cap. Examine after 2, 6, and 24 hours for results. If a positive reaction is not seen after this time, examine daily for up to six days to detect organisms that hydrolyze urea slowly.


A positive urease reaction is indicated by a color change to red-pink. Consult listed references for the identification of colony morphology and further biochemical tests required for identification.(1,2,4,7)


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.


Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Proteus mirabilis
ATCC ® 12453
E 18-24hr 35°C Aerobic Positive: growth; pink color change
Escherichia coli
ATCC ® 25922
E 18-24hr 35°C Aerobic Negative: growth; yellow color change

User Quality Control


Urea Agar, Slants should appear opalescent, and light orange in color.

P. mirabilis growing on Urea Agar

Proteus mirabilis (ATCC ® 12453) growing on Urea Agar (Cat. no. R42). The pink color change was indicative as positive for urea hydrolysis. Incubated aerobically for 24 hours at 35ºC.

E. coli growing on Urea Agar

Escherichia coli (ATCC ® 25922) growing on Urea Agar (Cat. no. R42). The absence of a pink color change was indicative as negative for urea hydrolysis. Incubated aerobically for 24 hours at 35ºC.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Christensen, W.B. J. Bacteriol.; 52:461.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. King, E.O. 1960. The Identification of Unusual Pathogenic Gram Negative Bacteria, U.S.D.H.E.W., CDC, Atlanta, GA.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

9. Webb, C.D., et al. 1973. Identification of Yeasts, U.S.D.H.E.W., CDC, Atlanta, GA.