Urea Media

Urea Agar Base • Urea Agar Base Concentrate 10

Urea Agar • Urea Broth • Urease Test Broth
Urease Broth Concentrate 10
Intended Use morganii, Providencia rettgeri, and a few Providencia stuartii
Urea Agar and Urease Test Broth are used for the differentiation strains with the reclassification of the members of the Proteeae.
of organisms, especially the Enterobacteriaceae, on the basis of Urease base is also supplied as a filter sterilized 10 concentrated
urease production. solution for use in preparing Urease Test Broth in the laboratory.

Summary and Explanation Principles of the Procedure

Urea Agar was devised by Christensen for use as a solid The urea medium of Rustigian and Stuart3 is particularly suited
medium for the differentiation of enteric bacilli.1 It differentiates for the differentiation of Proteus species from other gram-
between rapid urease-positive Proteeae organisms (Proteus spp., negative enteric bacilli capable of utilizing urea;1 the latter
Morganella morganii subsp. morganii, Providencia rettgeri, are unable to do so in Urease Test Broth because of limited
and some Providencia stuartii) and other urease-positive nutrients and the high buffering capacity of the medium. To
organisms: Citrobacter, Enterobacter and Klebsiella and provide a medium with greater utility, Urea Agar was devised
bacteria other than Enterobacteriaceae; i.e., some Bordetella by Christensen1 with peptone and dextrose included and
and Brucella spp.2 reduced buffer content to promote more rapid growth of many
The base is also supplied as a filter-sterilized 10 concentrated of the Enterobacteriaceae and permit a reduction in incubation
solution in tubes for use in preparing Urea Agar slants in the time. The complete Urea Agar contains 15.0 g/L of agar in
laboratory. addition to the ingredients in the base medium.
Urease Test Broth was developed by Rustigian and Stuart.3 When organisms utilize urea, ammonia is formed during
It may be used for the identification of bacteria on the basis incubation which makes the reaction of these media alkaline,
of urea utilization and it is particularly recommended for the producing a red-pink color. Consequently, urease production
differentiation of members of the genus Proteus from those of may be detected by the change in the phenol red indicator.
Salmonella and Shigella in the diagnosis of enteric infections.4
The medium is positive for Proteus, Morganella morganii subsp.

Difco™ & BBL™ Manual, 2nd Edition

 User Quality Control
NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the
development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications Identity Specifications

Difco™ Urea Broth BBL™ Urea Agar Base
Dehydrated Appearance: Light orange to light pink, homogeneous, Dehydrated Appearance: Fine, homogeneous, free of extraneous
inherently lumpy. material.
Solution: 3.87% solution, soluble in purified water. Solution: 29 g/100 mL solution, soluble in puri-
Solution is orange-yellow, clear. fied water. Complete medium is light to
Prepared Appearance: Orange-yellow, clear. medium, orange, clear to slightly hazy.
Reaction of 3.87% Prepared Appearance: Complete medium is light to medium,
Solution at 25°C: pH 6.8 ± 0.1 orange, clear to slightly hazy.
Reaction of 2.9%
Solution at 25°C: pH 6.8 ± 0.2
Cultural Response
Difco™ Urea Broth
Cultural Response
Prepare the medium per label directions. Inoculate with fresh cultures
and incubate at 35 ± 2°C for 8-48 hours.
BBL™ Urea Agar Base
Prepare the medium per label directions. Inoculate with fresh cultures
ORGANISM ATCC™ UREASE REACTION (2 heavy loopfuls) and incubate at 35 ± 2°C for 24 hours.
Enterobacter aerogenes 13048 – ORGANISM ATCC™ UREASE REACTION
Escherichia coli 25922 – Proteus vulgaris 8427 +
Proteus mirabilis 25933 + Salmonella enterica
Proteus vulgaris 13315 + subsp. enterica
Salmonella enterica serotype Typhimurium 13311 –
subsp. enterica
serotype Typhimurium 14028 –

Uninoculated Proteus vulgaris Escherichia coli Uninoculated Proteus vulgaris Escherichia coli
Tube ATCC™ 13315 ATCC™ 25922 Tube ATCC™ 13315 ATCC™ 25922
Urea Broth Urea Agar

Formulae Directions for Preparation from

BBL™ Urea Agar Base Dehydrated Product
Approximate Formula* Per Liter BBL™ Urea Agar Base
Pancreatic Digest of Gelatin ........................................ 1.0 g
Dextrose ..................................................................... 1.0 g 1. Dissolve 29 g of the powder in 100 mL of purified water.
Sodium Chloride ......................................................... 5.0 g Mix thoroughly. Sterilize by filtration.
Potassium Phosphate .................................................. 2.0 g 2. Suspend 15 g of agar in 900 mL of purified water.
Urea.......................................................................... 20.0 g
Phenol Red................................................................ 12.0 mg 3. Autoclave at 121°C for 15 minutes.
Difco™ Urea Broth 4. Cool to 50°C and add 100 mL of the sterile Urea Agar Base.
Approximate Formula* Per Liter 5. Mix thoroughly and dispense aseptically in sterile tubes.
Yeast Extract ............................................................... 0.1 g 6. Cool tubed medium in a slanted position so that deep butts
Monopotassium Phosphate ......................................... 9.1 g are formed.
Dipotassium Phosphate ............................................... 9.5 g
Urea.......................................................................... 20.0 g 7. Do not remelt the complete medium.
Phenol Red.................................................................. 0.01 g 8. Test samples of the finished product for performance using
*Adjusted and/or supplemented as required to meet performance criteria. stable, typical control cultures.

Difco™ & BBL™ Manual, 2nd Edition

BBL™ Urea Agar Base Concentrate 10 (Prepared Tubes) A negative reaction is no color change. The agar medium
1. To prepare Urea Agar medium, add 1.7 g of granulated agar remains pale yellow to buff; the broth remains yellowish-
to 100 mL of purified water. Heat with agitation and boil orange.
for 1 minute. For a listing of urease-positive organisms, consult appropriate
2. Dispense in 9 mL aliquots into tubes and autoclave at 121°C texts.2, 4-7
for 15 minutes.
3. Cool the agar to 45-50°C, and allow one tube of Limitations of the Procedure
concentrate to come to room temperature. Add 1 mL of Urea Agar Base
concentrate to each 9 mL of cooled agar solution and mix 1. The alkaline reaction produced in this medium after prolonged
thoroughly. incubation may not be caused by urease activity. False positive
4. Allow the tubes to cool in a slanted position so that slants reactions may occur due to the utilization of peptones
with deep butts are formed. (especially in slant agar by Pseudomonas aeruginosa, for
5. Test samples of the finished product for performance using example) or other proteins which raise the pH due to
stable, typical control cultures. protein hydrolysis and the release of excessive amino acid
Difco™ Urea Broth residues. To eliminate possible protein hydrolysis, perform a
1. Equilibrate the medium to room temperature before control test with the same test medium without urea.7
opening. The presence of urea in this medium renders it 2. Do not heat or reheat the medium because urea decomposes
inherently lumpy. This condition will not adversely affect a very easily.
properly stored medium. 3. Urea Agar detects rapid urease activity of only the urease-
2. Dissolve 38.7 g of the powder in 1 L of purified water. Mix positive Proteus species. For results to be valid for the
thoroughly to completely dissolve the powder. detection of Proteus, the results must be read within the first
3. Filter sterilize. DO NOT BOIL OR AUTOCLAVE THE 2-6 hours after incubation. Urease-positive Enterobacter,
MEDIUM. Citrobacter or Klebsiella, in contrast, hydrolyze urea much
4. Test samples of the finished product for performance using more slowly, showing only slight penetration of the alkaline
stable, typical control cultures. reaction into the butt of the medium in 6 hours and requiring
3-5 days to change the reaction of the entire butt.
BBL™ Urease Broth Concentrate 10 (Prepared Tubes)
Urea Broth
1. To prepare medium, aseptically add 10 mL of the concentrate
to 90 mL of cold sterile purified water. Mix thoroughly. 1. To rule out false positives due to protein hydrolysis (as op-
2. Dispense aseptically in 1-3 mL amounts, in small sterile test posed to urea hydrolysis) that may occur in the medium after
tubes. prolonged incubation, perform a control test with the same
test medium without urea.7
Procedure 2. Do not heat or reheat the medium because urea decomposes
If Urea Agar Base Concentrate 10 or Urease Broth Con- very easily.
centrate 10 is being used, prepare the complete medium as 3. The high buffering system in this medium masks ure-
described above. If crystals form in the concentrate, they will ase activity in organisms that are delayed positive. This
usually dissolve at room temperature, or in a few minutes in a medium is therefore recommended for the detection of
40°C water bath. urease activity in all Proteus spp., Providencia rettgeri and
urease-positive Providencia stuartii.1 M. morganii slowly
Using a heavy inoculum (2 loopfuls) of growth from an 18- to
hydrolyzes urea and may require approximately a 36 hour
24-hour pure culture (TSI Agar or other suitable medium),
incubation for a strong urease-positive reaction to occur.1 If
inoculate the broth or agar (streaking back and forth over the
in doubt as to a result, compare with an uninoculated tube
entire slant surface). Do not stab the butt since it serves as a
or incubate for an additional 24 hours.
color control. For broth, shake tubes gently to suspend the
4. Variations in the size of the inoculum can affect the time
bacteria. Incubate tubes with loosened caps at 35 ± 2°C in an
required to reach positive (alkaline, pH 8.1) results.
incubator or water bath. Observe reactions after 2, 4, 6, 18,
24 and 48 hours. For agar, continue to check every day for
References
a total of 6 days; even longer incubation periods may be 1. Christensen. 1946. J. Bacteriol. 52:461.
2. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams
necessary. & Wilkins, Baltimore, Md.
3. Rustigian and Stuart. 1941. Proc. Soc. Exp. Biol. Med. 47:108.
4. Ewing. 1985. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Expected Results Publishing Co, Inc., New York, N.Y.
5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
The production of urease is indicated by an intense pink-red 9th ed. Williams & Wilkins, Baltimore, Md.
6. Farmer. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
(red-violet) color on the slant or throughout the broth. The 7th ed. American Society for Microbiology, Washington, D.C.
color may penetrate into the agar (butt); the extent of the color 7. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1.
Williams & Wilkins, Baltimore, Md.
indicates the rate of urea hydrolysis.5

Difco™ & BBL™ Manual, 2nd Edition

Availability Difco™ Urea Broth
BBL™ Urea Agar Base AOAC BAM COMPF SMD

BAM CCAM ISO USDA

Cat. No. 227210 Dehydrated – 500 g*
Cat. No. 211795 Dehydrated – 500 g* BBL Urease Test Broth
™

BBL™ Urea Agar Base Concentrate 10 AOAC BAM COMPF SMD

Cat. No. 221100 Prepared Tubes – Pkg. of 10* Cat. No. 221719 Prepared Tubes, 3 mL – Pkg. of 10*

BBL™ Urea Agar BBL Urease Broth Concentrate 10

™

Cat. No. 221096 Prepared Slants – Pkg. of 10* Cat. No. 221098 Prepared Tubes – Pkg. of 10*
221097 Prepared Slants – Ctn. of 100* *Store at 2-8°C.

Difco™ & BBL™ Manual, 2nd Edition