Section III

C CLED Agar

CLED Agar
Intended Use CLED Agar is recommended for use in plates or in urine
CLED Agar is used for the isolation, enumeration and presump- dipsticks for detecting significant bacteriuria by quantitative
tive identification of microorganisms from urine. culture of urine. For reliable results, inoculation of the
medium must occur as soon after collection as possible.
Summary and Explanation Confluent or semiconfluent growth of bacteria will occur on
In 1960, Sandys reported on the development of a new method of the surface of the dipstick medium when bacterial counts are
preventing the swarming of Proteus on solid media by restricting greater than 105 per mL of urine, as confirmed by plates inocu-
the electrolytes in the culture medium.1 Previous chemical lated by the calibrated-loop or duplicate-dilution pour-plate
methods used to inhibit swarming by Proteus included the methods.4 Once the medium has been inoculated by immersion
addition of chloral hydrate, alcohol, sodium azide, surface-active of the dipstick or by pouring the urine over the surface of the
agents, boric acid and sulfonamides to the culture medium.1 medium if only a small volume is available, the dipstick may be
held 48 hours or longer, refrigerated or at room temperature
This electrolyte-deficient medium of Sandys was modified by
until received in the laboratory. On receipt, the dipstick should
Mackey and Sandys2 for use in urine culture by substituting
be incubated at 35 ± 2°C for 18-24 hours, to allow colonies to
lactose and sucrose for the mannitol and increasing the
develop on the medium.
concentrations of the bromthymol blue indicator and of the
agar. These two investigators further modified the medium by
Principles of the Procedure
the incorporation of cystine in order to enhance the growth of
The nutrients in CLED Agar are supplied by peptones, pancreatic
cystine-dependent “dwarf colony” coliforms and by deletion
digests of gelatin and casein, and beef extract. Lactose is included
of sucrose.3 They designated the new medium as Cystine-
to provide an energy source for organisms capable of utilizing it
Lactose-Electrolyte-Deficient (CLED) medium and reported it
by a fermentative mechanism. The cystine permits the growth
to be ideal for dip-inoculum techniques and for urinary
of “dwarf colony” coliforms. Bromthymol blue is used as a pH
bacteriology in general.
Staphylococcus aureus Proteus vulgaris
User Quality Control ATCC™ 25923 ATCC™ 8427

Identity Specifications
BBL™ CLED Agar
Dehydrated Appearance: Fine, homogenous, free of extraneous
material.
Solution: 3.6% solution, soluble in purified
water upon boiling. Solution is medium,
yellow green to blue green, clear to
slightly hazy, with up to a large amount
of minute suspended insolubles.
Prepared Appearance: Medium, yellow green to blue green,
clear to slightly hazy.
Reaction of 3.6%
Solution at 25°C: pH 7.3 ± 0.2

Cultural Response
BBL™ CLED Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ±
2°C for 42-48 hours.
INOCULUM
ORGANISM ATCC™ CFU RECOVERY REACTION
Enterococcus faecalis 29212 103-104 Good Yellow
Escherichia coli 25922 103-104 Good Yellow Enterococcus
faecalis Escherichia coli
Klebsiella pneumoniae 33495 103-104 Good With or ATCC™ 29212 ATCC™ 25922
without green to
yellow reaction
Pseudomonas aeruginosa 10145 103-104 Good With or without
blue reaction
Staphylococcus aureus 25923 103-104 Good Yellow
Proteus vulgaris 8427 103-104 Good Blue

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 CLED Agar, cont.

indicator to differentiate lactose fermenters from lactose Typical colonial morphology on CLED Agar is as follows:
nonfermenters. Organisms that ferment lactose will lower the Escherichia coli ............................... Yellow colonies, opaque,
pH and change the color of the medium from green to yellow. center slightly deeper yellow
Electrolyte sources are reduced in order to restrict the swarming of Klebsiella ........................................ Yellow to whitish-blue
Proteus species. colonies, extremely mucoid
Proteus .......................................... Translucent blue colonies
Bacteriuria is determined by inoculating the surface of an agar
medium using 0.1 mL of a 10-2 dilution of the urine sample or Pseudomonas aeruginosa ................ Green colonies with typical
matted surface and rough
using a calibrated loop (0.001 mL) of the undiluted sample.5 periphery
Current guidelines are that for a single isolate a density of >105 Enterococci .................................... Small yellow colonies,
CFU/mL indicates infection, <104 CFU/mL indicates urethral about 0.5 mm in diameter
or vaginal contamination, and between 104 and 105 CFU/mL Staphylococcus aureus .................... Deep yellow colonies, C
needs to be evaluated based on clinical information.6 uniform in color
Staphylococci coagulase-negative .... Pale yellow colonies, more
Formula opaque than E. faecalis
BBL™ CLED Agar
Approximate Formula* Per Liter Limitations of the Procedure
Pancreatic Digest of Gelatin ....................................... 4.0 g Factors that may cause urine counts from infected patients to
Pancreatic Digest of Casein ........................................ 4.0 g be low include: rapid rate of urine flow, prior initiation of
Beef Extract ................................................................ 3.0 g
Lactose ..................................................................... 10.0 g antimicrobial therapy, a urine pH of less than 5 and a specific
L-Cystine ................................................................ 128.0 mg gravity of less than 1.003.7
Bromthymol Blue ....................................................... 0.02 g
Agar ......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
References
1. Sandys. 1960. J. Med. Lab. Technol. 17:224.
2. Mackey and Sandys. 1965. Br. Med. J. 2:1286.
Directions for Preparation from 3.
4.
Mackey and Sandys. 1966. Br. Med. J. 1:1173.
Benner. 1970. Appl. Microbiol. 19:409.
Dehydrated Product 5. Barry, Smith and Turck. 1975. Cumitech 2, Laboratory diagnosis of urinary tract infections. Coord.
ed., Gavan. American Society for Microbiology, Washington, D.C.
1. Suspend 36 g of the powder in 1 L of purified water. Mix 6. Clarridge, Pezzlo and Vosti. 1987. Cumitech 2A, Laboratory diagnosis of urinary tract infections.
Coordinating ed., Weissfeld. American Society for Microbiology, Washington, D.C.
thoroughly. 7. Finegold and Martin. 1982. Bailey & Scott’s diagnostic microbiology, 6th ed. The C.V. Mosby
Company, St. Louis, Mo.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. Availability
3. Autoclave at 121°C for 15 minutes. BBL™ CLED Agar
4. Test samples of the finished product for performance using Cat. No. 212218 Dehydrated – 500 g
stable, typical control cultures. United States and Canada
Cat. No. 221850 Prepared Plates – Pkg. of 10*
Procedure 221530 Prepared Plates – Ctn. of 100*
Inoculate the medium as soon as possible after the specimen is Europe
received in the laboratory. It is recommended that quantitative Cat. No. 254003 Prepared Plates – Pkg. of 20*
methods be used for culturing urine specimens.5 Incubate at 254070 Prepared Plates – Ctn. of 120*
35 ± 2°C for 24-48 hours. Japan
Cat. No. 251953 Prepared Plates – Pkg. of 20*
251530 Prepared Plates – Ctn. of 100*
Expected Results *Store at 2-8°C.
Count the number of colonies on the plate or dipstick. Multiply
by an appropriate number to convert the count to CFU per
mL of sample.
Contaminant bacteria usually appear in low numbers which
vary in colonial morphology. Urinary pathogens will usually
yield high counts having uniform colonial morphology and should
be subcultured directly to routine media for identification and
susceptibility testing.5,7

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