INSTRUCTIONS FOR USE –
READY-TO-USE PLATED
MEDIA
PA-254003.06 Rev.: Oct 2012
BD CLED Agar
INTENDED USE
BD CLED Agar (Cystine-Lactose-Electrolyte-Deficient Agar) is a differential culture medium for
use in isolating and enumerating bacteria from urine. It supports the growth of urinary
pathogens and contaminants but prevents undue swarming of Proteus species due to its lack of
electrolytes.
PRINCIPLES AND EXPLANATION OF THE PROCEDURE
Microbiological method.
In 1960, Sandys reported on the development of a new method of preventing the swarming of
Proteus on solid media by restricting the electrolytes in the culture medium which was modified
later several times for use in urine culture.1-3 It was designated as Cystine-Lactose-Electrolyte-
Deficient (CLED) medium and reported to be ideal for dip-inoculum techniques and for urinary
bacteriology in general.
The nutrients in BD CLED Agar are supplied by the gelatin and casein peptones, and beef
extract. Lactose is included to provide an energy source for organisms capable of utilizing it by a
fermentative mechanism. Bromthymol blue is used as a pH indicator to differentiate lactose
fermenters from lactose-nonfermenters. Organisms which ferment lactose will lower the pH and
change the color of the medium from green to yellow. The cystine permits the growth of "dwarf
colony" coliforms.3 Electrolyte sources are reduced in order to minimize the swarming of Proteus
species. Thus, the medium allows quantitative determination of urinary pathogens including
Proteus when calibated loops are used for inoculation.
REAGENTS
BD CLED Agar
Formula* Per Liter Purified Water
Pancreatic Digest of Gelatin 4.0 g
Pancreatic Digest of Casein 4.0
Beef Extract 3.0
Lactose 10.0
L-Cystine 0.128
Bromthymol Blue 0.02
Agar 15.0
pH 7.3 +/- 0.2
*Adjusted and/or supplemented as required to meet performance criteria.
PRECAUTIONS
. For professional use only.
Do not use plates if they show evidence of microbial contamination, discoloration, drying,
cracking or other signs of deterioration.
Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures,
biohazards, and disposal of used product.
STORAGE AND SHELF LIFE
On receipt, store plates in the dark at 2 to 8° C, in their original sleeve wrapping until just prior to
use. Avoid freezing and overheating. The plates may be inoculated up to the expiration date
(see package label) and incubated for the recommended incubation times.
Plates from opened stacks of 10 plates can be used for one week when stored in a clean area
at 2 to 8° C.
PA-254003.06 -1-
USER QUALITY CONTROL
Inoculate representative samples with the following strains (for details, see GENERAL
INSTRUCTIONS FOR USE document). Incubate plates at 35 ± 2°C in an aerobic atmosphere.
Examine plates at 18 to 24 h for amount of growth, pigmentation, colony size and inhibition of
Proteus swarming/spreading.
Strains Growth Results
Escherichia coli ATCC 25922 Growth; colonies yellow, medium yellow
Proteus vulgaris ATCC 8427 Growth; colonies colorless to blue; swarming
inhibited; slight spreading acceptable
Enterococcus faecalis ATCC 29212 Growth; colonies colorless to yellow; medium yellow
Staphylococcus aureus ATCC 25923 Growth; colonies small, yellow; medium yellow
Staphylococcus saprophyticus Growth; colonies small, white to yellowish; medium
ATCC 15305 yellow
Uninoculated Green to blue-green
PROCEDURE
Materials Provided
BD CLED Agar (90 mm Stacker plates). Microbiologically controlled.
Materials Not Provided
Ancillary culture media, reagents and laboratory equipment as required.
Specimen Types and Collection of Specimens
This medium is exclusively used for enumerating and differentiating bacteria in urine. Midstream
or catheter urine, or urine collected by suprapubic bladder puncture can be used (see also
PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE). Observe
aseptic techniques for collecting urine specimens. Urine must be directly streaked onto the
medium not later than 2 hours after collection or must be kept refrigerated (not longer than 24
hours) to avoid overgrowth of the infectious agents or contaminants before inoculation of this
medium.2-5
Test Procedure
Collect a sample of the undiluted, well-mixed urine using a calibrated loop (0.01 or 0.001 ml).
Ensure proper loading of the loop with the specimen. Inoculate the sample down the middle of
the plate in a single streak from which additional spreading of the inoculum is performed. 4 ,5
Incubate plates in ambient air at 35 ± 2°C for 24 to 48 h.
Results
Typical colonial morphology on BD CLED Agar is as follows:
Organisms Growth Results
Escherichia coli Yellow colonies, opaque; yellow medium
Klebsiella, Enterobacter Yellow to whitish-blue colonies, often mucoid; yellowish medium
Proteus Translucent blue colonies; blue-green to blue medium
Pseudomonas aeruginosa Green colonies with typical matted surface and rough periphery; blue medium
Enterococci Small yellow colonies, about 0.5 mm in diameter; yellow medium
Staphylococcus aureus Deep yellow colonies, uniform in color; yellow medium
Coagulase negative staphylococci Pale yellow colonies, more opaque than Enterococcus faecalis
Calculation and Interpretation of Results
Count the number of colonies (CFU) on the plate. If a 0.01 ml loop was used, each resultant
colony is representative of 100 CFU/ml; if a 0.001 ml loop was used, each colony corresponds
to 1000 CFU/ml of urine.5
Midstream and catheter urine: Current guidelines indicate that for a single isolate a density of
105 CFU/ml indicates infection, <105 CFU/ml indicates urethral or vaginal contamination, and
between 104 to 105 CFU/ml needs to be re-evaluated based on clinical information.6
Contaminant bacteria usually appear in low numbers which vary in colonial morphology.
PA-254003.06 -2-
Urine collected by suprapubic bladder puncture: Since the bladder is sterile in non-infected
individuals, any CFU detected indicates an infection.
Urinary pathogens will usually yield high counts having uniform colonial morphology and must be
subcultured directly to routine media for identification and susceptibility testing.5,6
PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE
BD CLED Agar is suitable for the isolation and counting of many aerobically growing
microorganisms, such as Enterobacteriaceae, Pseudomonas and other non-fermenting Gram
negative rods, enterococci, staphylococci, Candida species, and many others from urine
specimens.
Streptococci and other organisms requiring blood or serum for growth may only be insufficiently
recovered on this medium or may need extended incubation. Therefore, the specimen should
also be cultivated onto a blood agar plate if such organisms are expected.
Genitourinary pathogens such as Neisseria gonorrhoeae, Gardnerella vaginalis, Chlamydia,
Ureaplasma, or other fastidious organisms do not grow on this medium. Consult the references
for the appropriate detection techniques of these organisms.4-6
Although a differentiation according to lactose fermentation and certain other diagnostic tests
may be performed directly on this medium, biochemical and, if indicated, serological testing
using pure cultures is necessary for complete identification.
REFERENCES
1. Sandys, G.H. 1960. A new method of preventing swarming of Proteus sp. with a description
of a new medium suitable for use in routine laboratory practice. J. Med. Lab. Technol. 17:224-
233.
2. Mackey, J.P., and G.H. Sandys. 1965. Laboratory diagnosis of infection of the urinary tract in
general practice by means of a dip-inoculum transport medium. Br. Med. J. 2:1286-1288.
3. Mackey, J.P., and G.H. Sandys. 1966. Diagnosis of urinary infections. Br. Med. J. 1:1173.
4. Barry, A.L., P.B. Smith, and M. Turck. 1975. Cumitech 2, Laboratory diagnosis of urinary tract
infections. Coordinating ed., T.L. Gavan. American Society for Microbiology, Washington,
D.C.
5. Thomson, R.B., and J.M. Miller. 2003. Specimen collection, transport, and processing:
bacteriology. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken
(ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology,
Washington, D.C.
6. Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Cumitech 2A, Laboratory diagnosis of
urinary tract infections. Coordinating ed., A.S. Weissfeld. American Society for Microbiology,
Washington, D.C.
PACKAGING/AVAILABILITY
BD CLED Agar
Cat. No. 254003 Ready-to-use plated media, 20 plates
Cat. No. 254070 Ready-to-use plated media, 120 plates
FURTHER INFORMATION
For further information please contact your local BD representative.
Becton Dickinson GmbH
Tullastrasse 8 – 12
D-69126 Heidelberg/Germany
Phone: +49-62 21-30 50 Fax: +49-62 21-30 52 16
Reception_Germany@europe.bd.com
PA-254003.06 -3-
http://www.bd.com
http://www.bd.com/europe/regulatory/
ATCC is a trademark of the American Type Culture Collection
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2012 BD
PA-254003.06 -4-
