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BBL CLED Agar

L007367 Rev. 04 May 2006
QUALITY CONTROL PROCEDURES

INTRODUCTION CLED Agar (Cystine-Lactose-Electrolyte-Deficient Agar) is a differential culture medium for use in enumerating bacteria in urine. It supports the growth of urinary pathogens and contaminants but prevents undue swarming of Proteus species due to its lack of electrolytes. PERFORMANCE TEST PROCEDURE 1. Inoculate representative samples with dilutions of the cultures listed below. a. Spread-inoculate with 103104 CFU for all organisms. b. Incubate plates at 35 2C in an aerobic atmosphere. c. Include Trypticase Soy Agar with 5% Sheep Blood plates as nonselective controls for all organisms. 2. Examine plates at 1824 and 48 h for growth, pigmentation, colony size and inhibition of Proteus swarming/spreading. 3. Expected Results CLSI Organisms *Escherichia coli *Proteus vulgaris *Staphylococcus aureus Additional Organism P. mirabilis ATCC 25922 8427 25923 12453 Recovery Growth Growth Growth Growth Colony Color Yellow with deep yellow centers Blue Uniform deep yellow Blue

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*Recommended organism strain for User Quality Control.

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ADDITIONAL QUALITY CONTROL 1. Examine plates as described under Product Deterioration. 2. Visually examine representative plates to assure that any existing physical defects will not interfere with use. 3. Determine the pH potentiometrically at room temperature for adherence to the specification of 7.4 0.2. 4. Note the firmness of plates during the inoculation procedure. 5. Incubate uninoculated representative plates aerobically at 35 2C for 72 h and examine for microbial contamination.

PRODUCT INFORMATION
IV V INTENDED USE CLED Agar is used for the isolation, enumeration and presumptive identification of microorganisms from urine. SUMMARY AND EXPLANATION 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.1 Previous chemical methods used to inhibit swarming by Proteus included the addition of chloral hydrate, alcohol, sodium azide, surface active agents, boric acid or sulfonamides to the culture medium.1 This electrolyte-deficient medium of Sandys was modified by Mackey and Sandys2 for use in urine culture by substituting lactose and sucrose for the mannitol and increasing the concentrations of the bromthymol blue indicator and of the agar. These two investigators further modified the medium by the incorporation of cystine in order to enhance the growth of cystine dependent dwarf colony coliforms and by deletion of sucrose.3 They designated the new medium as Cystine-LactoseElectrolyte-Deficient (CLED) medium and reported it to be ideal for dip-inoculum techniques and for urinary bacteriology in general. PRINCIPLES OF THE PROCEDURE The nutrients in CLED Agar are supplied by the peptones, pancreatic digests of gelatin and casein, and beef extract. Lactose is included to provide an energy source for organisms capable of utilizing it by a fermentative mechanism. The cystine permits the growth of dwarf colony coliforms. 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. Electrolyte sources are reduced in order to restrict the swarming of Proteus species. 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 using a calibrated loop (0.001 mL) of the undiluted sample.4 Current guidelines are that for a single isolate a density of >105 CFU/mL indicates infection, <104 CFU/mL indicates urethral or vaginal contamination, and between 104 and 105 CFU/mL needs to be evaluated based on clinical information.5 REAGENTS CLED Agar Approximate Formula* Per Liter Purified Water Pancreatic Digest of Gelatin..................................4.0 g Pancreatic Digest of Casein ..................................4.0 g Beef Extract ............................................................3.0 g Lactose ..................................................................10.0 g L-Cystine..................................................................0.128 g Bromthymol Blue ..................................................0.02 g Agar ......................................................................15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

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Warnings and Precautions: For in vitro Diagnostic Use. If excessive moisture is observed, invert the bottom over an off-set lid and allow to air dry in order to prevent formation of a seal between the top and bottom of the plate during incubation. Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. "Standard Precautions"6-9 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids. After use, prepared plates, specimen containers and other contaminated materials must be sterilized by autoclaving before discarding. Storage Instructions: On receipt, store plates in the dark at 28C. Avoid freezing and overheating. Do not open until ready to use. Minimize exposure to light. Prepared plates stored in their original sleeve wrapping at 28C until just prior to use may be inoculated up to the expiration date and incubated for recommended incubation times. Allow the medium to warm to room temperature before inoculation. Product Deterioration: Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking or other signs of deterioration. VIII SPECIMEN COLLECTION AND HANDLING Specimens suitable for culture may be handled using various techniques. For detailed information, consult appropriate texts.10,11 Specimens should be obtained before antimicrobial therapy has been administered. Provision must be made for prompt delivery to the laboratory. IX PROCEDURE Material Provided: CLED Agar Materials Required But Not Provided: Ancillary culture media, reagents, quality control organisms and laboratory equipment as required. Test Procedure: Observe aseptic techniques. The agar surface should be smooth and moist, but without excessive moisture. Inoculate the medium as soon as possible after the specimen is received in the laboratory. It is recommended that quantitative methods be used for culturing urine specimens.5 Incubate plates aerobically at 35 2C for 2448 h. User Quality Control: See Quality Control Procedures. Quality Control requirements must be performed in accordance with applicable local, state and/or federal regulations or accreditation requirements and your laboratorys standard Quality Control procedures. It is recommended that the user refer to pertinent CLSI (formerly NCCLS) guidance and CLIA regulations for appropriate Quality Control practices. RESULTS Count the number of colonies on the plate. Multiply by an appropriate number to convert the count to CFU per mL. 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.4,5 Typical colonial morphology on CLED Agar is as follows: Escherichia coli ......................Yellow colonies, opaque, center slightly deeper yellow Klebsiella ................................Yellow to whitish-blue colonies, extremely mucoid Proteus ..................................Translucent blue colonies Pseudomonas aeruginosa ....Green colonies with typical matted surface and rough periphery Enterococci ............................Small yellow colonies, about 0.5 mm in diameter Staphylococcus aureus ..........Deep yellow colonies, uniform in color Staphylococci, ........................Pale yellow colonies, more opaque than E. faecalis coagulase negative LIMITATIONS OF THE PROCEDURE Factors which may cause urine counts from infected patients to be low include: rapid rate of urine flow, prior initiation of antimicrobial therapy, a urine pH of less than 5 and specific gravity of less than 1.003.12 For identification, organisms must be in pure culture. Morphological, biochemical and/or serological tests should be performed for final identification. Consult appropriate texts for detailed information and recommended procedures.10,11,13-16 A single medium is rarely adequate for detecting all organisms of potential significance in a specimen. It should be recognized that organisms generally susceptible to the antimicrobial agent in a selective medium may be completely or only partially inhibited depending upon the concentration of the agent, the characteristics of the microbial strain and the number of organisms in the inoculum. Organisms that are generally resistant to the antimicrobial agent should not be inhibited. Cultures of specimens grown on selective media should, therefore, be compared with specimens cultured on nonselective media to obtain additional information and help ensure recovery of potential pathogens. AVAILABILITY Cat. No. 221850 221530 XIII REFERENCES
1. 2. 3. 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. 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. Mackey, J.P., and G.H. Sandys. 1966. Diagnosis of urinary infections. Br. Med. J. 1:1173.

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Description BBL CLED Agar, Pkg. of 10 plates BBL CLED Agar, Ctn. of 100 plates

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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. 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. National Committee for Clinical Laboratory Standards. 2001. Approved Guideline M29-A2. Protection of laboratory workers from occupationally acquired infections, 2nd ed. NCCLS, Wayne, PA. Garner, J.S. 1996. Hospital Infection Control Practices Advisory Committee, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Guideline for isolation precautions in hospitals. Infect. Control Hospital Epidemiol. 17:53-80. U.S. Department of Health and Human Services. 1999. Biosafety in microbiological and biomedical laboratories, HHS Publication (CDC), 4th ed. U.S. Government Printing Office, Washington, D.C. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC). Official Journal L262, 17/10/2000, p. 0021-0045. Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R. H. Yolken (ed.). 2003. Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 2002. Bailey and Scott's diagnostic microbiology, 11th ed. Mosby, Inc., St. Louis. Finegold, S.M., and W.J. Martin. 1982. Bailey and Scotts diagnostic microbiology, 6th ed. The C.V. Mosby Company, St. Louis. Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams (ed.). 1994. Bergey's Manual of determinative bacteriology, 9th ed. Williams & Wilkins, Baltimore. MacFaddin, J.F. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams & Wilkins, Baltimore. Koneman, E.W., S.D. Allen, W.M. Janda, P.C. Schreckenberger, and W.C. Winn, Jr. 1997. Color atlas and textbook of diagnostic microbiology, 5th ed. Lippincott-Raven, Philadelphia. Isenberg, H.D. (ed.). 2004. Clinical microbiology procedures handbook, vol. 1, 2 and 3, 2nd ed. American Society for Microbiology, Washington, D.C.

Becton, Dickinson and Company 7 Loveton Circle Sparks, Maryland 21152 USA 800-638-8663 ATCC is a trademark of the American Type Culture Collection. BD, BD Logo, BBL and Trypticase are trademarks of Becton, Dickinson and Company. 2006 BD.

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