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BBL CLED AgarL007367 Rev. 04 May 2006

QUALITY CONTROL PROCEDURES

I 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 lackof electrolytes.

II 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 ATCC Recovery Colony Color

*Escherichia coli 25922 Growth Yellow with deep yellow centers

*Proteus vulgaris 8427 Growth Blue

*Staphylococcus aureus 25923 Growth Uniform deep yellow

Additional Organism

P. mirabilis 12453 Growth Blue

*Recommended organism strain for User Quality Control.

III 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 INTENDED USE

CLED Agar is used for the isolation, enumeration and presumptive identification of microorganisms from urine.

V SUMMARY AND EXPLANATION

In 1960, Sandys reported on the development of a new method of preventing the swarming of Proteus on solid media byrestricting the electrolytes in the culture medium.1 Previous chemical methods used to inhibit swarming by Proteus included theaddition 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 cystinedependent dwarf colony coliforms and by deletion of sucrose.3 They designated the new medium as Cystine-Lactose-Electrolyte-Deficient (CLED) medium and reported it to be ideal for dip-inoculum techniques and for urinary bacteriology ingeneral.

VI PRINCIPLES OF THE PROCEDURE

The nutrients in CLED Agar are supplied by the peptones, pancreatic digests of gelatin and casein, and beef extract. Lactose isincluded to provide an energy source for organisms capable of utilizing it by a fermentative mechanism. The cystine permitsthe growth of dwarf colony coliforms. Bromthymol blue is used as a pH indicator to differentiate lactose fermenters fromlactose-nonfermenters. Organisms which ferment lactose will lower the pH and change the color of the medium from green toyellow. 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 orusing 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,

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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 aseal between the top and bottom of the plate during incubation.

Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinicalspecimens. "Standard Precautions"6-9 and institutional guidelines should be followed in handling all items contaminated withblood and other body fluids. After use, prepared plates, specimen containers and other contaminated materials must besterilized by autoclaving before discarding.

Storage Instructions: On receipt, store plates in the dark at 28C. Avoid freezing and overheating. Do not open until ready touse. Minimize exposure to light. Prepared plates stored in their original sleeve wrapping at 28C until just prior to use may beinoculated up to the expiration date and incubated for recommended incubation times. Allow the medium to warm to roomtemperature before inoculation.

Product Deterioration: Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking orother signs of deterioration.

VIII SPECIMEN COLLECTION AND HANDLING

Specimens suitable for culture may be handled using various techniques. For detailed information, consult appropriatetexts.10,11 Specimens should be obtained before antimicrobial therapy has been administered. Provision must be made forprompt 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 asrequired.

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 quantitativemethods 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 oraccreditation requirements and your laboratorys standard Quality Control procedures. It is recommended that the user refer topertinent CLSI (formerly NCCLS) guidance and CLIA regulations for appropriate Quality Control practices.

X 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 yieldhigh counts having uniform colonial morphology and should be subcultured directly to routine media for identification andsusceptibility 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. faecaliscoagulase negative

XI LIMITATIONS OF THE PROCEDURE

Factors which may cause urine counts from infected patients to be low include: rapid rate of urine flow, prior initiation ofantimicrobial 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 performedfor 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 recognizedthat organisms generally susceptible to the antimicrobial agent in a selective medium may be completely or only partiallyinhibited depending upon the concentration of the agent, the characteristics of the microbial strain and the number oforganisms in the inoculum. Organisms that are generally resistant to the antimicrobial agent should not be inhibited. Culturesof specimens grown on selective media should, therefore, be compared with specimens cultured on nonselective media toobtain additional information and help ensure recovery of potential pathogens.

XII AVAILABILITY

Cat. No. Description

221850 BBL CLED Agar, Pkg. of 10 plates

221530 BBL CLED Agar, Ctn. of 100 plates

XIII 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 routinelaboratory 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-inoculumtransport 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.

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4. Barry, A.L., P.B. Smith, and M. Turck. 1975. Cumitech 2, Laboratory diagnosis of urinary tract infections. Coordinating ed., T.L. Gavan. AmericanSociety for Microbiology, Washington, D.C.

5. 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.

6. National Committee for Clinical Laboratory Standards. 2001. Approved Guideline M29-A2. Protection of laboratory workers fromoccupationally acquired infections, 2nd ed. NCCLS, Wayne, PA.

7. Garner, J.S. 1996. Hospital Infection Control Practices Advisory Committee, U.S. Department of Health and Human Services, Centers forDisease Control and Prevention. Guideline for isolation precautions in hospitals. Infect. Control Hospital Epidemiol. 17:53-80.

8. U.S. Department of Health and Human Services. 1999. Biosafety in microbiological and biomedical laboratories, HHS Publication (CDC), 4thed. U.S. Government Printing Office, Washington, D.C.

9. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related toexposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC). OfficialJournal L262, 17/10/2000, p. 0021-0045.

10. 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 forMicrobiology, Washington, D.C.

11. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 2002. Bailey and Scott's diagnostic microbiology, 11th ed. Mosby, Inc., St. Louis.

12. Finegold, S.M., and W.J. Martin. 1982. Bailey and Scotts diagnostic microbiology, 6th ed. The C.V. Mosby Company, St. Louis.

13. 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.

14. MacFaddin, J.F. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams & Wilkins, Baltimore.

15. 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.

16. Isenberg, H.D. (ed.). 2004. Clinical microbiology procedures handbook, vol. 1, 2 and 3, 2nd ed. American Society for Microbiology,Washington, D.C.

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