CLED Agar

Cat. no. G223 CLED Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J21 CLED Agar / EMB Agar, Levine, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J212 CLED Agar / Mueller Hinton Agar, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J315 Blood Agar / CLED Agar / MacConkey Agar, 15x100mm Triplate, 7ml/section 10 plates/bag
Cat. no. J316 Blood Agar / CLED Agar / EMB Agar, Levine, 15x100mm Triplate, 7ml/section 10 plates/bag

INTENDED USE

Hardy Diagnostics CLED Agar is recommended for the isolation, enumeration, and presumptive identification of urinary pathogens on the basis of lactose fermentation, while controlling the swarming of Proteus spp.

SUMMARY

In the mid 1960s, Sandys and Mackey reported on the laboratory diagnosis of urinary tract infections using a new medium Sandys had developed to prevent the swarming of Proteus spp. (7,8,10) Previous culture methods used to inhibit the swarming of Proteus included adding chloral hydrate, alcohol, sodium azide, surface-active agents, boric acid, and sulfonamides to the medium. (10) However, Mackey and Sandys' modified medium replaced mannitol with lactose, discontinued the use of sucrose, increased the indicator strength of bromothymol blue and the concentration of agar, and incorporated the use of cystine in order to enhance the growth of cystine-dependent "dwarf colony" coliforms. (8) They named their final medium Cystine Lactose Electrolyte-Deficient (CLED) Agar and reported it as ideal for dip-slide techniques and for general urinary bacteriology and colony differentiation. CLED Agar also lacks sodium chloride, which helps to prevent the swarming of Proteus spp.

CLED Agar supports the growth of all potential urinary pathogens, and a number of contaminants such as diphtheroids, lactobacilli, and micrococci. Urine samples containing mixed flora are typical of urethral or vaginal contamination. Research demonstrates the best results are obtained when inoculation occurs as soon after sample collection as possible. (2,8) Otherwise, confluent or semiconfluent growth may occur when CFU counts exceed 10 5 per ml of urine.

Hardy Diagnostics CLED Agar is recommended for use in the spread plate technique for detection of bacteria in urine. The medium contains enzymatic digest of casein, enzymatic digest of gelatin, and beef extract, which provide nitrogen, vitamins, and carbon to support microbial growth. Lactose is added as the carbohydrate source. L-cystine is a growth supplement for cystine-dependent coliforms. Organisms capable of fermenting lactose will lower the pH and change the color of the medium to yellow. Consequently, bromothymol blue is the pH indicator. Agar acts as the solidifying agent.

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Pancreatic Digest of Gelatin 4.0gm
Pancreatic Digest of Casein 4.0gm
Beef Extract 3.0gm
L-Cystine 0.128gm
Bromothymol Blue 0.02gm
Agar 15.0gm

Final pH 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

It is recommended that quantitative methods be used for cultivating specimens obtained from urine. For best results, the medium should be inoculated with a 1ul loop of a freshly voided, "clean catch" mid-stream urine as soon as possible after receipt. If there is a delay in getting the specimen to the lab, it should be refrigerated until inoculation to the media is possible. Incubate the media at 35 +/- 2ºC. for 24-48 hours. Consult the appropriate references for additional information on inoculation and incubation methods as established by laboratory policy. (1,3,4,9)

If only a small volume of urine is available, a small sample (approximately 1ml) may be poured directly over the medium, and the plate swirled to coat the entire surface for inoculation.

INTERPRETATION OF RESULTS

Count the number of colonies on the plate. Multiply the result by the appropriate number to convert the count to the number of CFU per ml of urine sample.

Potential bacterial contaminants usually appear in low numbers and vary in morphology. Typical urinary pathogens will usually yield high counts, have uniform colony morphologies, and should be subcultured directly to routine media for further identification and susceptibility testing.

Typical colony morphology on CLED Agar is as follows:

Escherichia coli Opaque yellow colonies with a slightly deeper yellow center
Klebsiella Yellow to whitish-blue colonies, extremely mucoid (7)
Proteus Translucent blue colonies
Pseudomonas aeruginosa Green colonies with typical matted surface and rough periphery
Enterococci Small yellow colonies, about 0.5mm in diameter
Staphylococcus aureus Deep yellow colonies, uniform in color
Staphylococci coagulase-negative Pale yellow colonies, more opaque than Enterococcus faecalis

LIMITATIONS

Limiting factors in low urine counts from infected patients include: rapid rate of urine flow, prior initiation of antimicrobial treatment, urine with a pH less than 5 and a specific gravity less than 1.003. (8)

The nutritional requirements of organisms vary and some strains may grow poorly or fail to grow entirely on this medium.

CLED Agar is a non-selective medium. However, the growth of some Shigella species may be inhibited due to electrolyte exclusion in the formula. (6)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Enterococcus faecalis
ATCC ® 29212
A 24hr 35°C Aerobic Growth; small yellow colonies
Escherichia coli
ATCC ® 25922
A 24hr 35°C Aerobic Growth; yellow colonies, opaque, center slighlty deeper yellow
Proteus mirabilis
ATCC ® 12453
A 24hr 35°C Aerobic Growth; translucent blue colonies
Staphylococcus aureus
ATCC ® 25923
A 24hr 35°C Aerobic Growth; deep yellow colonies
Pseudomonas aeruginosa
ATCC ® 27853
A 24hr 35°C Aerobic Growth; green colonies with matte surface and rough periphery
Klebsiella pneumoniae
ATCC ® 13883
A 24hr 35°C Aerobic Growth; yellow to whitish-blue colonies, mucoid

USER QUALITY CONTROL

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Benner, E.J. 1970. Simple Disposable Method for Quantitative Cultures of Urine. Appl. Micro. Vol. 19, No. 3.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Medical Bacteria. Vol I. Williams & Wilkins, Baltimore, MD.

7. Mackey, J.P and G.H. Sandys. 1965. Laboratory Diagnosis of Infections of the Urinary Tract in General Practice by Means of a Dip-inoculum Transport Medium. Brit. Med. J.; 2:1286-1288.

8. Mackey, J.P. and G.H. Sandys. 1966. Diagnosis of Urinary Infections. Brit. Med. J.; 1:1173.

9. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

10. Sandys, G.H. 1960. A new method of preventing swarming of Proteus spp. with a description of a new medium suitable for use in routing laboratory practice. J. Med. Lab. Technol.; 17:224.


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121415hh