CLED Agar Composition, Principle, Preparation, Results, Uses

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Cystine-Lactose-Electrolyte-Deficient (CLED) medium, first described by Sandys and later modified by Mackey and Sandys, is generally used for diagnostic routine urinary bacteriology as a non-selective medium capable of supporting the growth of most urinary pathogens. CLED Agar, a differential medium for the isolation and counting of bacteria from urine, is used. It supports the growth all potential urinary pathogens. The medium also provides distinct colony morphology. It is suitable for the growth of all urinary pathogens, contaminants, and provides good colonial differentiation. However, it does not allow for the spread of Proteus species because of its low electrolytes.

Composition of CLED Agar

Constituents        gm/Litre
Lactose      10.0
Enzymatic Digest of Gelatin       4.0
Enzymatic Digest of Casein       4.0
Beef extract       3.0
L-Cystine      0.128
Bromothymol Blue      0.02
Agar      15.0

pH 7.3 +/- 0.2

Principle of CLED Agar

CLED is made up of an enzymatic digestion of Casein, an enzyme digest of gelatin and beef extract. These provide the essential nutrients, vitamins, and minerals for growth. L-cystine is essential for the development of the cysteine dependent dwarf coliform colony. Lactose, a fermentable carbohydrate, provides energy and carbon. The pH of organisms that can ferment lactose will be lower and the medium’s color will change from green to yellow. Bromthymol Blue, which is a pH indicator, indicates the color change. The solidifying agent is the agar in the medium.


Preparation of CLED Agar

  1. In one liter of distilled tap water, dissolve 36 grams of the medium.
  2. Combine well, heat with constant agitation, and boil for 1 minute to dissolve.
  3. For 15 minutes, autoclave at 121°C
  4. Allow to cool to 50°C. Mix well and then dispense onto plates. To avoid moisture buildup, invert the plates once the medium has solidified. Keep the prepared medium at 8-15°C

Colony characteristics of various organisms on CLED Agar

Different microorganisms can show different cultural responses to CLED Agar. These reactions are after incubation at the right temperature and atmosphere for 18-24 hours.

OrganismColony Morphology
Escherichia coliLarge, elevated, yellow, opaque colonies with a center more intense yellow; yellowish medium
Klebsiella speciesextremely mucoid colonies varying in color from yellow to whitish-blue. yellowish medium.
Proteus speciestranslucent blue colonies are usually smaller than Escherichia coli. blue-green to blue medium.
Salmonella speciesflat blue colonies
Pseudomonas aeruginosagreen colonies with a typical matte surface and rough periphery. “Sweet” odor. Blue-green agar
Enterococcus faecalisyellow colonies about 0.5 mm in diameter. yellow medium
Staphylococcus aureusdeep yellow colonies about 0.75 mm diameter, uniform in color. yellow medium
Corynebacteriavery small grey colonies
Lactobacillisimilar to corynebacteria but with a rougher surface

Quality Control of CLED Agar

CLED agar quality control should be performed by performing performance and sterility testing.

  • Sterility testing: For 18-24 hours, incubate 3-4 plates of uninoculated medium at37. The medium should not be considered positive if it shows signs of growth.
  • Perform performance testing by inoculating one to three standard strains onto the prepared media and incubating for 18-24 hours at 35 +-2degC in an oxygenated atmosphere. After overnight incubation, observe for growth, pigmentation, colony sizes, and inhibition of Proteus spreading/swarming.
OrganismDesired characteristics
Escherichia coli ATCC 25922Luxuriant Growth; colonies are yellow, medium yellow
Proteus vulgaris ATCC 8427Good growth; colonies are colorless to blue; swarming is inhibited; however, slight spreading is  acceptable
Enterococcus faecalis ATCC 29212Growth; small colonies ,colorless to yellow
Staphylococcus aureus ATCC 25923Good growth; colonies small, yellow to yellowish
Uninoculated platesGreen to blue-green

Application of CLED Agar

  • It can be used to culture routine urine and for the growth and enumeration urinary tract organisms.
  • It aids in the differentiation between lactose fermenter, and lactose not-fermenter.
  • It can be used to isolate and count many aerobically-growing microorganisms such as Enterobacteriaceae and Pseudomonas.

Advantages of CLED Agar

  • Excellent discrimination of gram negative bacteria based on lactose fermentation, colony appearance and other factors
  • Inhibits the swarming Proteus species (Proteus mirabilis, Proteus vulgaris) are often involved in urinary tract infections.
  • Low cost (compare to the combined use blood agar/MacConkey agar urine culture).

Note: Proteus spp. can be swarming in MacConeky medium that contains bile salts

Limitations of CLED Agar

  • Streptococci or other organisms that require blood or serum to grow may not be easily recovered on this medium and may need to be incubated for extended periods. If you suspect that such organisms may be present, you should inoculate an additional plate of blood agar.
  • This medium is not suitable for the recovery of genitourinary parasites like Neisseria vaginalis, Gardnerella vaginalis or Chlamydia. These organisms cannot grow on this medium.
  • This medium may not be suitable for Shigella species.
  • It is a differential medium so colonies cannot be used directly for serological testing.
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