microbeonline

CLED Agar: composition, uses and typical colony characteristics

CLED (cysteine-, lactose-, and electrolyte-deficient) agar is a differential culture medium for use in isolating and enumerating bacteria in urine from the suspected cases of Urinary Tract Infection. CLED Agar supports the growth of all potential urinary pathogens, and a number of contaminants such as diphtheroids, lactobacilli, and micrococci.

For routine urine culture, inoculation of specimens in a combination Blood agar (BA) and MacConkey agar is commonly used approach whereas CLED agar can be used as a sole medium, reducing the cost without compromising the quality.

Many researchers found that the productivity of CLED is similar to that of standard procedures (Blood agar and MacConkey agar combined).  It’s economy and convenience (less time, easy morphological differentiation) make it a medium worth using for routine culture of urine samples.

Benefits and Disadvantages of Using CLED Agar for Urine Culture

  1. Good discrimination of gram-negative bacteria on the basis of lactose fermentation and colony appearance;
  2. Inhibits swarming of Proteus spp (Proteus mirabilis and Proteus vulgaris are  frequently involved in urinary tract infection );
  3. Relatively low cost (compared with combined use of Blood Agar and MacConkey agar for urine culture).

Note: MacConeky medium containing bile salts also prevent the swarming of Proteus spp.

Disadvantages: poor growth of some gram-positive bacteria.

Composition

Major constituents of CLED Agar are Lactose, Pancreatic digest of gelatin and casein, Beef extract, Cystine, pH indicator Bromothymol blue and Agar.

Ingredients per liter of de-ionized water and their main use are as follows:

  1. Lactose (10.0gm):
    • Lactose is an energy source for organisms capable of utilizing it by a fermentative mechanism..
  2. Pancreatic Digest of Gelatin (4.0gm)
  3. Pancreatic Digest of Casein (4.0gm)
  4. Beef Extract (3.0gm)
    • Enzymatic Digest of Casein, Enzymatic Digest of Gelatin, and Beef Extract provide the nitrogen, vitamins, and carbon in CLED Agar.
  5. L-Cystine (0.128gm):
    • L-Cystine is added as a growth supplement for cystine-dependent coliforms.  It permits the growth of “dwarf colony” coliforms.
  6. Bromothymol Blue (0.02gm)
    • Bromthymol Blue is the pH indicator to differentiate lactose fermenters from lactose-nonfermenters. Organisms capable of fermenting lactose will lower the pH and change color of the medium from green to yellow.
  7. Agar (15.0 gm): Agar is the solidifying agent.

Final pH 7.3 +/- 0.2 at 25°C

Electrolyte sources are reduced in order to minimize the swarming of Proteus species.

Lactose fermenting (Yellow colonies) and Lactose Non fermenting colonies in CLED

Typical colony morphology on CLED Agar is as follows:

  1. Escherichia coli: Opaque yellow colonies with a slightly deeper yellow center
  2. Klebsiella spp: Yellow to whitish-blue colonies, extremely mucoid
  3. Proteus spp: Translucent blue colonies
  4. Pseudomonas aeruginosa: Green colonies with typical matted surface and rough periphery
  5. Enterococci: Small yellow colonies, about 0.5mm in diameter
  6. Staphylococcus aureus: Deep yellow colonies, uniform in color
  7. Coagulase Negative Staphylocci (CONS): Pale yellow colonies, more opaque than Enterococcus faecalis