CLED Agar: Composition, Uses, Colony Characteristics

Last updated on June 17th, 2021

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

CLED (cysteine-, lactose-, and electrolyte-deficient) agar is a differential culture medium primarily used for isolation and enumeration of bacteria especially from urine samples.

CLED is preferred over a combination of blood agar (BA) and MacConkey agar for routine urine culture because it supports the growth of all potential urinary pathogens, and a number of contaminants such as diphtheroids, lactobacilli, and micrococci. It also differentiates between lactose fermenting and non-lactose fermenting colonies and inhibits the swarming of Proteus spp. With all these benefits, CLED agar can be used as a sole medium, reducing the cost without compromising the quality.

Principle

Major constituents of CLED agar are lactose, pancreatic digest of gelatin, pancreatic digest of casein, beef extract, vitamins, and carbon. Lactose is an energy source for organisms capable of utilizing it by a fermentative mechanism. Enzymatic digest of casein, enzymatic digest of gelatin, and beef extract provide the nitrogen, vitamins, and carbon. L-Cystine is added as a growth supplement for cystine-dependent coliforms. It permits the growth of “dwarf colony” coliforms.

Bromthymol blue is used as a pH indicator to differentiate lactose fermenters from non-fermenters. Lactose fermenters will lower the pH of the medium by producing acid, and change the color of the indicator from blue to yellow. L-cystine is added as a growth supplement for cystine-dependent coliforms. Electrolytes are reduced to restrict the swarming of Proteus species. Agar is used as a solidifying agent.

Composition of CLED agar

Ingredientsgm/L
Lactose10.0
Pancreatic digest of gelatin4.0
Pancreatic digest of casein4.0
Beef extract3.0
L-Cystine0.128
Bromothymol Blue0.02
Agar15.0

Final pH 7.3 +/- 0.2 at 25°C

Preparation of CLED agar

  1. Weigh and suspend 36 grams of the dehydrated medium in one liter of distilled water.(follow manufactures instructions)
  2. Mix well, heat with frequent agitation, and boil for one minute until the media is completely dissolved.
  3. Autoclave at 121°C for 15 minutes.
  4. Cool to 50°C, mix well and dispense into sterile Petri plates.
  5. When the medium is solidified, label the plates with the name of the media and date of preparation.
  6. Store the plates in an inverted position (to avoid excess moisture) at 2-8°C until use.

Result Interpretation:

Lactose fermenting colonies appear yellow but have distinguishable colony morphology based on size, consistency, etc. Non-lactose fermenting colonies appear blue. Colonies of Gram-positive cocci appear yellow but smaller in size as compared to Gram-negative ones.

Colony morphology of some common organisms are enlisted below

Name of the organismColony morphology
E. coliOpaque yellow colonies with a slightly deeper yellow center
Klebsiella spp.Yellow to whitish-blue colonies, extremely mucoid
Proteus sppTranslucent blue colonies
Pseudomonas aeruginosaGreen colonies with typical matted surface and rough periphery
EnterococciSmall yellow colonies, about 0.5mm in diameter
Staphylococcus aureusDeep yellow colonies, uniform in color
Coagulase Negative Staphylocci (CONS)Pale yellow colonies, more opaque than Enterococcus faecalis

Quality Control of CLED Agar

QC of CLED agar should be done by performing sterility testing and performance testing.

  1. Sterility testing: Incubate 3-5 plates of uninoculated medium at 37℃ for 18-24 hrs. Any growth in the medium should be regarded positive and the whole lot should be discarded.
  2. Performance testing should be performed by inoculating one or more standard strains onto the prepared medium and incubating at 35 ± 2°C in an aerobic atmosphere for 18-24 hours. Observe for the growth, pigmentation, colony size, and inhibition of Proteus swarming/spreading after overnight incubation.
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

Advantages of CLED Agar

  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 the combined use of blood agar and MacConkey agar for urine culture).

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

Limitations of CLED Agar

  1. Streptococci and other organisms requiring blood or serum for growth may be scarcely recovered on this medium or may need extended incubation. Therefore, an additional blood agar plate should also be inoculated if such organisms are suspected.
  2. Not suitable for recovery of genitourinary pathogens such as Neisseria gonorrhoeae, Gardnerella vaginalis, Chlamydia, Ureaplasma, or other fastidious organisms as these organisms do not grow on this medium.
  3. Shigella species may not grow on this medium.
  4. Since it is a differential medium, colonies from the media could not be directly used for serological testing.
About Acharya Tankeshwar 473 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

23 Comments

  1. what is the advantage of using CLED Medium over Mac.conkey
    it prevents proteus swarming
    it shows pseudomonas growth better
    it also grows staph and candida
    this was a qsn asked in AIIMS pg entrance

    • Nithya Ji
      Thank you for your question. Is it the single best question? If this question is the exact replica of the question asked, i label it as a tricky question with a trap. CLED prevents swarming of Proteus (this is benefit over using Blood Agar).MacConkey agar with bile salts also prevents swarming of Proteus spp. There is no evidence that Pseudomonas grow grow better in CLED. Yes CLED supports the growth of Staph (MacConeky does not).

  2. hi,you are strong welcome how help me your well prepared lesson so,l want to ask you aquestion your lesson. my question is about benefits of cled please explain cled inhibits swarming of proteus spp?

  3. During culturing of urine a found a grey swarming colonies .in gram reaction are gram positive rods,some with bipolar spores what are those?

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