CLED (cysteine-, lactose-, and electrolyte-deficient) agar is a differential culture medium primarily used to isolate and enumerate 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.
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 a pH indicator to differentiate lactose fermenters from non-fermenters. Lactose fermenters will lower the pH of the medium by producing acid and changing the indicator’s color 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
|Pancreatic digest of gelatin||4.0|
|Pancreatic digest of casein||4.0|
Final pH 7.3 +/- 0.2 at 25°C
Preparation of CLED agar
- Weigh and suspend 36 grams of the dehydrated medium in one liter of distilled water. (follow manufacturer instructions)
- Mix well, heat with frequent agitation, and boil for one minute until the media is completely dissolved.
- Autoclave at 121°C for 15 minutes.
- Cool to 50°C, mix well, and dispense into sterile Petri plates.
- When the medium is solidified, label the plates with the name of the media and the date of preparation.
- Store the plates in an inverted position (to avoid excess moisture) at 2-8°C until use.
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 organism||Colony morphology|
|E. coli||Opaque yellow colonies with a slightly deeper yellow center|
|Klebsiella spp.||Yellow to whitish-blue colonies, extremely mucoid|
|Proteus spp||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|
|Coagulase Negative Staphylococci (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.
- 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.
- 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.
|Escherichia coli ATCC 25922||Luxuriant Growth; colonies are yellow, medium yellow|
|Proteus vulgaris ATCC 8427||Good growth; colonies are colorless to blue; swarming is inhibited; however, slight spreading is acceptable|
|Enterococcus faecalis ATCC 29212||Growth; small colonies, colorless to yellow|
|Staphylococcus aureus ATCC 25923||Good growth; colonies small, yellow to yellowish|
|Uninoculated plates||Green to blue-green|
Advantages of CLED Agar
- Good discrimination of gram-negative bacteria on the basis of lactose fermentation and colony appearance;
- Inhibits swarming of Proteus spp (Proteus mirabilis and Proteus vulgaris are frequently involved in urinary tract infection);
- Relatively low cost (compared with the combined use of blood agar and MacConkey agar for urine culture).
Note: MacConeky medium containing bile salts also prevents the swarming of Proteus spp.
Limitations of CLED Agar
- 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.
- 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.
- Shigella species may not grow on this medium.
- Since it is a differential medium, colonies from the media could not be directly used for serological testing.
References and further readings
- Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
- Review of Medical Microbiology and Immunology, Lange Medical Books, 13th edition
- Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology), 7th edition