CLED Agar: Composition, Principle, Uses, and Colony Characteristics

A 28-year-old woman presents with dysuria, frequency, and suprapubic pain. Her urine dipstick shows nitrites and leukocytes. The clinician sends a midstream urine sample for culture. In the laboratory, the technician has two options: plate it onto two separate media (blood agar plus MacConkey agar), or use a single plate of CLED agar.

The CLED plate costs less, takes up half the bench space, and in 18–24 hours produces colonies that are color-coded by lactose fermentation — yellow for fermenters like E. coli, blue-green for non-fermenters like Pseudomonas — without any concern that Proteus will swarm across the plate and overgrow everything else.

That combination of economy, safety, and clarity is why CLED agar became the standard medium for urine culture in many diagnostic microbiology laboratories worldwide.

CLED (Cystine-Lactose-Electrolyte-Deficient) agar is a non-selective, differential culture medium used for the isolation and enumeration of bacteria in urine specimens from suspected cases of urinary tract infection (UTI). Each word in the name describes a functional component of the medium:

• Cystine — added as a growth supplement to support the growth of cystine-dependent "dwarf colony" coliforms that would otherwise be missed
• Lactose — the fermentable carbohydrate that drives color differentiation between uropathogens
• Electrolyte-Deficient — deliberately low in electrolytes (sodium chloride and other salts) to prevent Proteus species from swarming
• Agar — the solidifying agent

CLED agar supports the growth of all potential urinary pathogens as well as contaminants such as diphtheroids, lactobacilli, and micrococci.

Figure: Lactose fermenting (yellow colonies) and Lactose Non-fermenting colonies in CLED

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 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

Final pH 7.3 +/- 0.2 at 25°C

Ingredients	gm/L
Lactose	10.0
Pancreatic digest of gelatin	4.0
Pancreatic digest of casein	4.0
Beef extract	3.0
L-Cystine	0.128
Bromothymol Blue	0.02
Agar	15.0

Why Electrolyte-Deficient? The Proteus Swarming Problem

Why Electrolyte-Deficient? The Proteus Swarming Problem

Proteus mirabilis is a common cause of complicated urinary tract infections, particularly in catheterised patients and those with urinary tract abnormalities. It has an unusual property: on standard media containing electrolytes, it differentiates from normal short rods into hyperflagellated, elongated swarmer cells that spread rapidly in concentric waves across the entire plate surface.

The result on a standard blood agar or nutrient agar plate is a thin, spreading film of Proteus that overgrows and obscures all other colonies — making it impossible to identify co-infecting organisms or perform accurate colony counting.

CLED agar addresses this in a straightforward way: electrolytes (primarily sodium chloride) are a required trigger for the swarming differentiation. By keeping the electrolyte concentration very low, the medium prevents the morphological switch from vegetative cells to swarmer cells. Proteus still grows on CLED agar — producing its characteristic translucent blue colonies — but it stays confined to discrete colonies rather than spreading.

Key exam point: CLED agar inhibits Proteus swarming by electrolyte deficiency. MacConkey agar also inhibits swarming, but by a different mechanism — bile salts at the concentration used in MacConkey inhibit the surface translocation required for swarming, rather than acting through electrolyte depletion. On blood agar, Proteus will swarm freely — which is one reason CLED is preferred over blood agar for urine culture..

Preparation of CLED Agar

1. Weigh 36 grams of dehydrated CLED agar powder and suspend in one liter of distilled or deionised water. Follow the manufacturer's instructions — formulations vary slightly between manufacturers (Oxoid, HiMedia, Mast, BD).
2. Mix well and heat with frequent agitation, boiling for one minute until the powder is completely dissolved. The medium will appear green at this stage due to the bromothymol blue indicator.
3. Sterilize by autoclaving at 121°C for 15 minutes.
4. Cool to 50–55°C before pouring to avoid condensation on plates.
5. Dispense approximately 20 mL into sterile Petri plates under aseptic conditions.
6. Allow to solidify on a level surface, label with medium name and date of preparation.
7. Store inverted at 2–8°C. Allow plates to reach room temperature before use.

Important: Do not over-incubate CLED plates beyond 24 hours. If lactose-fermenting organisms predominate, continued acid production can turn the entire medium yellow, masking non-lactose fermenters present in lower numbers.

Colony Characteristics

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.

The bromothymol blue indicator in CLED agar gives a clear color signal: lactose fermenters produce acid, lowering the pH and turning colonies yellow. Non-lactose fermenters produce alkaline or neutral reactions, giving blue or blue-green colonies. This color logic applies across all organisms and makes reading CLED plates fast and intuitive.

Organism	Colony color	Colony characteristics	Lactose fermentation
Escherichia coli	Yellow	Opaque, slightly deeper yellow center, 2–3 mm	Yes
Klebsiella pneumoniae	Yellow to whitish-yellow	Extremely mucoid, large, dome-shaped	Yes
Enterococcus faecalis	Yellow	Small (0.5 mm), entire margin	Yes (slowly)
Staphylococcus aureus	Deep yellow	Uniform, opaque, 1–2 mm	Yes
Coagulase-negative Staphylococci	Pale yellow	More opaque than Enterococcus, smaller than S. aureus	Variable
Proteus mirabilis	Translucent blue	Discrete colonies (no swarming), 1–2 mm	No
Pseudomonas aeruginosa	Green to blue-green	Matted surface, rough irregular periphery; may produce pyocyanin (blue-green diffusible pigment)	No
Candida albicans	White to cream	Raised, opaque, yeast-like; may show foot-like projections at margins on prolonged incubation	No
Streptococcus agalactiae (GBS)	Yellow	Tiny colonies; may be missed if mixed with larger coliforms	Yes

Reading tip: On a typical UTI culture plate, a pure growth of large yellow mucoid colonies strongly suggests Klebsiella. Pure yellow non-mucoid colonies with a deeper center suggest E. coli. Blue-green colonies with a matted surface and characteristic sweet/grape-like odour suggest Pseudomonas. Translucent blue discrete colonies in a patient with a urinary catheter suggest Proteus.

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.

Organism	Desired characteristics
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

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 prevents the swarming of Proteus spp.

Urine Culture: Quantitative Interpretation on CLED Agar

A critical and often under-taught aspect of CLED agar use is that it is designed for quantitative culture — not just presence or absence of growth. A calibrated bacteriological loop (1 µL or 10 µL) is used to inoculate the plate, which allows colony counts to be converted to colony-forming units per milliliter (CFU/mL) of urine.

Standard interpretation thresholds:

Colony count (CFU/mL)	Interpretation
≥ 10⁵ (100,000)	Significant bacteriuria — likely true infection in symptomatic patient
10⁴ – 10⁵	Borderline — repeat culture or interpret with clinical context
< 10⁴	Likely contamination in most clinical contexts

Important caveats:

• A count of ≥ 10² CFU/mL may be significant in catheter specimens or in symptomatic men (lower contamination risk)
• A count of ≥ 10³ CFU/mL may be significant in suprapubic aspirate specimens (any growth is significant)
• Single organism growth supports infection; three or more species suggests contamination in most cases

This quantitative approach is one of CLED agar's key practical advantages over non-calibrated plating: the same plate used to identify the organism also provides the count needed for clinical interpretation.

Choosing Your Urine Culture Medium

The three media commonly used for urine culture each have distinct strengths. The table below clarifies when each is most appropriate — and why many laboratories have moved to CLED as a single-medium approach.

Feature	CLED Agar	MacConkey Agar	Blood Agar
Gram-positive growth	Good	Inhibited (selective for GN)	Good
Gram-negative growth	Good	Good	Good
Lactose differentiation	Yes (yellow vs. blue-green)	Yes (pink vs. colourless)	No
Proteus swarming	Inhibited (electrolyte deficiency)	Inhibited (bile salts)	Not inhibited — will swarm
Candida growth	Good	Poor	Good
Hemolysis visible	No	No	Yes
Quantitative culture	Yes (with calibrated loop)	Yes	Yes
Cost as sole medium	Low (single plate)	Moderate (needs BA companion)	Moderate (needs MAC companion)
Best for	Routine urine culture (sole medium)	GN organism isolation and differentiation	Broad-spectrum, hemolysis assessment

Practical note for resource-limited laboratories: CLED agar as a sole medium has been validated in multiple studies as equivalent to the BA + MacConkey combination for routine urine culture. In settings where cost or plate availability is a constraint, CLED alone is an evidence-based choice.

How to Remember

Use the name as your framework. CLED — Cystine, Lactose, Electrolyte-Deficient — is not just an abbreviation, it is a three-part description of how the medium works:

• C = Cystine supports dwarf coliforms (without it, some E. coli strains produce tiny colonies easily missed)
• L = Lactose drives the yellow/blue color split — the most useful visual cue on the plate
• ED = Electrolyte-Deficient stops Proteus from swarming

Color logic — a simple rule:

Yellow colony = acid from lactose = lactose fermenter (most common uropathogens: E. coli, Klebsiella, enterococci) Blue/green colony = no acid = non-fermenter (Proteus, Pseudomonas) White/cream = yeast (Candida)

A clinical anchor to make it stick: Think of CLED agar as the "urine culture plate that solved three problems at once" — it grew everything, told you what fermented lactose by color, and stopped Proteus from ruining the plate. Before CLED, laboratories needed two plates (blood agar + MacConkey) and still had the Proteus swarming problem on the blood agar. CLED was the single-medium solution.

Limitations

1. Poor discrimination of some Gram-positive organisms. While enterococci and staphylococci grow on CLED, their colonies can be small and overlooked if mixed with larger Gram-negative colonies. Blood agar gives better colony morphology for Gram-positive organisms.
2. No hemolysis visible. CLED does not contain blood, so haemolytic organisms (e.g., beta-haemolytic streptococci, S. aureus) cannot be identified by hemolysis pattern. Blood agar is required for this.
3. Over-incubation problem. Beyond 24 hours, acid accumulation from dominant lactose fermenters can turn the entire plate yellow, masking non-fermenters present in smaller numbers.
4. Not suitable for fastidious organisms. Haemophilus and Neisseria will not grow on CLED agar. For urethral/genital specimens where gonorrhoea is suspected, chocolate agar is required alongside CLED.
5. Cannot differentiate within lactose fermenters by color alone. Both E. coli and Klebsiella produce yellow colonies; colony morphology (mucoid vs. non-mucoid) and further biochemical testing are required for species-level identification.

References and further readings

1. Tille, P. M. (2017). Bailey and Scott's Diagnostic Microbiology (14th ed.). Elsevier.
2. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.
3. Sandys, G. H. (1960). A new method of preventing swarming of Proteus spp. with a description of a new medium suitable for use in routine laboratory practice. Journal of Medical Laboratory Technology, 17, 224–233.
4. Mackey, J. P., & Sandys, G. H. (1966). Laboratory diagnosis of infections of the urinary tract in general practice by means of a dip-inoculum transport medium. British Medical Journal, 2(5504), 1286–1288.
5. Clinical and Laboratory Standards Institute (CLSI). (2023). M35: Abbreviated Identification of Bacteria and Yeast (3rd ed.). CLSI.