CLED Agar: Composition, Function, and Use

• CLED agar, or Cystine Lactose Electrolyte-Deficient agar is a growth medium for isolation and enumeration of urinary tract bacteria.
• It is a non-inhibitory differential medium.
• CLED agar was developed by Sandys in 1960 at the Public Health Laboratory in Northampton.
• It allows growth of a wide range of bacteria while providing visual differentiation of colonies, based on lactose fermentation.

Composition and Function of Key Components

• Cystine encourages development of dwarf colony variants of coliforms, aiding in colony differentiation and enumeration.
• Lactose is a fermentable carbohydrate source; lactose fermentation leads to acid production, which is detected by bromothymol blue.
• Deficiency of electrolytes inhibits swarming of Proteus species, which can obscure other colonies on regular media.
• Bromothymol blue is a pH indicator that changes color depending on the acidity or alkalinity of the medium.
• At neutral or alkaline pH, bromothymol blue appears blue, whereas at acidic pH, it turns yellow.

Principle of CLED Agar

• The main principle relies on its ability to support the growth of various bacteria while differentiating them based on lactose fermentation.
• Absence of electrolytes prevents the swarming phenomenon of Proteus, enabling better visualization of mixed cultures.
• Lactose-fermenting bacteria produce acid, lowering the pH of the surrounding medium.
• This pH change is detected by bromothymol blue; colonies of lactose fermenters then appear yellow.
• Bacteria that do not ferment lactose produce alkaline or neutral byproducts, resulting in blue or green colonies.

Uses of CLED Agar

• Primarily used for the cultivation and enumeration of microorganisms in urine samples.
• Useful in diagnostic laboratories for identifying urinary tract infections (UTIs).
• Can differentiate between lactose-fermenting and non-lactose-fermenting bacteria.
• Can be part of the routine workup when performing a urine culture.
• Helps in isolating and counting the different types of bacteria present in a sample, providing valuable information for diagnosis and treatment.

Interpretation of Results

• Yellow colonies indicate lactose-fermenting bacteria such as Escherichia coli and Klebsiella pneumoniae.
• The yellow color results from acid production due to lactose fermentation, which changes the bromothymol blue indicator.
• Blue/Green colonies indicate non-lactose-fermenting bacteria such as Proteus, Pseudomonas, and Enterococcus faecalis.
• These bacteria either do not utilize lactose or produce alkaline compounds.
• Colony Morphology: Colony size, shape, and texture on CLED agar can also provide additional clues for bacterial identification.
• Pinpoint colonies might suggest the presence of certain fastidious organisms.

Advantages of CLED Agar

• Non-inhibitory: Allows a wide range of bacteria to grow, making it suitable for general use in urine cultures.
• Differential: Enables easy differentiation between lactose fermenters and non-lactose fermenters, aiding in the preliminary identification of bacteria.
• Anti-swarming: Absence of electrolytes prevents swarming of Proteus species, providing a clearer view of the mixed culture.
• Ease of Use: Simple to prepare and use in routine laboratory settings.

Limitations of CLED Agar

• Limited Identification: While it differentiates based on lactose fermentation, further biochemical tests are needed for definitive species identification.
• Not Suitable for Fastidious Organisms: May not support the growth of highly fastidious organisms that require specific growth factors not present in CLED agar.
• Color Interpretation: Color changes can sometimes be subtle and may require experience to interpret accurately, especially with mixed cultures.

Quality Control

• Appearance: Dehydrated medium should be homogeneous, free-flowing, and beige. Prepared medium should be clear with a green-blue hue.
• Growth Performance: Should support good growth relevant microorganisms; lactose-fermenting strains should produce yellow colonies and non-lactose-fermenting strains should produce blue/green colonies.
• Sterility: Should be free from contamination; incubate uninoculated plates to ensure no growth appears.

Storage

• Store dehydrated medium in a tightly closed container in a cool, dry place, protected from light.
• Prepared agar plates should be stored at 2-8°C and used before the expiration date.
• Proper storage ensures the integrity and performance of the media.

Procedure for Using CLED Agar in Urine Culture

• Collect Urine Sample: Collect a midstream urine sample aseptically.
• Inoculation: Use a calibrated loop to streak a known volume of urine onto the CLED agar plate for semi-quantitative analysis.
• Incubation: Incubate the inoculated plate at 35-37°C for 18-24 hours.
• Observation: Observe the growth and colony morphology; note the color of the colonies to differentiate lactose fermenters from non-lactose fermenters.
• Enumeration: Count the number of colonies to estimate the bacterial load in colony-forming units per milliliter (CFU/mL).
• Identification: Perform further tests, like Gram stains and biochemical tests, to identify the bacterial species.

Variations and Modifications

• Some formulations may include additional supplements to enhance the growth of specific organisms; these are not standard, but can be tailored for specific diagnostic needs.
• Chromogenic CLED agar is a modified version that includes chromogenic substrates for specific enzymes.
• This allows for more specific and rapid identification of certain urinary pathogens directly on the plate, based on the color reactions of colonies.

Common Organisms Isolated on CLED Agar

• Escherichia coli: A common lactose-fermenting bacterium that produces yellow colonies and a frequent cause of UTIs.
• Klebsiella pneumoniae is another lactose fermenter that produces mucoid, yellow colonies and can cause UTIs and other infections.
• Proteus mirabilis: A non-lactose fermenter that typically produces blue/green colonies, known for its ability to cause UTIs and produce urease.
• Enterococcus faecalis: A non-lactose fermenter that appears as blue or green colonies, and is an opportunistic pathogen in UTIs.
• Pseudomonas aeruginosa: A non-lactose fermenter that forms blue-green colonies and can cause UTIs, especially in catheterized patients.

Troubleshooting Common Issues

• No Growth: Check the viability of the inoculum; ensure proper incubation temperature and duration; verify that the CLED agar is not expired or improperly stored.
• Swarming: If swarming occurs, it may indicate that the medium formulation is incorrect or that the electrolyte balance is off; ensure a CLED agar is in use to prevent swarming.
• Poor Color Differentiation: Ensure the pH indicator is functioning correctly; check the age and storage conditions of the agar; use a control strain to verify color reactions.

Importance in Clinical Microbiology

• CLED agar is a valuable tool in clinical microbiology laboratories for the diagnosis of UTIs.
• Its ability to support diverse bacterial growth along with differential characteristics makes it an essential medium for urine culture workup.
• By providing a clear and easily interpretable growth pattern, it aids in the rapid identification and enumeration of urinary pathogens, contributing to timely and effective patient care.

Cystine Lactose Electrolyte-Deficient (CLED) agar is a growth medium for isolating and counting urinary tract bacteria. It differentiates colonies via lactose fermentation, indicated by bromothymol blue. The absence of electrolytes prevents Proteus swarming.