CLED Agar- Composition, Principle, Preparation, Results, Uses

CLED Agar is an acronym for Cystine Lactose Electrolyte Deficient Agar. It is a type of differential and selective culture medium that is commonly used for the isolation and enumeration of bacteria from urine specimens. It is especially useful for the detection of urinary tract infections (UTIs) caused by members of the family Enterobacteriaceae, such as Escherichia coli, Klebsiella, Proteus, and others.

CLED Agar was first developed by Sandys in 1960 as a modification of MacConkey Agar, which is another differential and selective medium for enteric bacteria. The main difference between CLED Agar and MacConkey Agar is that CLED Agar does not contain any electrolytes (such as sodium chloride) or bile salts. These components are known to inhibit the growth of some urinary pathogens, such as Proteus species, which can produce urease and alkalize the medium. CLED Agar also contains L-cystine, which enhances the growth of some coliforms that require this amino acid.

CLED Agar has a green color due to the presence of bromthymol blue, which is a pH indicator that changes color depending on the acidity or alkalinity of the medium. The medium turns yellow when lactose-fermenting bacteria grow on it, indicating acid production. The medium remains green or turns blue when non-lactose-fermenting bacteria grow on it, indicating no acid production or alkaline reaction. CLED Agar also allows the differentiation of some bacteria based on their colony morphology and size. For example, E. coli produces large yellow colonies with a dark center, while Proteus produces small translucent colonies with a characteristic swarming pattern.

CLED Agar is widely used in clinical microbiology laboratories as a primary screening tool for urine culture. It can help identify the presence and type of bacteria in urine samples and guide further diagnostic tests and treatment options. CLED Agar is also suitable for long-term storage and transport of urine specimens without compromising their viability or quality. However, CLED Agar has some limitations that need to be considered when interpreting the results. For instance, some bacteria that cause UTIs may not grow well or at all on CLED Agar, such as streptococci, Neisseria gonorrhoeae, and other fastidious organisms. Therefore, other culture media and methods may be required to detect these pathogens. Moreover, CLED Agar does not provide sufficient information for the complete identification of bacteria, which may require additional biochemical and serological tests.

CLED Agar is a complex medium that contains the following ingredients:

• Enzymatic digest of casein: This is a source of nitrogen, vitamins, minerals, and amino acids that are essential for the growth of bacteria. Casein is a protein found in milk that is hydrolyzed by enzymes to produce smaller peptides and amino acids.
• Enzymatic digest of gelatin: This is another source of nitrogen, vitamins, minerals, and amino acids that are essential for the growth of bacteria. Gelatin is a protein derived from animal collagen that is hydrolyzed by enzymes to produce smaller peptides and amino acids.
• Beef extract: This is a source of organic nitrogen compounds, carbohydrates, salts, and other nutrients that are essential for the growth of bacteria. Beef extract is obtained by boiling beef in water and concentrating the resulting broth.
• L-cystine: This is a sulfur-containing amino acid that supports the growth of cysteine-dependent dwarf coliforms. These are strains of Escherichia coli that require cysteine for their growth and produce small colonies on CLED Agar.
• Lactose: This is a fermentable carbohydrate that provides carbon and energy for the bacteria. Lactose is a disaccharide composed of glucose and galactose that is found in milk and dairy products.
• Bromthymol blue: This is a pH indicator that changes color depending on the acidity or alkalinity of the medium. Bromthymol blue is green at neutral pH (7.0), yellow at acidic pH (below 6.0), and blue at alkaline pH (above 7.6).
• Agar: This is a polysaccharide extracted from red algae that acts as a solidifying agent. Agar forms a gel-like matrix when dissolved in water and cooled, which provides a stable surface for bacterial growth.

The final pH of CLED Agar is 7.3 +/- 0.2, which is slightly alkaline and inhibits the growth of some bacteria that prefer acidic conditions. The medium also lacks electrolytes, which prevents the swarming of Proteus species. CLED Agar can be supplemented with blood or serum if desired to enhance the growth of some fastidious organisms.

CLED Agar is a differential and non-selective medium that allows the growth and enumeration of most urinary tract pathogens. It also helps to differentiate them based on their ability to ferment lactose.

The principle of CLED Agar is based on the following components:

• Enzymatic digest of casein, enzymatic digest of gelatin, and beef extract: These ingredients provide the nitrogen, vitamins, minerals, and amino acids essential for the growth of bacteria.
• L-cystine: This amino acid supports the growth of cysteine-dependent dwarf coliforms, which are strains of Escherichia coli that produce small colonies on CLED Agar due to their reduced ability to synthesize cysteine.
• Lactose: This is the fermentable carbohydrate that provides carbon and energy for the bacteria. Lactose-fermenting bacteria lower the pH of the medium and change its color from green to yellow. Lactose-nonfermenting bacteria do not affect the pH or color of the medium.
• Bromthymol blue: This is the pH indicator that changes color from green to yellow in acidic conditions. It helps to differentiate lactose-fermenting bacteria from lactose-nonfermenting bacteria on CLED Agar.
• Agar: This is the solidifying agent that gives the medium its gel-like consistency.

The principle of CLED Agar is similar to that of Eosin Methylene Blue (EMB) Agar, which is another differential medium for urinary tract pathogens. However, CLED Agar has some advantages over EMB Agar, such as:

• CLED Agar does not contain any inhibitory substances, such as eosin and methylene blue, that may suppress the growth of some bacteria.
• CLED Agar does not produce metallic sheen colonies, which may be difficult to distinguish from other colony types on EMB Agar.
• CLED Agar allows the growth of both Gram-positive and Gram-negative bacteria, whereas EMB Agar is more selective for Gram-negative bacteria.

Therefore, CLED Agar is a more suitable medium for routine urine culture and enumeration of urinary tract pathogens.

To prepare CLED Agar, you will need the following materials and equipment:

• CLED Agar powder
• Distilled water
• Measuring cylinder
• Weighing balance
• Magnetic stirrer and bar
• Erlenmeyer flask
• Autoclave
• Petri dishes
• Laminar flow hood

The steps for preparing CLED Agar are as follows:

1. Measure 1 liter of distilled water using a measuring cylinder and pour it into an Erlenmeyer flask.
2. Weigh 36 grams of CLED Agar powder using a weighing balance and add it to the flask.
3. Place a magnetic stirrer bar in the flask and place the flask on a magnetic stirrer. Mix well until the powder is completely dissolved.
4. Heat the flask with frequent agitation and boil for one minute to sterilize the medium.
5. Transfer the flask to an autoclave and autoclave at 121°C for 15 minutes.
6. After autoclaving, cool the flask to 50°C in a water bath or on a cooling rack.
7. Working under a laminar flow hood, mix the medium well and dispense into sterile Petri dishes. Pour about 15-20 ml of medium per dish and swirl gently to spread evenly.
8. Allow the medium to solidify at room temperature and then invert the plates to avoid excess moisture.
9. Store the prepared plates at 8-15°C until use.

The prepared CLED Agar plates should have a green color and a clear appearance. They should be used within one month of preparation.

Various cultural responses are shown by different microorganisms on CLED Agar at the appropriate atmosphere and temperature after 18 – 24 hours incubation are as follows:

Some examples of colony characteristics on CLED Agar are shown below:

CLED Agar is a widely used medium for the isolation and enumeration of urinary tract pathogens. It has several advantages over other media for this purpose, such as:

• It is non-inhibitory to most urinary tract organisms, including gram-positive and gram-negative bacteria, yeasts, and some fastidious bacteria. It does not contain any bile salts, dyes, or antimicrobial agents that may interfere with the growth of some organisms.
• It is differential for lactose fermentation. Lactose-fermenting organisms produce yellow colonies on CLED Agar, while non-lactose-fermenting organisms produce blue-green colonies. This helps in the preliminary identification of common urinary tract pathogens, such as Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Proteus mirabilis.
• It is transparent and allows the detection of hemolysis and pigment production by some organisms. For example, hemolytic streptococci produce clear zones around their colonies, while Pseudomonas aeruginosa produces greenish pigments on CLED Agar.
• It is low in electrolytes and prevents the swarming of Proteus species. Proteus species are notorious for their motility and ability to spread over the surface of agar plates, obscuring other organisms. CLED Agar inhibits this phenomenon by limiting the availability of electrolytes for the flagellar movement of Proteus species.
• It is easy to prepare and has a long shelf life. CLED Agar can be prepared by simply dissolving the dehydrated powder in distilled water and autoclaving. It can be stored at room temperature for up to two years without losing its performance.

CLED Agar is suitable for the isolation and counting of many aerobically growing microorganisms from urine specimens, such as:

• Enterobacteriaceae: E. coli, K. pneumoniae, Enterobacter spp., Citrobacter spp., Serratia spp., etc.
• Non-fermenting gram-negative rods: P. aeruginosa, Acinetobacter spp., Stenotrophomonas spp., etc.
• Enterococci: E. faecalis, E. faecium, etc.
• Staphylococci: Staphylococcus aureus, S. epidermidis, S. saprophyticus, etc.
• Yeasts: Candida albicans, C. tropicalis, C. glabrata, etc.

CLED Agar can also be used for the detection of urinary tract infections in animals and for environmental monitoring of water quality.

CLED Agar is a useful and convenient medium for the isolation and enumeration of urinary tract pathogens. However, it also has some limitations that should be considered when using it for urine culture. Some of these limitations are:

• Streptococci and other organisms requiring blood or serum for growth may only be insufficiently recovered on this medium or may need extended incubation. Therefore, the specimen should also be cultivated onto a blood agar plate if such organisms are expected. Blood agar can enhance the growth and hemolysis of streptococci and other fastidious bacteria that may cause urinary tract infections (UTIs).
• Genitourinary pathogens such as Neisseria gonorrhoeae, Gardnerella vaginalis, Chlamydia, Ureaplasma, or other fastidious organisms do not grow on this medium. These organisms require special media and conditions for their cultivation and detection. For example, Neisseria gonorrhoeae needs a selective medium such as Thayer-Martin agar or chocolate agar, and an increased CO2 atmosphere. Gardnerella vaginalis needs a pH indicator such as phenolphthalein to show its characteristic clue cells. Chlamydia and Ureaplasma need cell culture or molecular methods for their identification.
• Serological tests using pure cultures are necessary for complete identification. CLED Agar can differentiate between lactose fermenters and non-fermenters, but it cannot provide further information on the species or serotype of the isolated bacteria. Therefore, additional tests such as biochemical reactions, antigen detection, or molecular typing are needed to confirm the identity and epidemiology of the urinary tract pathogens.

These limitations should not discourage the use of CLED Agar for urine culture, but rather remind the users to supplement it with other methods and media to ensure a comprehensive and accurate diagnosis of UTIs. CLED Agar is still a valuable tool for the initial screening and enumeration of urinary tract organisms.

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