CLED AGAR: RATIONALE, USES, AND PREPARATION - BIOLOGY - 2025

The CLED agar (Cystine-Lactose-Electrolyte-Deficient) is a solid culture medium differential, used for diagnosis of urinary tract infections. The composition of the culture medium is designed for the good growth of urinary pathogens and is ideal for the quantification of colony forming units (CFU).

The CLED culture medium is non-selective, since Gram negative and Gram positive microorganisms can grow in it. But this is not a problem, since most UTIs are caused by only one type of microorganism.

CLED agar

In case of polymicrobial infections, 2 or 3 different bacteria can be obtained, but it is very rare and most of the time it is contaminated samples.

Among the Gram negative bacteria that can grow in this medium are the bacilli belonging to the Enterobacteriaceae family and other enteric bacilli, the uropathogens most frequently isolated in urine samples being the following: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Morganella morganii, Pseudomonas aeruginosa, among others.

Likewise, among the Gram positive bacteria that can grow in this medium are Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus faecalis, Streptococcus agalactiae, Corynebacterium sp, Lactobacillus sp and even yeasts, such as the Candida albicans complex, can grow.

However, due to the chemical composition of the medium, it does not allow the growth of some demanding genitourinary pathogens, such as Neisseria gonorrhoeae, Gardnerella vaginalis, among others.

CLED Agar Rationale

The CLED culture medium has meat extract, pancreatic hydrolyzate of casein and hydrolyzate of gelatin as an energy source. They provide the nutrients for the development of undemanding bacteria.

It also contains cystine, an amino acid that allows the growth of coliforms, distinguishable by their small size.

Likewise, it contains lactose as a fermentable carbohydrate, for this reason this medium is differential; being able to distinguish the fermenting bacteria from the non-lactose fermenting.

Fermenting bacteria cause the pH of the medium to change by the production of acids, developing yellow colonies, while non-fermenting bacteria do not generate changes in the medium, therefore they take on the color of the original agar, green.

The fermentation reaction is revealed thanks to the presence of the pH indicator, which in this medium is bromothymol blue.

On the other hand, the low electrolyte concentration of the medium inhibits the typical invasive growth of the genus Proteus, called the swarming effect. This generates an advantage over other media, since it allows the counting of CFUs, including if the Proteus genus is present.

However, the low concentration of electrolytes inhibits the growth of some species of the genus Shigella, this being a disadvantage compared to other media.

Rationale for CLED agar (Bevis)

There is a variant or modification of this medium made by Bevis, who incorporated acid fuchsin (Andrade's indicator) into the original composition. It works in conjunction with bromothymol blue to differentiate fermenting from non-fermenting bacteria.

The difference between conventional and modified medium is the color that the colonies take on. In the case of lactose-fermenting bacteria, the colonies turn reddish-orange with a pink or red halo, while the non-fermenting ones are bluish-gray.

Applications

CLED agar is used exclusively for the seeding of urine samples. The use of this medium is especially frequent in European laboratories, while in America it is less used.

Sample collection must meet certain parameters to obtain reliable results, including:

• Not be taking antibiotics before sampling.
• Preferably take the urine first thing in the morning, since it is more concentrated, when it is not possible to take the sample by invasive methods.
• Wash the genitals well before taking the sample.
• Discard the first stream of urination and then place the container.
• Collect 25 to 30 ml of urine in a well-labeled sterile container.
• Take immediately to the laboratory surrounded in ice.
• It must be processed within 2 hours of issue or refrigerated at 4 ° C for a maximum of 24 hours.

Seeding urine samples

The urine sample should be diluted 1:50.

For dilution, place 0.5 ml of patient urine and dilute with 24.5 ml of sterile physiological solution.

Measure out 0.1 ml of the diluted urine and surface spread with a drigalski spatula on the CLED medium. This is the best seeding method for counting colonies. For this reason it is used in urine samples, since the results must be expressed in CFU / ml.

To quantify the colonies obtained, proceed as follows: count the colonies on the plate and multiply by 10 and then by 50. This gives you the amount of CFU / ml of urine.

Interpretation

Counts above 100,000 CFU / ml -– Indicates urinary infection

Counts below 1000 CFU / ml-– No infection

Counts between 1000-10,000 CFU / ml -– Doubtful, possible contamination, repeat sampling.

ID

The colonies grown on CLED agar should have a Gram and depending on the morphotintorial characteristics of the microorganism, a certain subculture is carried out.

For example, if it is a Gram negative bacillus, it will be sown on a MacConkey agar, where the fermentation or not of lactose is corroborated. In addition, a nutrient agar is attached to perform the oxidase test.

In case the Gram reveals Gram positive cocci, it can be subcultured on salty mannitol agar and on nutrient agar. In the latter, the catalase test is carried out. Finally, if yeasts are observed, it will be sown on Sabouraud agar.

Many laboratories avoid the use of CLED medium and prefer only to use blood agar, MacConkey and nutrient agar to seed urine samples.

Preparation

In a flask with one liter of distilled water dissolve 36.2 g of CLED agar powder. After 5 minutes' standing, heat the resuspended agar, mixing constantly to boil for 1 minute.

Then sterilize at 121 ° C for 15 minutes in the autoclave. At the end of the time, it is removed from the autoclave and allowed to cool down to a temperature of 45 ° C. Subsequently, 15-20 ml is served in each sterile Petri dish.

The plate serving procedure must be carried out inside a laminar flow hood or in front of the Bunsen burner to avoid contamination.

The plates served are left to solidify, arranged in an inverted rack and stored in a refrigerator (2-8 ° C) until use.

The final pH of the prepared medium should be 7.3 ± 0.2.

References

1. Recommendations for the microbiological diagnosis of urinary infection. chil. infectol. 2001; 18 (1): 57-63. Available at: scielo.org.
2. Panchi J. Identification of the microbial agent causing urinary tract infections in inpatients undergoing bladder catheterization. 2016. Undergraduate work to qualify for the Bachelor's degree in Clinical Laboratory. Technical University of Ambato. Ecuador.
3. Britannia Laboratories. CLED medium. Available at: britanialab.com.
4. Renylab Laboratories. Instructions for use, CLED Agar. 2013 Available at: es.renylab.ind.br.
5. Cultimed Laboratories. Basic Manual of Microbiology. Available at: ictsl.net.
6. Muñoz P, Cercenado E, Rodríguez-Créixems M, Díaz MD, Vicente T, Bouza E. The CLED agar option in urine culture routine. A prospective and comparative evaluation. Diagn Microbiol Infect Dis. 1992; 15 (4): 287-90.
7. García P, Paredes F, Fernández del Barrio M. (1994). Practical clinical microbiology. University of Cadiz, 2nd edition. UCA Publications Service.