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Introduction of CLED Agar

CLED stands for cysteine, lactose and electrolyte- deficient. CLED agar or medium is a differential culture medium using for isolation and enumeration of bacteria in urine specimen from the suspected cases of urinary tract infection (UTI). It favors the growth of all potential urinary pathogens as well as a number of contaminants such as diphtheroid, lactobacilli, and micrococci. Candida also enjoys this medium.

This is a sole medium alternate for routine urine culture, inoculation of specimens in a combination blood agar (BAP) and MacConkey agar (MAC) without compromising the quality and reducing the cost.

Composition

CLED agar  typically contains-

Peptone : 4 g/L

“Lab Lemco” Powder (meat extract): 3g/L

Tryptone: 4g/L

Lactose:  10g/L

L-Csteine: 0.128g/L

Bromothymol blue:  0.02g/L

Agar: 15 g/L

Final pH 7.3 +/- 0.2 at 25°C

Preparation of CLED Agar

CLED agar is available in dehydrated powder form by various manufacturers like Mast, Oxoid, Difco, Himedia and so on.

• Follow the instructions of the manufacturer to prepare the medium i.e. 36.13gm dehydrated medium in 1000mL distilled or deionized  water , mix properly and finally Sterilize by autoclaving at 121°C for 15 minutes.
• After cooling to 50-55°C, mix well, and dispense it aseptically in sterile petri dishes. Date the medium and give it a batch number.
• Store the plates at 2-8°C preferably in plastic bags to prevent loss of moisture.

Shelf life

It can be used for Several weeks but should be free from of any change in the appearance of the medium showing contamination, deterioration, or alteration of pH.

Working

Reduced electrolyte prevents the swarming of Proteus whereas Cystine promotes the formation of cystine-dependent dwarf colonies. Bromothymol blue is the indicator used in the agar, it changes to yellow in case of acid production during fermentation of lactose or changes to deep blue in case of alkalization. Lactose fermenting bacteria build yellow colonies. Bacteria which decarboxylate L-Cystine cause an alkaline reaction and build deep blue colonies.

Typical Colony Morphology on CLED Agar is as follows:

1. Staphylococcus aureus: Deep yellow colonies of uniform in color
2.  S. saprophyticus and other Coagulase Negative Staphylococci (CoNS): Pale yellow to white colonies, more opaque than Enterococcus faecalis
3.  Enterococci: Small yellow colonies, about 0.5mm in diameter
4.  Escherichia coli: Opaque yellow colonies with a slightly deeper yellow center
5. Klebsiella species: Large mucoid yellow to whitish-blue colonies
6. Proteus spp: Translucent blue-grey colonies
7. Pseudomonas aeruginosa: Green colonies with typical matted surface and rough periphery

Modification

Note: CLED medium with modification i.e. CLED Medium with Andrade’s Indicator now uses most commonly.

Composition is same with slightly increment of Andrade’s indicator i.e. Andrade indicator (0.100 gm/L)

CLED Medium is further modified by Bevis by incorporation of Andrade’s indicator. This medium provides sharper differentiation between lactose-fermenters (LF) and lactose-non-fermenters (NLF). Addition of Andrade’s indicator enhances the appearance of colony and aids in the identification of microorganisms. At different pH values, the color of the medium varies from the standard medium, which is well documented by Bevis .

Color of CLED medium

pH               Color

• 7.4               deep blue
• 7.0               bluish grey
• 6.8              pale grey
• 6.6               pinkish grey
• 6.4                bright red with whitish tinge
• 6.0               bright red

For better results, the medium should not be incubated for more than 24 hours because if lactose fermenters predominate, the entire medium may turn pink masking the presence of non-lactose fermenters. Inoculate the medium immediately after urine collection.

Typical colony morphology on CLED  Medium with Andrade’s Indicator is as follows:

Cultural characteristics after incubation at 35-37°C for 18-24 hours

Klebsiella aerogenes ATCC 13048 – greyish green, mucoid

Escherichia coli ATCC 25922  -bright pink with pink halo

 Enterococcus faecalis ATCC 29212 -orange-yellow or greenish

Proteus mirabilis ATCC 25933  -blue-green

Staphylococcus aureus ATCC 25923 –golden-yellow

Streptococcus pyogenes ATCC 19615 -greyish green

Advantages

(CLED Agar for Urine Culture)

1. Good discrimination of gram-negative bacteria on the basis of lactose fermentation and colony appearance.
2. It inhibits swarming of Proteus species (Proteus mirabilis and Proteus vulgaris are frequently involved in urinary tract infection) due to electrolyte deficient.
3. Relatively low cost as compared to combined use of blood agar and MacConkey agar for urine culture.

Limitations

1. This medium is recommended for urine infection. Low urine count may be a result of antibiotic therapy, low pH of urine.
2. Recovery depends on the urine count.
3.  Inoculate the medium immediately after urine collection.
4. Shigella species may not grow on this medium.
5. For better results, the medium should not be incubated for more that 24 hours because if lactose fermenters predominate, the entire medium may turn pink masking the presence of non-lactose fermenters.

Bacillus species colony characteristics on CLED agar and its Gram stain pictures as shown below video clip-

Fungal (Candida albicans) growth  on CLED agar and its Gram stain pictures as shown below video-

Bibliography