CLED Agar is an abbreviation for Cystine Lactose Electrolyte-Deficient Agar. It is a type of differential medium recommended for diagnostic urinary bacteriology. The medium supports the growth of all urinary potential pathogens and provides distinct colony morphology.
CLED Agar also supports the growth of a number of contaminants such as diphtheroids, lactobacilli, and micrococci. It is electrolyte deficient to prevent the swarming of Proteus species.
Composition
- L-Cystine 0.128 gm
- Bromothymol Blue 0.02 gm
- Agar 15 gm
| Constituents | Amount in g/L |
|---|---|
| Lactose | 10 gm |
| Enzymatic Digest of Gelatin | 4 gm |
| Enzymatic Digest of Casein | 4 gm |
| Beef extract | 3 gm |
- Enzymatic Digest of Casein, Enzymatic Digest of Gelatin, and Beef Extract provide the nitrogen, vitamins, and carbon in CLED Agar.
- L-Cystine is added as a growth supplement for cystine-dependent coliforms.
- Lactose is the carbohydrate. Organisms capable of fermenting lactose will lower the pH and change color of the medium from green to yellow.
- Bromthymol Blue is the pH indicator.
- Agar is the solidifying agent.
Preparation
- Suspend 36 g of the medium in one liter of purified water.
- Heat with frequent agitation and boil for one minute to completely dissolve the medium.
- Autoclave at 121°C for 15 minutes.
- Cool to 50°C, mix well and dispense into plates. When the medium is solidified, invert the plates to avoid excess moisture. The prepared medium should be stored at 8-15°C .
Appearence:
Dehydrated Appearance: Light blue-green, homogeneous, free flowing powder
Prepared Appearance: Prepared medium is trace to slightly hazy and light grey-green.
pH
7.3 ± 0.2 @ 25°C
Procedure for Use:
Inoculate the specimen. Incubate plates at 35 ± 2°C in an aerobic atmosphere. Examine plates at 18 to 24 h for amount of growth, pigmentation, colony size and inhibition of Proteus swarming/spreading.
Colony Characteristics of Various Organisms
Various cultural responses shown by different microorganisms on CLED Agar at the appropriate atmosphere and temperature after 18 – 24 hours incubation are as follows :
| Organism | Colony Morphology |
|---|---|
| Escherichia coli | yellow, opaque colonies with a slightly deeper coloured centre about 1.25 mm diam. (Non-lactose fermenting strains – blue colonies). yellow medium. |
| Klebsiella species | extremely mucoid colonies varying in colour from yellow to whitish-blue. yellowish medium. |
| Proteus species | translucent blue colonies usually smaller than Escherichia coli. blue-green to blue medium. |
| Salmonella species | flat blue colonies |
| Pseudomonas aeruginosa | green colonies with typical matte surface and rough periphery. “Sweet” odor. Blue-green agar |
| Enterococcus faecalis | yellow colonies about 0.5 mm diameter. yellow medium |
| Staphylococcus aureus | deep yellow colonies about 0.75 mm diameter, uniform in colour. yellow medium |
| negative staphylococci | pale yellow or white, more opaque than Enterococcus faecalis, often with paler periphery |
| Corynebacteria | very small grey colonies |
| Lactobacilli | similar to corynebacteria but with a rougher surface |
Limitations
Although CLED agar is suitable for the isolation and counting of many aerobically growing microorganisms, such as Enterobacteriaceae, Pseudomonas and other non-fermenting Gram negative rods, enterococci, staphylococci, Candida species, and many others from urine specimens, some common 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.
- Genitourinary pathogens such as Neisseria gonorrhoeae, Gardnerella vaginalis, Chlamydia, Ureaplasma, or other fastidious organisms do not grow on this medium.
- Although a differentiation according to lactose fermentation and certain other diagnostic tests may be performed directly on this medium, biochemical and, if indicated, serological testing using pure cultures is necessary for complete identification.
why the color turns blue in case of (salmonella spp.)?
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