Tuesday, April 10, 2018

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Cystine Lactose Electrolyte-Deficient Agar(CLED Agar)  is a type of differential medium recommended for diagnostic urinary infections. The medium supports the growth of all urinary potential pathogens and provides distinct colony morphology.It 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.

Cled is used for Routine urine culture, inoculation of specimens in a combination Blood agar (BA) and MacConkey agar is commonly used approach whereas CLED agar can be used as a sole medium, reducing the cost without compromising the quality.
CLED Agar is constitute of Lactose, Pancreatic digest of gelatin and casein, Beef extract, Cystine, pH indicator Bromothymol blue and Agar.
Ingredients per liter of de-ionized water and their main use are as follows:

COMPÖSITION


  • Lactose (10.0gm): Lactose is an energy source for organisms capable of utilizing it by a fermentative mechanism..
  • Pancreatic Digest of Gelatin (4.0gm)
  • Pancreatic Digest of Casein (4.0gm)
  • Beef Extract (3.0gm)  
 Enzymatic Digest of Casein, Enzymatic Digest of Gelatin, and Beef Extract provide the nitrogen,                vitamins, and carbon in CLED Agar.
  • L-Cystine (0.128gm): L-Cystine is added as a growth supplement for cystine-dependent coliforms.  It permits the growth of “dwarf colony” coliforms.
  • Bromothymol Blue (0.02gm)  Bromthymol Blue is the pH indicator to differentiate lactose fermenters from lactose-nonfermenters. Organisms capable of fermenting lactose will lower the pH and change color of the medium from green to yellow.
  • Agar (15.0 gm): Agar is the solidifying agent.
  • Final pH 7.3 +/- 0.2 at 25°C
Electrolyte sources are reduced in order to minimize the swarming of Proteus species.

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:

Cled Agar

Dehydrated Appearance: Light blue-green, homogeneous, free flowing powder
Prepared Appearance: Prepared medium is trace to slightly hazy and light grey-green.

Procedure for Use:

Inoculate the specimen and 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 on CLED Agar

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

OrganismColony Morphology
Escherichia coliyellow, opaque colonies with a slightly deeper coloured centre about 1.25 mm diam. (Non-lactose fermenting strains – blue colonies). yellow medium.
Klebsiella speciesextremely mucoid colonies varying in colour from yellow to whitish-blue. yellowish medium.
Proteus speciestranslucent blue colonies usually smaller than Escherichia coli. blue-green to blue medium.
Salmonella speciesflat blue colonies
Pseudomonas aeruginosagreen colonies with typical matte surface and rough periphery. “Sweet” odor. Blue-green agar
Enterococcus faecalisyellow colonies about 0.5 mm diameter. yellow medium
Staphylococcus aureusdeep yellow colonies about 0.75 mm diameter, uniform in colour. yellow medium
negative staphylococcipale yellow or white, more opaque than Enterococcus faecalis, often with paler periphery
Corynebacteriavery small grey colonies
Lactobacillisimilar to corynebacteria but with a rougher surface

Advantages and Benefits of CLED Agar


  • Cled provides a Good discrimination of gram-negative bacteria on the basis of lactose fermentation and colony appearance;
  • It Inhibits swarming of Proteus spp (Proteus mirabilis and Proteus vulgaris are  frequently involved in urinary tract infection );
  • Relatively low cost (compared with combined use of Blood Agar and MacConkey agar for urine culture).

Disadvantages: 

  • CLED Agar provides poor growth of some gram-positive bacteria.



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