CLED Agar Composition, Preparation and Colony growth Characteristics

cled agar MLTGEEKS

Cystine Lactose Electrolyte-Deficient Agar(CLED Agar)  is a type of differential culture media in microbiology recommended for the diagnosis of urinary tract 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 because it support the growth of most urinary tract potential pathogens. Inoculation of specimens in a combination with Blood agar (BA) and MacConkey agar is a commonly used approach whereas CLED agar can be used as a sole medium, reducing the cost without compromising the quality and providing reliable result.

Composition of CLED Agar

Enzymatic Digest of Gelatin4g
Enzymatic Digest of Casein4g
Beef Extract3g
Lactose10g
L-cystine0.128g
Bromothymol blue0.02g
Agar15g
Final pH at temperature 25°C7.3 +/- 0.2

Principle of cystine lactose electrolyte deficient agar

  • 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 cysteine-dependent coliforms.  It permits the growth of “dwarf colony” coliforms.
  • Bromothymol Blue (0.02gm)  Bromothymol 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.
  • Also electrolyte sources are reduced in order to minimize the swarming of Proteus species.
Also read  ONPG test principles,Composition and Cultural response

How to prepare CLED Agar culture media

  1. Start by suspending 36 g of the medium in one liter of purified water.
  2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
  3. Autoclave at 121°C for 15 minutes.
  4. Cool to 50°C, mix well and dispense into plates. When the medium is solidified, invert the plates to avoid excess moisture.
  5. The prepared medium should be stored at 8-15°C .

Appearance of CLED Agar after preparation

It appears trace to slightly hazy and light grey-green in color.

Uninoculated Cled agar

How to inoculate on CLED Agar

  1. Inoculate the specimen and Incubate plates at 35 ± 2°C in an aerobic atmosphere.
  2. Examine plates after 18 to 24 hours for growth, pigmentation, colony size and inhibition of Proteus swarming/spreading.

Plate reading and Colonies morphology(diagnostic properties of 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
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
Lactobacillisimilar to corynebacteria but with a rougher surface
Corynebacteriavery small grey colonies
Also read  Loa Loa Filariasis Laboratory diagnosis and treatment

Benefits and Advantages of CLED Agar(cled agar uses)

  • 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 of CLED Agar

  • CLED Agar provides poor growth of some gram-positive bacteria.
E.coli on Cled agar
E.coli on Cled agar
Klebsiella on CLED Agar
Klebsiella on CLED Agar
Staphylococcus aureus on Cled Agar
Staphylococcus aureus on Cled Agar

Source : Medical Laboratory Expo

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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