Cystine-Lactose-Electrolyte-Deficient (CLED) Agar Preparation

Cystine-lactose-electrolyte-deficient (CLED) agar is a differential culture media that is used to isolate uropathogens (i.e., pathogens of the urinary tract system) from urine samples. CLED agar preparation will be elaborated in this section. CLED agar is deficient in electrolytes; and that is why it is so named (as: Cystine-lactose-electrolyte-deficient agar). Because electrolytes is absent in CLED agar, some contaminants such as Proteus species (which is a swarming organism) can hardly grow on CLED agar. However, Proteus species can still grow on CLED agar, with a distinct morphology, but its swarming nature will be largely inhibited by CLED agar – since CLED agar is deficient in electrolytes. CLED agar is important for the isolation of urinary pathogens. CLED agar supports the growth of all urinary pathogens; and it also gives a good colonial differentiation and clear diagnostic characteristics for the isolated uropathogens including E. coli, Klebsiella, Enterococcus, Pseudomonas and Proteus. Fermenting E. coli strains produces yellow, opaque colonies with a slightly deeper coloured centre on CLED agar. Non-lactose fermenting strains of E. coli produces blue colonies on CLED agar. Klebsiella species produces extremely mucoid colonies varying in colour from yellow to whitish-blue on CLED agar. Proteus species produces translucent, non-swarming blue colonies on CLED agar; and their colonies are usually smaller than colonies of E. coli. Pseudomonas aeruginosa produces green colonies with typical sweet odour, matte surface and rough periphery on CLED agar. Enterococcus faecalis produces yellow colonies on CLED agar.

This image shows how CLED agar looks like after preparation

Components of CLED agar base

The components of CLED agar base required for CLED agar preparation include:

  1. CLED agar base- which is the solidifying agent
  2. Beef/meat extract – which provide sources of nitrogen, carbon, and vitamins
  3. Pancreatic digest of gelatin
  4. Pancreatic digest of casein
  5. Lactose
  6. L-cystin
  7. Bromothymol blue – which is used as a pH indicator to differentiate lactose
    fermenters from lactose-non-fermenters. Bacteria which ferment lactose will lower the pH of the medium and change the color of the medium from green/blue to yellow.
  8. pH – which is usually adjusted to 7.3 at 25 °C (or 77 °F)

MATERIALS

You require these materials to prepare your CLED agarCLED agar base (usually comes in 500 g), autoclave, conical flask, measuring cylinder, beaker, stirring rod, Bunsen burner, incubator, refrigerator, wire gauze, spatula, weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish

STEP BY STEP PROTOCOL TO PREPARE CLED AGAR

  1. Weigh out 36.2 g of CLED agar powder using the weighing balance.
  2. Suspend the 36.2 g of CLED agar powder in 1 litre (1000 ml) of distilled water in a conical flask.
  3. Mix the solution by stirring to dissolve the agar.
  4. Bring the mixture to boil, by mild boiling of the mixture over a Bunsen burner flame. This helps to dissolve the agar completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Transfer the conical flask containing the boiled/mixed agar suspension to the autoclave.
  6. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  7. At the end of sterilization, allow the autoclave to return to normal (zero point) before opening. Otherwise the pressure built up in the autoclave will affect the quality and quantity of the prepared molten medium. More so, you may be affected by the steam from the autoclave due to the high pressure built up in the autoclave.
  8. Allow molten CLED agar medium to cool to about 45-50 degrees Celsius.
  9. Pour prepared molten medium into sterile Petri dish plates.
  10. Allow the poured plates on the bench to solidify.
  11. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  12. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  13. Absence of colony on the plate means that your sterilization is good.
  14. You can now use your prepared CLED agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

REFERENCES

  1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.

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