Harlequin™ CLED

HAL 7

Description

Harlequin™ C.L.E.D. is a chromogenic medium for the improved isolation and differentiation of urinary tract pathogens. The superior growth qualities of Harlequin™ C.L.E.D. mean that it can also be used to improve differentiation of mixed growth in specimens where more fastidious organisms may be expected. Escherichia coli is responsible for the majority of urinary tract infections (U.T.I.'s), with other Gram-negative organisms such as Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Morganella spp., Enterobacter spp., and Citrobacter spp. often encountered. Grampositive organisms involved in U.T.I.'s include Staphylococcus aureus, Staphylococcus saprophyticus, enterococci, and Lancefield Group B streptococci. Traditional C.L.E.D. (Cystine Lactose Electrolyte Deficient) formulations rely upon Lactose fermentation to differentiate organisms. However this does not allow discrimination between E. coli and the other coliforms that cause U.T.I.'s. In Harlequin™ C.L.E.D., lactose has been replaced by a double chromogen system, which allows clear differentiation of many of the key pathogens involved.

X-glucuronide is incorporated to produce characteristic green E. coli colonies (96-97% of strains are positive for ß-glucuronidase, the enzyme required to split this chromogenic compound). Other coliforms will produce colourless colonies or, if they possess the ßglucosidase enzyme, black colonies will be produced. This colour is due to the action of this enzyme on the second chromogenic substrate CHE-glucoside. Glucosidase positive organisms include enterococci and Klebsiella spp. Further discrimination is achieved by the addition of phenylalanine which is deaminated by Proteus spp. and related organisms, producing brown colonies (may also have a brown halo surrounding). In parallel trials Harlequin™ C.L.E.D. isolated more clinically significant isolates than traditional C.L.E.D. formulation and an alternative chromogenic formulation(1).

Formula g/litre
Balanced Peptone No.1 6.5
Beef Extract 4.0
Meat Peptone 4.0
Yeast Extract 7.0
L-Cystine 0.11
CHE-glucoside 0.2
X-glucuronide 0.075
Ferrous Gluconate 0.95
Phenylalanine 0.2
Agar 20.0

Method for reconstitution
Weigh 43.0 grams of powder, disperse in 1 litre of deionised water and allow the mixture to soak for 10 minutes. Swirl to mix and sterilise at 121°C for 15 minutes. Cool to 47°C and pour into sterile Petri dishes. Allow the medium to set. Dry the surface prior to inoculation.

Appearance:
Tan, clear gel.

pH:
7.3 ± 0.2

Minimum Q.C. organisms:
Escherichia coli NCIMB 50034
Staphylococcus aureus NCIMB 50080

Storage of Powdered Medium:
Plates - up to 7 days at 2 - 8°C in the dark.

Inoculation:
Surface inoculation, either spreading for single colonies, or using a 1μl loop for semi-quantitative enumeration.

Incubation:
18 - 24 hours at 37° C

References:
1) Perry, J.D. - Personal Communication (data on file)