Harlequin™ E. coli/Coliform Medium
HAL 8

Description
This dual chromogenic substrate medium has been developed for the simultaneous enumeration of Escherichia coli and coliforms in food and environmental samples. The different colony types are simple to distinguish allowing rapid counting of both E. coli and coliforms on a single medium. Based upon the formulation of Tryptone Bile Agar, LAB 072, the medium has been modified by the addition of two chromogenic substrates, one to detect the ß -glucuronidase enzyme (X-glucuronide) and another to detect the b -galactosidase enzyme (magenta-b-gal). Typical E. coli strains possess both enzymes and produce blue-purple colonies (a combination of the blue and magenta pigments produced by the cleavage of the chromogenic compounds). Typical coliforms, however, possess only the b -galactosidase enzyme and produce red-magenta colonies. The colony types are easily distinguishable, even in the presence of other organisms, or when large numbers are observed, making simultaneous enumeration of E. coli and coliforms a quick and simple procedure.

Formula g/litre
Tryptone 20.0
Bile Salts No.3 1.5
X-glucuronide 0.075
Magenta- b -galactoside 0.1
Agar 15.0

Method for reconstitution
Weigh 36.6 grams of powder, disperse in 1 litre of deionised water and allow the medium to soak for 10 minutes. Swirl to mix and sterilise at 121°C for 15 minutes. Cool to 47°C and pour in to sterile Petri dishes. Allow the medium to set. Dry the surface prior to inoculation.

Appearance:
Straw, clear gel.

pH:
7.2 ± 0.2

Minimum Q.C. organisms:
Escherichia coli NCIMB 50034
Enterobacter aerogenes NCIMB 50029
Staphylococcus aureus NCIMB 50080 (Inhibition)

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

Inoculation:
Inoculate 0.5 ml of a 1:10 dilution of the sample and spread over the entire surface of the plate. Further dilution may be necessary if large numbers of E. coli and/or coliforms are present, to ensure colonies can be easily counted.
Incubation: 18 - 24 hours at 37° C

Interpretation:
Count all blue-purple colonies as presumptive E. coli, and calculate the cfu/g. Count all blue-purple and magenta colonies as presumptive coliforms, and calculate cfu/g.

References
1) Baylis, C.L., Patrick, M. (1999). Comparison of a range of Chromogenic media for enumeration of total Coliforms and Escherichia coli in foods. Leatherhead International Technical Notes. No.135: 99.