Harlequin™ TBGA (TBX)

(Tryptone Bile Glucuronide Agar)

HAL 3

Description

A medium developed for the simple enumeration of E. coli without the need for membranes, or pre-incubation on Minerals Modified Glutamate Medium. It is based upon the formulation of Tryptone Bile Agar, LAB 072, the medium has been modified by the addition of a chromogenic substrate to detect the ß-glucuronidase enzyme, which is highly specific for E. coli*, and is detected by the MUG reagent in other formulations. The advantage of the chromogenic substrate is that it requires no UV lamp to visualise the reaction, and it is concentrated within the colony, facilitating easier enumeration in the presence of other organisms, or when large numbers are present on the plate.

Formula g/litre

Tryptone 20.0
Bile Salts No.3 1.5
X-glucuronide 0.075
Agar 15.0

Method for reconstitution

Weigh 36.5 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 in to Petri dishes. Dry the surface prior to inoculation.

Appearance:
Straw, clear gel.

pH:
7.2 ± 0.2

Minimum QC organisms:
Escherichia coli NCIMB 50034 (blue/green)

Enterobacter aerogenes NCIMB 50029 (cream)

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 are present, to ensure colonies can be easily counted.

Incubation:
30°C for 4 hours, followed by 18 hours at 44°C.

Interpretation:
Count all blue/green colonies as presumptive E. coli, calculate the cfu/g in the original material. Asimple indole test can be performed by placing one drop of Kovac’s reagent onto a colony and if positive, a red halo will appear in the medium around the colony. If negative, then the halo will be white. *96-97% of E. coli strains positive. A notable exception is E. coli 0157:H7.

References

Dibb, W.L. and Bottolfsen, K.L. (1984). Evaluation of Rosco Diagnostic ß-glucuronidase Tablets in the dentification of Urinary Isolates of Escherichia coli. Acta Path.Microbiol. Immunol. Scand. Sect. B 92 261-264. Hansen, W. and Yourassowsky, E. (1984). Detection of ß-glucuronidase in Lactose Fermenting Members of the Family Enterobacteriaceae and its Presence in Bacterial Urine Cultures. J. Clin. Micro.20 (6) 1177-1179. Robinson, B.J. (1984). Evaluation of a Fluorogenic Assay for Detection of E. coli. App & Env. Microbiol.48 (2) 285-288. Perez, J.L., Berrocal, C.I. and Berrocal, L. (1986). Evaluation of a Commercial ß-glucuronidase Test for the Rapid and Economical Identification of Escherichia coli. J.App.Bacteriol. 61 541-545. Raghubeer, E. and Matches, J.R. (1990). Temperature Range for Growth of Escherichia coli Serotype 0157:H7 and Selected Coliforms in E. coli Medium. J.Clin. Micro. 28 (4) 803-805. Bolton, F.J. (1995) Personal Communication.