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TRYPTONE BILE X-GLUCURONIDE MEDIUM (TBX)

Code: CM0945

A selective, chromogenic medium for the detection and enumeration of Escherichia coli in food and animal feed. This medium meets the formulation and performance criteria laid down in ISO 16649^1,2,3 and ISO 11133:2014^4.

* Adjusted as required to meet performance standards

Directions
Suspend 36.6g of TBX Medium in 1 litre of distilled water. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and pour the medium into sterile Petri dishes.

Description
TBX Medium is based on Tryptone Bile Agar (CM0595). Tryptone Bile Agar was originally formulated to improve on earlier methods used to detect Escherichia coli in foods^5.6 in terms of speed, reliability, better recovery from frozen samples and the detection of poor lactose fermenters.
TBX Medium builds on these advantages through the addition of a chromogenic agent - X-glucuronide - which detects glucuronidase activity. This is the same enzyme detected by MUG reagent⁷, and has been shown to be highly specific for E. coli⁸. However, approximately 3-4% of E. coli are glucuronidase negative, notably E. coli O157 strains⁹.

Most E. coli strains can be differentiated from other coliforms by the presence of the enzyme glucuronidase. The chromogen in TBX Medium is 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-glucuronide), and is targeted by this enzyme. E. coli cells are able to absorb this complex intact and intracellular glucuronidase splits the bond between the chromophore and the glucuronide. The released chromophore is coloured and builds up within the cells, causing E. coli colonies to be coloured blue/green.

Technique
Dry the surface of the medium of the prepared plates. Dilute the food sample according to the method being followed for example 1:10 with Maximum Recovery Diluent (CM0733). Homogenise in a stomacher or a laboratory blender. Further dilute the sample as required.

The following incubation techniques may be used (consult the relevant standard for the complete method):
1. Pipette 0.1ml of the homogenate on to the plate and spread over the surface with a sterile spreader. Incubate the plates for 24 hours at 37°C¹⁰.
2. Pipette 0.5ml of the homogenate on to the plate and spread over the surface with a sterile spreader. Incubate the plates for 4 hours at 30°C, then 18 hours to 24 hours at 44°C¹¹.
3. Place a cellulose membrane on to the surface of a Minerals Modified Glutamate Agar plate. Pipette 1ml of the homogenate on to the membrane. Incubate for 4 hours at 37°C. Transfer the membrane to a TBX plate and incubate for 18 hours to 24 hours hours at 44°C¹.
4. Pipette 1ml of the homogenate into a sterile Petri dish. Add TBX Medium, cooled to 45°C. Mix well and allow to set. Incubate for 18 hours to 24 hours at 44°C. If the presence of stressed cells is suspected pre-incubate the plates for 4 hours at 37°C².

Multiply the numbers of blue/green colonies by the dilution factor and express the result as the number of E. coli per gram of food.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

References
1. ISO 16649-1:2001 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 1: Colony-count technique at 44 degrees C using membranes and 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
2. ISO 16649-2:2001 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
3. ISO 16649-3:2015 Microbiology of the food chain - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide
4. ISO 11133:2014 Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media
5.Gross R.J. and Rowe B. (1985) J. Hyg. Lond. 95. 531-550.
6. Anderson J.M. and Baird-Parker A.C. (1975) J. Appl. Bact. 39. 111-117.
7. Feng P.C.S. and Hartman P.A. (1982) Appl. Environ. Microbiol. 43. 1320-1329.
8. Hansen W. and Yourassowsky E. (1984) J. Clin. Microbiol. 20. 1177-1179.
9. Ratnam S., March S.B., Almed R., Bezanson G.S. and Kasatiya S. (1988) J. Clin. Microbiol. 26. 2006-2012.
10. Donovan T.J. et al (1998) Communicable Disease and Public Health 1 : 188-196.
11. PHLS Standard Methods for Microbiological Examination of Food, Dairy and Water Samples. F20: Direct Enumeration of Escherichia coli.