CM0945, TBX Agar | Oxoid - Product Detail

Directions
Suspend 36.6 g 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^1,2 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 reagent³, and has been shown to be highly specific for Escherichia coli⁴. However, approximately 3-4% of Escherichia coli are glucuronidase negative, notably Escherichia coli O157 strains⁵.
Unlike MUG, where the flurophore leaches out of the cell into the surrounding agar, the released chromophore in TBX Medium is insoluble and accumulates within the cell. This ensures that coloured target colonies are easy to identify.
Most Escherichia 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. Escherichia 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 Escherichia coli colonies to be coloured blue/green.

Technique
Dry the surface of the medium in the prepared plates. Dilute the food sample according to the method being followed e.g. 1:10 with Maximum Recovery Diluent CM0733. Homogenise in a stomacher or a laboratory blender.
The following incubation techniques may be used (consult the relevant standard for the complete method):
1. Pipette 0.1 ml of the homogenate on to the plate and spread over the surface with a sterile glass spreader. Incubate the plates for 24 hours at 37°C⁶.
2. Pipette 0.5 ml of the homogenate on to the plate and spread over the surface with a sterile glass spreader. Incubate the plates for 4 hours at 30°C, then 18-24 hours at 44°C⁷.
3. Place a cellulose membrane on to the surface of a Minerals Modified Glutamate Medium CM0607 prepared plate. Pipette 1 ml of the homogenate on to the membrane. Incubate for 4 hours at 37°C. Transfer the membrane to a TBX prepared plate and incubate for 18-24 hours at 44°C⁸.
4. Pipette 1 ml of the homogenate into a sterile Petri dish. Add TBX Medium, cooled to 45°C. Mix well and allow to set. Incubate for 18-24 hours at 44°C. If the presence of stressed cells is suspected pre-incubate the plates for 4 hours at 37°C⁹.
Multiply the numbers of blue/green colonies by the dilution factor and express the result as the number of Escherichia 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

Positive controls:	Expected results
Escherichia coli ATCC® 25922 *	Good growth; blue/green coloured colonies.
Negative control:
Klebsiella pneumoniae ATCC® 29665	Good growth; straw coloured colonies.

* This organism is available as a Culti-Loop®

References
1. Gross R.J. and Rowe B. (1985) J. Hyg. Camb. 95. 513-550.
2. Anderson J.M. and Baird-Parker A.C. (1975) J. Appl. Bact. 39. 111-117.
3. Feng P.C.S. and Hartmann P.A. (1982) Appl. Environ. Microbiol. 43. 1320-1329.
4. Hansen W. and Yourassowsky E. (1984) J. Clin. Microbiol. 20. 1177-1179.
5. Ratnam S., March S.B., Almed R., Bezanson G.S. and Kasatiya S. (1988) J. Clin. Microbiol. 26. 2006-2012.
6. Donovan T.J. et al (1998) Communicable Disease and Public Health 1 : 188-196.
7. PHLS Standard Methods for Microbiological Examination of Food, Dairy and Water Samples. F20: Direct Enumeration of Escherichia coli .
8. ISO 16649-1: 2001. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli . Part 1: Colony-count technique at 44°C using membranes and 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide.
9. ISO 16649-2: 2001. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli . Part 2: Colony-count technique a 44°C using 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide.