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TBX Chromogenic Agar (Tryptone Bile X-Glucuronide) is based on Tryptone Bile Salts Agar medium, used to detect and enumerate E. coli in foods, with the addition of a chromogenic agent, x-ß-D-Glucuronide, to detect the presence of the enzyme glucuronidase, which is highly specific for E. coli.
The released chromophore in TBX Agar is colored and target colonies are easily identified. E. coli absorbs the chromogenic agent x-ß-D-glucuronide, and the intracellular glucuronidase enzyme activity breaks the bond between the chromophore and the glucuronide. The released chromophore is colored and builds up within the cells, causing the E. coli colonies to be blue-green colored.
Casein peptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Bile Salts are inhibitors to other Gram-positive organisms and suppress coliform bacteria. Bacteriological agar is the solidifying agent.
ISO 16649 specifies a horizontal method for the enumeration of ß-glucuronidase-positive E. coli in products intended for human consumption or for the feeding of animals.
The negative b-ß-glucuronidase E. coli colonies are colorless, e.g. E. coli O157: H7. The high temperatures (44°C) inhibit the growth of E. coli O157: H7.

Principles and uses
Chromogenic Coliforms Agar (CCA) is a selective medium for the detection of E. coli and other coliforms in waters and foods. The recovery and enumeration of Escherichia coli and coliforms are important indicators of environmental and food hygiene. CCA is especially recomended for waters with low bacterial numbers, whether it is drinking water, disinfected pool water, or finished water from drinking water treatment plants.
The interaction of ingredients in the medium, such as peptone, sorbitol and pyruvate, grants a quick colony growth, including infectious coliforms and also permits the recovery of sublethal thermally injured coliforms. Tergitol-7 inhibits Gram-positive bacteria and some Gram-negative without affecting the coliform bacteria. Sodium chloride maintains the osmotic balance and phosphate salts act as a buffer system. Bacteriological agar is the solidifying agent.
Detection of ß-glucuronidase is widely used to differentiate Escherichia coli, as the enzyme is present in E. coli but not in other member of coliform group. The chromogenic mixture contains chromogenic substrates: Salmon-GAL and X-glucuronide. Coliform enzymes produced, ß-D-galactosidase and ß-D-glucuronidase, cleave these substrates resulting in the different coloration of bacteria colonies. The ß-D-galactosidase cleaves Salmon-GAL substrate, and gives a salmon-red color to the coliform colonies. The ß-D-glucuronidase, enzyme characteristic of E. coli, cleaves X-glucuronide, giving a blue color to these colonies. E. coli has the two enzymes and cleaves both chromogenic substances giving dark blue to violet colonies. Total coliforms are the sum of E. coli colonies plus salmon-red colonies. The addition of tryptophan to the medium allows the performance of the Indole test for further E.coli confirmation.

For the enumeration of E. coli and coliform bacteria according to ISO 9308:

• Filter sample through a membrane .
• Place the membrane filter over a E. Coli Coliforms Chromogenic Agar plate.
• Invert Petri dish and incubate at 36±2 ºC during 21±3 h.
• Count the ß-D-galactosidase colonies (pink to red in color) as presumptive coliform bacterias that are not E. coli
  -To avoid false positive results, caused by oxidase-positive bacteria, for example, Aeromonas spp, confirm bacterial colonies through an oxidase-negative reaction.
• The positive colonies ß-D-galactosidase and ß-D-glucuronidase (dark blue to violet) are counted as E. coli.
• The total coliform bacteria count is the sum of oxidase-negative colonies, ß-D-galactosidase-positive colonies (pink to red) and all colonies which dark blue to violet.
• Some Shigella strains contain the enzyme ß-D-glucuronidase and can grow as light blue colonies.