E. coli-Coliforms Chromogenic Medium

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 beta-glucuronidase is widely used to differentiate Escherichia coli, as the enzyme is present in E. coli but not in other members of the coliform group. The chromogenic mixture contains chromogenic substrates: Salmon-GAL and X-glucuronide. Coliform enzymes produced, beta-D-galactosidase and beta-D-glucuronidase, cleave these substrates resulting in the different coloration of bacteria colonies. The beta-D-galactosidase cleaves Salmon-GAL substrate, and gives a salmon-red color to the coliform colonies. The beta-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.

Note: Some Shigella strains contain the enzyme beta-D-glucuronidase and can grow as light blue colonies. The beta-glucuronidase-negative E. coli colonies are salmon, e.g. E. coli O157:H7.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 26.4 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Allow to cool to 45-50 °C and dispense in Petri dishes.

Instructions for use

Pour plate technique:

1. Deposit 1 mL of the initial suspension and/or diluted sample in an empty Petri dish.
2. Add 12-15 mL per plate of agar cooled to 44-47 °C in each Petri dish.
3. Invert the plates and incubate at 35 ± 2 °C for 18-24 hours.

Surface plating technique:

1. Inoculate 0.1 mL of the initial suspension and/or diluted sample.
2. Spread the inoculum on the surface of the agar plate.
3. Invert the plates and incubate at 35 ± 2 °C for 18-24 hours.

Filter-membrane technique:

1. Filter an appropriate volume of sample through the membrane.
2. Place the membrane onto the surface of an agar plate, avoiding the formation of air bubbles.
3. Invert the plates and incubate at 35 ± 2 °C for 18-24 hours.

• Alonso, J.L. Soriano, K., Amoros I., Ferrus, M.A. 1998 Quantitative determination of E. coli and fecal coliforms in water using a chromogenic medium. J. Environ. Sci Health 33.