E. coli-Coliforms Chromogenic Agar Base (BOE)

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. Selectivity is enhanced by cefsulodin and vancomycin, supplied by the E. coli-Coliforms Supplement (LC-6041), which act against Pseudomonas and Gram-negative, oxidase-positive bacteria, enterococci and other Gram-positive 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.

Preparation

Suspend 13.25 grams of the medium in 500 mL of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Cool to 45-50 °C and aseptically add one vial of the E. coli-Coliforms Supplement (LC-6041). Homogenize gently and dispense into Petri dishes.

Instructions for use

The following techniques may be used.

Spread plate method (Drigalski):

1. In a Petri dish, add 12-15 mL of molten agar and let it solidify.
2. Inoculate 0.1 mL of the initial suspension and/or diluted sample.
3. Spread the inoculum with a sterile loop on the agar surface.
4. Incubate the plates in an inverted position at a temperature of 36 ± 2 °C for 18-24 hours.

Pour plate method:

1. Deposit 1 mL of the initial suspension and/or diluted sample in an empty Petri dish.
2. Add 12-15 mL of agar cooled to 45 °C in each Petri dish and mix gently by moving the plate.
3. Allow the plates to solidify and incubate in an inverted position at a temperature of 36 ± 2 °C for 18-24 hours.

Filter membrane method:

1. Dry the surface of the prepared plates.
2. Filter an appropriate volume of the sample through the membrane.
3. Place the membrane on the surface of the agar plate, avoiding the formation of air bubbles.
4. Invert the plates and incubate at 36 ± 2 °C for 18-24 hours.

Incubate up to 24 hours to observe possible delayed beta-galactosidase and beta-glucuronidase reactions.

• 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.
• BOLETÍN OFICIAL DEL ESTADO. Num. 78 Martes 31 de marzo de 2009 Sec. I. Pág. 30417. Orden SCO/778/2009, de 17 de marzo, sobre métodos alternativos para el análisis microbiológico del agua de consumo humano.