Chromogenic Coliforms Agar (CCA) ISO

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.

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

Preparation

Suspend 26.45 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. Cool to 45-50 °C, homogenize gently and dispense into Petri dishes.

According to ISO 9308

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

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

• ISO 9308-1/2014 Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method for waters with low bacterial background flora.
• ISO 7218:2007, Microbiology of food and animal feeding stuffs - General requirements and guidance for microbiological examinations.
• Byamukama D., Kansiime F., Mach R.L., Farnleitner A.H. Determination of Escherichia coli Contamination with Chromocult Coliform Agar Showed a High Level of Discrimination Efficiency for Differing Fecal Pollution Levels in Tropical Waters of Kampala, Uganda. Appl. Environ. Microbiol. 2000, 66 pp. 864-868.
• Geissler K., Manafi M., Amoros I., Alonso J.L. Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J. Appl. Microbiol. 2000, 88 pp. 280-285.
• Ossmer R., Schmidt W., Mende U. Chromocult Coliform Agar - Influence of Membrane Filter Quality on Performance. Poster presentation, 1999. Congreso de la Sociedad Española de Microbiologia, Granada, Spain.
• USEPA: 40 CFR Part 141 (sec. 141.21) Federal Register/Vol. 67, No. 209, Tuesday October 29, 2002/Rules and Regulations.
• Lange B., Strathmann M., Ossmer R. Performance validation of chromogenic coliform agar for the enumeration of Escherichia coli and coliform bacteria. Lett. Appl. Microbiol. 2013, 57 pp. 547-553.