CCA Coliforms Chromogenic Agar

Description

The selectivity is attained, partially, by Tergitol 7, which inhibits the growth of Gram-positive bacteria and some Gram-negative bacteria without affecting the coliform bacteria.

The colonial differentiation is due to the chromogenic mixture, composed of two enzyme substrates: 6-chloro-3-indoxyl-beta-D-galactopyranoside (Salmon-GAL) and 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide (X-Glucuronide).

The first one is cleaved by the characteristic enzyme found in coliforms, beta-D-galactosidase, and gives a salmon-red colour to the coliform colonies. The second chromogenic substance is cleaved by the beta-D-glucuronidase enzyme characteristic of E. coli and turns the colonies of these bacteria a blue colour.

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. IPTG enhances the reactions described above.

Other Gram-negative bacteria produce colourless colonies, except some that possess glucuronidase activity (but not galactosidase), which produce light blue to turquoise colonies.

To confirm the E. coli colonies, a small amount of tryptophan is included in this medium to verify indole production: coat the blue-violet colonies with a drop of Kovac's Reagent. If the reagent turns a cherry-red colour in a few seconds, this confirms the production of indole and hence the presence of E. coli.

When the Chromogenic Agar for Coliform is used with the membrane filter method, the colour and growth of the colonies can be modified by the characteristics of the membrane filter. It is advisable to perform validation of the membrane filter type used.

The Spanish Health Ministry (Ministerio de Sanidad y Consumo) has officially adopted this medium as an alternative methodology for the microbiological analysis of water for human consumption, giving a new definition for Escherichia coli ("Enterobacteriaceae that express the beta-D-galactosidase and the beta-D-glucuronidase enzymes simultaneously") and for coliform bacteria ("Enterobacteriaceae that express the beta-D-galactosidase enzyme").

Limitation of the procedure

The production of beta-galactosidase, although common to all the coliforms, varies from one strain to another, being influenced by the temperature and incubation time. At temperatures above 37 °C its production decreases, causing a loss of reddish color intensity, while the bluish tones in the strains of Escherichia coli are accentuated.

If the membrane filtration method is used, it must be taken into account that the nature and characteristics of the filter membrane used also influence the size and color of the colonies grown on this culture medium.

Technique

The water sample is filtered through a membrane filter of 0.45 µm pore diameter validated according to the ISO Standard 7704:1985. The membrane is then placed on the surface of the CCA medium, avoiding entrapment of air bubbles between the membrane and agar surface. The Petri dish with the membrane is incubated for 18-24 hours at 36 ± 2 °C. If in 18 h there is growth of red or colourless colonies, extend the incubation until 24 h to include late reactions of beta-galactosidase or beta-glucuronidase. Count beta-galactosidase positive and beta-glucuronidase negative colonies (all colonies coloured from salmon-rose to red) as coliform bacteria other than E. coli. Count beta-galactosidase positive and beta-glucuronidase positive colonies (all colonies coloured from deep blue to violet) as E. coli.

The total coliform count is obtained by adding the salmon-rose to red colonies and the deep blue to violet colonies.

Physical/chemical control

Microbiological control

Membrane filtration / practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity).

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Aerobiosis. Incubation at 36 ± 2 °C, reading at 18-24 h.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ADAMS, M., R. GRUBB, S.M. HAMER & A. CLIFFORD (1990) Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity. Appl. Environ. Microbiol. 56:2021.
• ISO 7704 Standard (1985) Water Quality - Evaluation of membrane filters used for microbiological analyses.
• ISO 9308-1:2014/Amd.1:2016(E) Water quality. Enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method for waters with low bacterial background flora.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• KILIAN, M. & P. BÜLOW (1976) Rapid Diagnostic of Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B 84:245-251.
• MANAFI, M. & W. KNEIFEL (1989) A combined chromogenic-fluorogenic medium for the simultaneous detection of total coliform and E. coli in water. Zentralbl. Hyg. 189:225-234.
• UNE-EN ISO 11133 (2014). Microbiología de los alimentos para consumo humano, alimentación animal y agua - Preparación, producción, conservación y ensayos de rendimiento de los medios de cultivo.
• TURNER, K.M., L. RESTAINO & E.W. FRAMPTON (2000) Efficacy of Chromocult Coliform Agar for coliform and Escherichia coli detection in Foods. J. Food Protect. 63(4):539-541.