M-CC Broth

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. Selectivity is enhanced by cefsulodin and vancomycin, which act against Pseudomonas and Gram-negative oxidase-positive bacteria, enterococci and other Gram-positive 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.

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").

Technique

If the membrane filtration (MF) method is used to concentrate the sample, impregnate a sterile pad with the medium and place the membrane filter on the pad. Incubate for 21-24 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. Calculate the concentration of coliform bacteria and E. coli in 100 mL from the initial volume of water filtered and the number of characteristic colonies counted on the membrane. The results are expressed as Colony Forming Units per millilitre (CFU/mL).

According to the internal regulations or methodologies of each laboratory, temperature and incubation time can vary.

Physical/chemical control

Microbiological control

Membrane filtration / practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity) / ≥ 10³ CFU (specificity).

Pad impregnated with the culture medium.

Aerobiosis. Incubation at 36 ± 2 °C, reading at 21-24 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/DIS 9308-1 Standard (2014)/Amd.1:2016 Water quality. Detection and enumeration of E. coli and coliform bacteria, Part 1: Membrane filtration method for waters with low bacterial background flora.
• 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.
• MINISTERIO DE SANIDAD Y CONSUMO (2009) Orden SCO/778/2009 de 17 de marzo sobre métodos alternativos para el análisis microbiológico del agua de consumo humano. BOE. n.º 78 de 31-04-2009. Sección I, Págs. 30417-30420. Madrid.