Chromogenic medium for detection and enumeration of E. coli and coliform bacteria in water, according to ISO 9308-1

DESCRIPTION
ChromaticTM Coliform Agar ISO is a selective and differential chromogenic medium used with supplements for the detection and enumeration of Escherichia coli and coliform bacteria in water samples with low bacterial background flora, according to ISO 9308-1.

PRINCIPLE
Enzymatic digest of casein provides amino acids, nitrogen, carbon, vitamins and minerals for organisms growth. Yeast extract is a source of vitamins, particularly of B-group. Sodium chloride maintains the osmotic balance of the medium. Phosphates act as buffer. Sodium pyruvate enhances recovery of injured organisms. Sorbitol is the fermentable carbohydrate. Tryptophan is incorporated into the medium to make possible performing indole test for confirmation of E. coli. Salmon®-GAL (6-Chloro-3-indolyl-β-D-galactopyranoside) is the substrate of β-D-galactosidase, an enzyme typically found in coliform bacteria. X-Glucuronide (5-bromo-4-chloro-3indoxyl-β-Dglucuronide) is the other chromogenic substrate cleaved by the β-D-glucuronidase enzyme characteristic of E. coli. The combination of these two substrates allows to differentiate E. coli from other coliforms and gram-negative bacteria on the basis of the color of the colonies. IPTG (isopropyl-β-D-thiogalactopyranoside) is an inducer for the expression of β-D-galactosidase. Agar is the solidifying agent.

Supplementation with Tergitol 1.5% Supplement serves to inhibit Gram-positive bacteria.

TYPICAL FORMULA (g/l)

PREPARATION
Suspend 31.3 g of powder in 1 liter of deionized or distilled water. Add 10 ml of Tergitol 1.5% Supplement (ref. 80042). Bring to boil and shake until completely dissolved. DO NOT AUTOCLAVE. Cool up to 45-50°C. Aseptically, pour in Petri dishes.

TECHNIQUE
ISO 9308-1 recommends to filter the water sample trough a filter membrane (0.45 μm pore diameter), transfer the membrane onto a ChromaticTM Coliform Agar ISO plate and incubate aerobically at 36 ± 2°C for 18-24 hours. Alternatively, samples can be inoculated by spread plating, pour plating or by direct streaking on the medium surface.

INTERPRETATION OF RESULTS
Most E. coli giving β-D-galactosidase and β-glucuronidase positive reaction produce dark-blue to violet colonies*. Other coliform bacteria cultivate with pink to red colonies. Carry out an oxidase test (ref. 88029) to confirm oxidase-negative coliforms. Other bacteria (if not inhibited) are colorless.
* β-glucuronidase-negative E. coli strains, such as E. coli O157, are pink to red on this medium
A few strains of Shigella and Salmonella which produce the enzyme β-glucuronidase can grow as light blue colonies.

STORAGE
The powder is very hygroscopic, store the powder at 10-30°C, in a dry environment, in its original container tightly closed and use it before the expiry date on the label or until sings of deterioration or contamination are evident. Store prepared plates at 2-8°C away from light.

WARNING AND PRECAUTIONS
The product does not contain hazardous substances in concentrations exceeding the limits set by current legislation and therefore is not classified as dangerous. it is nevertheless recommended to consult the safety data sheet for its correct use. The product is designed for professional use only and must be used by properly trained operators.

DISPOSAL OF WASTE
Disposal of waste must be carried out according to the national and local regulations in force.

PRESENTATION
Dehydrated medium

STORAGE
10-30°C

APPEARANCE OF THE MEDIUM
Powder medium
Appearance: free-flowing, homogeneous
Colour: light beige
Ready-to-use medium
Appearance: slightly opalescent
Colour: light amber

SHELFLIFE
2 years

QUALITY CONTROL

1. Control of general characteristics, label and print
2. Microbiological control
   Inoculum for productivity: 50-100 CFU
   Inoculum for selectivity: 10⁴-10⁶ CFU
   Inoculum for specificity: 10³-10⁴ CFU
   Incubation Conditions: 18-24 h at 36 ± 2°C, in aerobiosis

REFERENCES

1. 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.
2. Quantitative determination of Escherichia coli in water using CHROMagar E.coli. Jose L.Alonso et al. Journal of Microbiological Methods, 25, 1996, p.309-315.

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