A chromogenic medium for the enumeration of Escherichia coli and total coliforms in 100 mL water samples: validation of an MPN-based method according to ISO 9308-2.

Microbiological monitoring of water quality relies mainly on the detection and quantification of Escherichia coli, as required by international and European regulatory frameworks. Among the methods commonly applied for E. coli enumeration, ISO 9308-2 describes a Most Probable Number approach based on the detection of β-D-glucuronidase activity using the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide. However, fluorescence detection requires ultraviolet illumination at 365 nm, implying additional equipment requirements and potential occupational exposure concerns. Here, the ENUMERA®Coli100 method for E. coli and Total Coliforms in 100 mL is presented as a validated chromogenic alternative to ISO 9308-2. The method incorporates a chromogenic glucuronic acid derivative that produces a visible green colour reaction under ambient light, eliminating the need for UV irradiation. The dehydrated medium is integrated into a sealed bottle cap through a "twist-and-release" mechanism that minimises handling steps and reduces contamination risks during sample preparation. Performance was evaluated against ISO 9308-2 using 120 water samples representing five matrices: drinking water, surface water, wastewater, reclaimed water, and seawater. Both methods yielded statistically equivalent results for E. coli and Total Coliforms across all matrices. ENUMERA®-Coli100 demonstrated 100% sensitivity, high specificity and selectivity comparable to ISO 9308-2, excellent linearity, low limits of detection and quantification, and satisfactory accuracy. Validation was performed following ISO 16140-2 and ISO 13843 criteria. ENUMERA®-Coli100 constitutes a robust, reliable, and operationally practical alternative for the enumeration of E. coli and total coliforms in water samples, simplifying result interpretation, improving laboratory safety, streamlining workflow, and eliminating UV detection equipment.