Emerging investigator series: optimisation of drinking water biofilm cell detachment and sample homogenisation methods for rapid quantification via flow cytometry

Understanding biofilm microbial loads and viability within drinking water pipes is critical to inform sustainable management of ageing infrastructure to protect future water quality. This study establishes an optimised method for robustly harvesting and quantifying cells of biofilms sampled from drinking water systems. Extensive research was conducted to determine the best way to remove biofilms of diverse ages (3-9 months) from different sampling surfaces (pipe sections or coupons) and create homogenised samples for rapid cell enumeration using flow cytometry. Utilising a standardised brushing technique, the optimised approaches delivered the greatest yield of biofilm cells (nine times more cells removed than using sonication) and simultaneously homogenized samples without affecting integrity of intact cells. The optimal brushing strategy differed slightly between sampling surfaces (15 brush strokes for pipe sections, 30 for coupons). When applied to biofilms from a full-scale pipe system, the optimised sampling and flow cytometry methods consistently showed the same trends in biofilm cell concentrations as obtained via molecular analysis (qPCR), but more quickly and from a smaller sample area. Application of the optimised biofilm preparation approach to samples from operational DWDS will ensure that greater yield and more representative samples are collected and analysed, which is critical for any downstream biofilm characterisation or assessment of operational performance.