Emerging investigator series: Optimisation of Drinking Water Biofilm Cell Detachment and Sample Homogenisation Methods for Rapid Quantification via Flow Cytometry
Emerging investigator series: Optimisation of Drinking Water Biofilm Cell Detachment and Sample Homogenisation Methods for Rapid Quantification via Flow Cytometry
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新兴研究者系列：优化饮用水生物膜细胞分离和样品均质方法，通过流式细胞仪进行快速定量
DOI：
10.1039/d3ew00553d
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发表时间：
2024
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了解饮用水管道内的生物膜微生物负荷和生存能力对老化基础设施的可持续管理至关重要，以保护未来的水质。本研究建立了一种优化的方法，用于从饮用水系统中取样的生物膜细胞的稳健收集和定量。我们进行了广泛的研究，以确定从不同采样表面（管道切片或片状）去除不同年龄（3-9个月）生物膜的最佳方法，并创建均质样品，用于流式细胞术快速细胞计数。利用标准化的涂刷技术，优化的方法提供了最大的生物膜细胞产量（比使用超声去除的细胞多9倍），同时均质样品，而不影响完整细胞的完整性。不同采样表面的最佳刷刷策略略有不同（管道部分刷刷15下，管材刷刷30下）。当应用于来自全尺寸管道系统的生物膜时，优化的采样和流式细胞术方法始终显示出与通过分子分析（qPCR）获得的生物膜细胞浓度相同的趋势，但速度更快，样品面积更小。将优化的生物膜制备方法应用于运行DWDS的样品将确保获得更高的产量，并收集和分析更多具有代表性的样品，这对于任何下游生物膜表征或操作性能评估至关重要。
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.