Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China; Shaanxi Key Laboratory of Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China.
Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China; Shaanxi Key Laboratory of Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China.
Chemosphere. 2020 Mar;243:125453. doi: 10.1016/j.chemosphere.2019.125453. Epub 2019 Nov 23.
Fungal contamination in drinking water has been becoming a hot topic. The routine enumeration method of fungal spores is heterotrophic plate counts (HPC). However, this method is time-consuming and labor-intensive and there is also the difficulty of enumerating viable but non-culturable cells. In this study, a rapid, simple and accurate method for quantifying fungal spores and discriminating their viability in water was established using flow cytometry (FCM) combined with fluorescence dyes. The optimal staining conditions are as follows: spores suspensions are sonicated at 495 W for 5 min as pretreatment, and then 10 μL of SYBR Green I (100×) and 30 mM Ethylene diamine tetraacetic acid are added to a 500 μL water sample, which incubate at 35 °C for 20 min in dark. The concentration of fungal spores measured by FCM was highly correlated with HPC results and microscope observations, with correlation coefficient of 0.996 and 0.988, respectively. This staining method can be widely applied to the enumeration and viability evaluation of fungal spores. In addition, chlorine-based inactivation of three genera of fungal spores was assessed by plating and FCM. The result showed that all three genera of fungal spores lost culturability firstly and then membrane integrity decreased, preliminarily revealing the inactivation mechanism. The inactivation rate constants of membrane damage varied in the following order: chlorine dioxide > chlorine > chloramine. This study concluded that FCM is an appropriate and alternative tool to detect fungal spores' viability and can be used for evaluating the fungal inactivation by disinfectants.
水中真菌污染已成为热门话题。真菌孢子的常规计数方法是异养平板计数（HPC）。然而，这种方法既耗时又费力，并且还存在难以计数活但非可培养细胞的问题。在这项研究中，使用流式细胞术（FCM）结合荧光染料建立了一种快速、简单、准确的定量水中真菌孢子并区分其活力的方法。最佳染色条件如下：孢子悬浮液用 495 W 的功率超声处理 5 分钟作为预处理，然后向 500 μL 水样中加入 10 μL SYBR Green I（100×）和 30 mM 乙二胺四乙酸，在黑暗中 35°C 孵育 20 分钟。FCM 测量的真菌孢子浓度与 HPC 结果和显微镜观察高度相关，相关系数分别为 0.996 和 0.988。这种染色方法可广泛应用于真菌孢子的计数和活力评估。此外，通过平板计数和 FCM 评估了三种真菌孢子对含氯消毒剂的灭活作用。结果表明，三种真菌孢子首先失去可培养性，然后膜完整性降低，初步揭示了灭活机制。膜损伤的灭活速率常数按以下顺序变化：二氧化氯>氯>氯胺。本研究得出结论，FCM 是一种检测真菌孢子活力的合适替代工具，可用于评估消毒剂对真菌的灭活作用。
