基于流式细胞术的活性多环芳烃降解菌检测技术  

Determination of Active Polycyclic Aromatic Hydrocarbons Degrading Bacteria Using Flow Cytometry

作　　者：李可欣 吴蔓莉 高欢 刘恒 LI Ke-Xin;WU Man-Li;GAO Huan;LIU Heng(Shaanxi Key Laboratory of Environmental Engineering,Key Laboratory of Northwest Water Resource,Environment and Ecology,School of Environmental and Municipal Engineering,Xi′an University of Architecture and Technology,Xi′an 710055,China)

机构地区：[1]西安建筑科技大学环境与市政工程学院,陕西省环境工程重点实验室,西北水资源与环境生态教育部重点实验室,西安710055

出　　处：《分析化学》2021年第8期1357-1365,共9页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.52070154,21577109)资助。

摘　　要：建立了流式细胞术快速检测多环芳烃降解菌数量和活性细菌的方法。用超纯水制备1×10^(3)-1×10^(5) cells/mL多环芳烃降解菌的菌悬液,利用SYBR Green/碘化丙啶(SG/PI)进行染色,采用流式细胞仪进行检测。以FL1和FL3为检测器检测荧光强度,设定阈值为600,菌悬液流速为33 mL/min,计数率保持在1000 cells/s以下,所得降解菌数量的测定结果与平板计数法测定结果之间具有很好的相关性,相关系数为0.9742-0.9962。在检测活性多环芳烃降解菌时,利用NaClO将处于生长期的多环芳烃降解菌灭活,并作为阴性对照,未灭活细菌作为阳性对照,SG/PI双染法进行染色后,采用流式细胞术进行测定,根据阳性和阴性对照菌在流式细胞仪检测结果图中出现的位置,确定了活性细菌区和死细菌区,通过区域划分可确定体系中活性细菌数量。平板计数法检测细菌需48 h,而本研究利用流式细胞法进行细菌的检测,测定时长仅2 h,极大地提高了检测效率。本研究为快速准确测定多环芳烃降解菌提供了新方法,同时为多环芳烃降解体系中微生物检测的实际应用提供了参考。A method for determination of the number of active polycyclic aromatic hydrocarbons(PAHs)-degrading bacteria using flow cytometry was developed.PAHs degrading bacteria suspension(1×10^(3)-1×10^(5) cells/mL)was prepared by using ultra-pure water as dilution.After stained using SG/PI and incubated at 35℃for 10 min in the dark,the number of PAHs-degrading bacteria was determined using flow cytometry.The parameters of flow cytometry were set as follows:the green(FL1)and red(FL3)fluorescent acted as detector and the threshold value was 600;the flow rate of bacteria suspension was 33 mL/min and the counting rate kept below 1000 cells/s.Experimental results showed that there was a good correlation relationship between the results of flow cytometry and the plate counting determination,and the correlation coefficients were 0.9742-0.9962.To determine PAH-active bacteria,the bacteria during the growth phase were sterilized using NaClO that acted as negative control,and the activated bacteria were used as positive control.Both negative and positive bacteria were determined using flow cytometry after stained using SYBR Green I and propidium iodide,and the active and inactive cells regions were respectively classified.Compared to the plate counting method,the flow cytometry for counting bacteria number could greatly reduce determination time and improve the accuracy.This study provided a fast and efficiency method for determination of active bacteria in pollutant degrading systems.

关 键 词：流式细胞术 多环芳烃降解菌 活性菌 液相降解 快速检测 

分 类 号：X172[环境科学与工程—环境科学] X835