應用螢光染色法以螢光顯微鏡與流式細胞儀 評估醫院污水處理廠水中微生物特性

Microorganism Characterization in the Hospital Wastewater by Epifluorescence Microscopy and Flow Cytometry with Fluorochrome

江婉嘉(Wan-Chia Chiang)

指導教授 ： 李芝珊

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01157

摘要

傳統的培養方法 (Cultural Method)一直是主要的微生物分析方法，但此法相當耗時，且部分菌種對培養條件相當挑剔，因此往往會造成定量結果上的誤差，有鑑於此，對於微生物的分析有必要建立一個正確且快速的非培養方法 (Non-culture method)，以提供更完整的資訊。由於醫院污水內含有大量致病性微生物，且可能在污水處理過程中因曝氣使大量微生物逸散於周圍空氣中，使處理人員及附近居民之健康受威脅，因此本研究選定醫院污水作為研究對象，以非培養方法來評估醫院污水處理廠水中微生物的濃度與活性，應可對此環境中的微生物有進一步的了解。
本研究第一部分在建立螢光染色法之最佳化條件，並以螢光顯微鏡 (Epifluorescence Microscopy)與流式細胞儀 (Flow Cytometry)來定量微生物。第二部分則將已建立之最佳化分析法應用於醫院污水處理廠水中的微生物，利用具有不同染色機制的染劑，即具細胞穿透性而可測定總微生物濃度之acridine orange (AO)、4’,6 – diamidino - 2 - phenylindole dihydrochloride (DAPI)與以細胞膜破損與否定義微生物活性的YO-PRO-1、Propidium iodide (PI)，以非培養方法 - 螢光顯微鏡與流式細胞儀來分析各處理單元水中微生物的濃度與活性。
在微生物分析方法的建立方面：不同菌種之DAPI最佳化染色條件為，染劑濃度15 µg/ml配合染色時間30分鐘。本研究發現，染劑- AO、DAPI、YO-PRO-1與PI若以EFM與FCM分析被染劑染上的樣本，可成左瑰野峏騛篕睎藿瓞迉誘仄L生物的濃度與活性的分析。而醫院污水處理廠各處理單元水中總微生物濃度約為1.1×107 ~ 2.4×108 cells/ml，且在終沉池，總微生物濃度降低約10倍左右。各處理單元除放流池以外，以非培養方法定量之濃度比培養方法高11 ~ 100倍左右。由FCM分析結果可知，各處理單元水中的細菌濃度高於真菌濃度28 ~ 449倍 (細菌濃度：1.09×107 ~ 1.68×108 cells/ml；真菌濃度：5.35×104 ~ 4.48×106 cells/ml)。在微生物活性方面，細菌與真菌在尚未進行廢水處理的污水池中活性較高 (YO-PRO-1：0.74，PI：0.86)，而放流池中真菌與細菌的活性均較低，以YOPRO-1及PI分析真菌的活性分別為0.38及0.39，而以YOPRO-1及PI分析細菌的活性分別為 0.18及0.23。在放流池中由於氯的作用使微生物的culturability及viability降低，顯示加氯消毒對微生物的控制具一定成效。

關鍵字

流式細胞儀 ； 螢光染劑 ； 螢光顯微鏡

並列摘要

Culture-based analysis remains the primary methods for environmental sample analysis. But, there are some limitations of this method. The results always time-consuming and it can only account for a small proportion of the total cell count .Thus, providing a rapid and accurate non-culture method is needed. This study aims to provide a non-culture method for detecting microorganisms and validating this technique to the hospital wastewater.
The nucleic-acid dyes acridine orange (AO) and 4’,6 – diamidino - 2 - phenylindole dihydrochloride (DAPI) penetrate all bacterial membranes, while YO-PRO-1 and Propidium iodide (PI) only penetrate cells with damaged membranes. After optimization, those nucleic-acid dyes were applied to estimate both total counts and viable of microorganisms in the hospital wastewater by using the epifluorescence microscopy and the flow cytometry.
The results indicated that AO, DAPI, YO-PRO-1 and PI were the suitable dyes for determining total concentration and viability of microorganisms in the hospital waste water. The fluorescent techniques in combination with FCM and EFM for assessment of total concentration and viability for microorganisms were demonstrated to successfully apply for environmental field samples. The total concentrations of microorganisms in the hospital wastewater were 1.1×107 ~ 2.4×108 cells/ml and the concentrations of bacteria were 28 ~ 449 – folds higher than those of fungi . Besides, total counts in the outgoing water from clarifier showed dramatic reduction of over one log. The standard culture method underestimated the microorganism concentration over 11 ~100 –folds when compared to non-culture method except the effluent tank. In addition, the viabilities of microorganisms were high before treatment by YO-PRO-1 (0.74) and PI (0.86). However, the low viabilities of both bacteria (YO- PRO-1：0.18、PI：0.23) and fungi (YO- PRO-1：0.38、PI：0.39) were observed after chlorination tank. Therefore, the chlorination is an effective way to induce a decrease in culturabilities and viabilities of microorganisms.

並列關鍵字

Epifluorescence Microscopy ； Fluorochrome ； Flow Cytometry

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