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研究生:陳佳莉
研究生(外文):Chia-Li Chen
論文名稱:應用螢光染色法監測溫泉水微生物特性研究
論文名稱(外文):Monitoring Microorganisms in Hot Spring Water by Fluorochrome Method
指導教授:李芝珊李芝珊引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:環境衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:123
中文關鍵詞:溫泉螢光染色法螢光顯微鏡流式細胞儀
外文關鍵詞:hot springfluorochrome methodepifluorescence microscopyflow cytometry
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應用螢光染色法配合螢光顯微鏡(EFM)及流式細胞儀(FCM)評估北部溫泉水樣,以五種螢光染劑( AO、DAPI、SYTO-13、PI、YOPRO-1)分析,且同時以非培養方法(Non-culture)與傳統培養方法(Culture)並行分析比較。
以非培養方法的EFM-AO,總微生物濃度為1.08x105∼6.16x106cells/ml,以EFM-DAPI分析為1.14x105∼6.80x106 cells/ml,以FCM-AO分析總濃度範圍為1.30×105∼1.04×107cells/ml,SYTO-13為2.51×105∼1.07×107cells/ml。而以培養方法測得細菌濃度範圍為0∼4.60x104 CFU/ml ,真菌濃度範圍為 0∼1.64x103 CFU/ml;另外,在活性方面,以PI、YOPRO-1所得活性範圍從0∼0.76, 0∼0.63,以培養方法評估的活性為0∼ 0.082,應用螢光顯微鏡配合螢光染色法所分析之溫泉微生物活性比傳統培養方法高。
研究結果明確地顯示出傳統的培養方法會低估溫泉水中總微生物及活性,因此,為了降低潛在的健康風險,對於溫泉水中的微生物,一個完整的監測是必須的,以提供更明確及代表性的資訊,在此研究中,以螢光染色法配合螢光顯微鏡及流式細胞儀(EFM/FL、FCM/FL)技術,成功地應用於定量溫泉水之微生物總濃度及活性,且能應用於不同的環境樣本。
Total concentration, viability, and culturability of microorganisms in 48 hot spring water samples were monitored by using epifluorescence microscopy and flow cytometry with fluorochrome (EFM/FL) with five fluorescent dyes (AO, DAPI, SYTO-13 PI, and YOPRO-1). Results from the non-culture-based method were then compared with those using a commonly used culture method. The total cell concentrations measured using the non-culture-based EFM/FL methods were from 1.08x105 to 6.16x106cells/ml with AO staining, and from 1.14x105 to 6.80x106 cells/ml with DAPI staining. And the total cell concentrations measured using the non-culture-based FCM/FL methods were from 1.30x105 to 1.04x107cells/ml with AO staining, and from 2.51x105 to 1.07x107cells/ml cells/ml with SYTO-13 staining. The Results compared with the concentration range of 0 to 4.60x104 CFU/ml for bacteria and 0 to 1.64x103 CFU/ml for fungi by the culture method. The viability ranged from 0 to 0.76 with PI staining, from 0 to 0.63 with YOPRO-1 staining, and from 0 to 0.082 by the culture method. The viability by EFM/FL was much higher than the culturability.
Our results clearly show that the traditional culture method underestimates the total concentration and viability of microorganisms in hot spring water. Therefore, to reduce health risks, a comprehensive microorganism monitoring in hot spring water is needed to provide more accurate and representative information. In our present study, EFM/FL and FCM/FL technique was successfully applied to quantify the total concentration and viability of microorganisms in hot spring water, and can be used for different environmental environments.
總 目 錄
摘要……………………………………………………………………..Ⅰ
Abstract………………………………………………………………….Ⅱ
總目錄…………………………………………………………………..Ⅲ
表目錄…………………………………………………………………..Ⅴ
圖目錄…………………………………………………………………..Ⅵ
第一章 前言……………………………………………………………1
1.1 研究緣起………………………………………………………….1
1.2 研究目的………………………………………………………….2
第二章 文獻回顧…………………………………………………3
2.1 溫泉水特性、健康影響及管理現況…………………………….3
2.1.1 台北地區溫泉之分佈及特性………………………………3
2.1.2 溫泉的健康影響……………………………………………5
2.1.3 北部溫泉管理調查現況……………………………………5
2.2溫泉水之相關研究………………………………………………..7
2.2.1 台灣溫泉相關之研究………………………………………7
2.2.2 國外溫泉之相關研究………………………………………9
2.3 微生物之分析…………………………………………………...11
2.3.1 培養方法…………………………………………………..12
2.3.2 非培養方法………………………………………………..12
2.3.2.1 螢光顯微鏡…………………………………...……13
2.3.2.2 流式細胞儀………………………………………...14
2.3.2.3 螢光染色法………………………………………...14
2.4 應用螢光染色法以螢光顯微鏡與流式細胞儀分析微生物之
研究……………………………………………………………..17
2.4.1 以螢光染色法利用螢光顯微鏡分析環境中之微生物…..17
2.4.2 以螢光染色法利用流式細胞儀分析環境中之微生物…..18
2.4.3 以螢光染色法利用螢光顯微鏡與流式細胞儀分析環境中
之微生物…………………………………………………..19
第三章 材料與方法……………………………………………..27
3.1 實驗儀器與材料………………………………………………...27
3.1.1 實驗儀器…………………………………………………..27
3.1.2 實驗材料…………………………………………………..27
3.1.3 實驗菌種…………………………………………………..30
3.2 實驗方法與步驟………………………………………………...32
3.2.1分析方法最佳化之建立……………………………………32
3.2.1.1 螢光顯微鏡………………………………………...32
3.2.1.2 流式細胞儀………………………………………...34
3.2.2 應用分析方法最佳化於溫泉水…………………………..38
3.2.2.1水樣收集…………………………………………….38
3.2.2.2水樣分析流程……………………………………….39
3.3 統計分析………………………………………………………...41
第四章 結果與討論……………………………………………..48
4.1 分析方法之建立………………………………………………...48
4.1.1 EFM最佳化條件結果…………………………………….48
4.1.2 FCM最佳化條件結果…………………………………….49
4.1.3 FCM及EFM遮蔽現象(Masking)………………………..50
4.2應用分析方法於環境水樣-溫泉水……………………………...50
4.2.1 北部各溫泉區的溫泉水質………………………………..50
4.2.2 溫泉水微生物之定性分析………………………………..51
4.2.2.1 總濃度之定性分析………………………………...51
4.2.2.2 活性之定性分析…………………………………...53
4.2.3 溫泉水微生物之定量分析………………………………..53
4.2.3.1 總濃度之定量分析………………………………..53
4.2.3.2 活性之定量分析…………………………………..62
第五章 結論與建議…………………………………………………69
5.1 結論…………………………………………………………….69
5.2 建議…………………………………………………………….70
參考文獻………………………………………………………….100
附錄………………………………………………………………107
口試委員意見之回覆……………………………………….…...119

表目錄
表2-1 北投溫泉特性..........................................................................22
表2-2 消基會溫泉測試結果表……………………………………..23
表2-3 利用傳統方法培養環境中微生物的可能性………………..25
表3-1 本研究中所使用之培養基與配方…………………………..42
表3-2 使用兩種不同菌種,以FCM 搭配各種染劑分析菌種濃度濃縮10倍與100倍之結果…………………………………….43
表4-1-1 EFM最佳化條件之結果……………………………………76
表4-2-1 溫泉浴池之基本參數……………………………………….82
表4-2-2 以EFM-AO、DAPI與FCM-AO、SYTO-13及Culture分析溫泉水平均總微生物濃度…………………………………88
表4-2-3 以FCM-AO、SYTO-13及Culture分析溫泉水細菌與真菌平均總微生物濃度…………………………………………89
表4-2-4 以EFM(PI、YOPRO-1)及Culture方法評估溫泉水中微生物之活性(Viability)及可培養性(Culturability)….90
表4-2-5 以總濃度及活性比較各分組(不同pH值、季節及水樣收集來源)………………………………………………………91
表4-2-6 以總濃度及活性評估季節差異……………………………92
表4-2-7 以總濃度評估水樣收集來源差異…………………………92
表4-2-8 分析方法之比較……………………………………………92








圖目錄
圖2-1 評估細菌活性之分子方法……………………………............26
圖2-2 FCM分析原理…………………………………………...........26
圖3-1 研究架構………………………………………………………44
圖3-2 以AO進行具100%活性的各種菌株的染色………………..45
圖3-3 以YOPRO-1進行具60%活性的各種菌株的染色………….46
圖3-4 以PI進行具60% 活性的各種菌株的染色………………….47
圖4-1-1 DAPI在不同pH值、不同最終濃度下所得之不同純菌濃度……………………………………………………………72
圖4-1-2 AO在不同pH值、不同最終濃度下所得之不同純菌濃度73
圖4-1-3 YOPRO-1在不同pH值、不同最終濃度下所得之不同
純菌濃度……………………………………………………74
圖4-1-4 PI在不同pH值、不同最終濃度下所得之不同純菌濃度75
圖4-1-5 以YOPRO-1、PI在 pH 7.7水樣分析60%活性的實驗
菌株…………………………………………………………77
圖4-1-6 以YOPRO-1、PI在 pH 3.48水樣分析60%活性的實驗
菌株…………………………………………………………78
圖4-1-7 以YOPRO-1、PI在 pH 1.56水樣加入60%活性的實驗
菌株…………………………………………………………78
圖4-1-8 夏季樣本之遮蔽現象(masking)…………………………79
圖4-1-9 以不同染劑分析夏季樣本之遮蔽現象(masking)………79
圖4-1-10 以PI分析夏季樣本不同濃度之遮蔽現象(masking)…..80
圖4-1-11 以SYTO-13、AO在 pH 1.56水樣分析60%活性的實驗
菌株…………………………………………………………81
圖4-2-1 EFM分析溫泉水中總微生物………………………………83
圖4-2-2 EFM分析溫泉水中不具活性之微生物……………………83
圖4-2-3 以FCM-SYTO-13分析夏季不同溫泉區之總微生物濃度..84
圖4-2-4 以EFM-DAPI分析夏季不同溫泉區之總微生物濃度……84
圖4-2-5 以FCM-AO分析冬季不同溫泉區之總微生物濃度……...85
圖4-2-6 以EFM-DAPI分析冬季不同溫泉區之總微生物濃度……85
圖4-2-7 以FCM-SYTO-13烏來溫泉winter-j-pool樣本之總微生物
濃度………………………………………………………....86
圖4-2-8 以EFM分析烏來溫泉winter-j--pool樣本之總微生物濃度
及活性………………………………………………………86
圖4-2-9 以FCM-AO分析summer-h-source樣本………………….87
圖4-2-10 以EFM、FCM、Culture分析不同pH值溫泉水微生物總濃度………………………………………………………..93
圖4-2-11 以FCM、Culture分析不同pH值溫泉水細菌及真菌微生物總濃度…………………………………………………..93
圖4-2-12 以EFM、FCM、Culture分析不同季節溫泉水微生物總濃度…………………………………………………………..94
圖4-2-13 以FCM、Culture分析不同季節溫泉水細菌及真菌微生物總濃度……………………………………………………..94
圖4-2-14 以EFM、FCM、Culture分析不同水樣收集來源之溫泉水
微生物總濃度……………………………………………..95
圖4-2-15 以FCM、Culture分析不同水樣收集來源溫泉水細菌及真菌微生物總濃度…………………………………………..95
圖4-2-16 EFM-AO與FCM-AO分析總濃度之相關性…………….96
圖4-2-17 EFM-DAPI與FCM-SYTO-13分析總濃度之相關性……96
圖4-2-18 以EFM、Culture分析不同pH值溫泉水之活性(Viability)
及可培養性(Culturability)……………………………..97
圖4-2-19 以EFM、Culture分析不同季節溫泉水之活性(Viability)及可培養性(Culturability)……………………………..97
圖4-2-20 以EFM、Culture分析不同水樣收集來源溫泉水之活性
(Viability)及可培養性(Culturability)……………….98
圖4-2-21 Viability-PI與Culturability之相關性…………………….99
圖4-2-22 Viability-YOPRO-1與Culturability之相關性…………...99
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