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研究生:黃敏男
研究生(外文):Min-Nan Huang
論文名稱:以流式細胞儀結合螢光原位雜交技術分析活性污泥之菌群結構
論文名稱(外文):Combining flow cytometry and fluorescence in-situ hybridization technique on the characterization of activated sludge microbial community
指導教授:洪俊雄洪俊雄引用關係李季眉李季眉引用關係
指導教授(外文):Chun-Hsiung HungChi-Mei Lee
學位類別:碩士
校院名稱:國立中興大學
系所名稱:環境工程學系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:128
中文關鍵詞:流式細胞儀螢光原位雜交微生物相PI染色雙重染色
外文關鍵詞:Flow CytometryFISHmicrobial communityPI staindual stain
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螢光原位雜交(Fluorescence in-situ hybridization, FISH)為近來菌相研究中最有力的分子生物技術之一,然而傳統FISH技術必須配合螢光顯微鏡照相,以人工的方式計算細胞的數量,相當費時費力,而且對於螢光較弱的細胞經常無法正確判定,人為的誤差較大。流式細胞儀(Flow Cytometry)可以克服這些缺點,快速地測量出單一細胞或生物顆粒上的化學及物理特性。本研究以Flow Cytometry結合FISH技術分析活性污泥中微生物之菌群結構,並針對活性污泥膠羽的打散,原位雜交過程中各個操作參數作最佳化分析[固定劑選擇、sodium dodecyl sulfate(SDS)濃度、探針濃度、pH值對fluorescein螢光之影響、FISH結合Propidium Iodide(PI)染色的運用等],以克服活性污泥中非細胞雜質所帶來的干擾,以期建立一個可以快速、精確地鑑定出複雜樣品中微生物相的有力工具。
以多聚甲醛與酒精兩種固定劑對E. coli (G-)、Bacillus subtilis (G+)的固定效果實驗中,由顯微鏡觀察後發現以酒精固定的部分經過打散之後,細胞受到較嚴重的傷害,而導致細胞的雜交螢光也相對減弱。因此,在液體雜交方面無論是固定劑對細胞結構之完整性或其對螢光強度的影響,多聚甲醛的固定效果皆比酒精為佳。
於SDS濃度的最佳化分析中,在E. coli方面,最佳濃度為0.05 %,而在低濃度0.01 % 時,推測因為濃度過低導致細胞的穿透性不佳,探針無法順利的完全進入細胞內進行雜交;而推測0.5 % SDS使探針螢光減弱的原因有兩個,一為此高濃度的SDS在數小時的雜交過程當中將細胞壁給部分溶解(lysis),使細胞內的物質(包含RNA)有外滲的現象;其次為SDS不僅為一種介面活性劑,也是一種核酸變性劑,當濃度太高時會改變雜交條件的嚴格度,使探針不容易與目標rRNA雜交上所致。在B. subtilis方面,SDS濃度的影響並不明顯,推論兩者不相同的原因為:E. coli是屬於革蘭氏陰性菌,其細胞壁含有大量的脂質,這些脂質較易受SDS的影響而分解,進而增加了細胞的穿透性;而B. subtilis屬於革蘭氏陽性菌,其細胞壁結構含有大量的呔聚醣(peptidoglycan),且幾乎不含脂質,這些由多層peptidoglycan所連接而成的網狀組織構成了厚且堅硬的細胞壁而不易受SDS的影響。
在探針濃度之實驗結果顯示:1. 在所控制的菌量下,探針濃度2 ng/μL明顯不足,而無法得到最強的螢光。2. 探針濃度由3 增加至10 ng/μL所獲得的平均螢光強度已不再明顯提升,最佳之探針濃度應在3 ~ 5 ng/μL之間。3. 加入高濃度的探針 (5 ng/μL以上) 並無法有效提高螢光強度,在螢光顯微鏡下觀察,發現反而會提高探針非專一性鍵結的機會。
在pH值對fluorescein螢光強度的影響實驗中,結果發現當雜交步驟完成之後細菌懸浮液之pH為7.2時螢光強度明顯弱於pH值8.4時,因此液體雜合之保存液PBS的pH值應調整為8~9之間。
活性污泥中含有許多非細胞之顆粒雜質,使用Flow Cytometry分析時也都會被一一的偵測到,產生出巨大的背景雜訊,本實驗成功測試出最佳之PI (會與雙股DNA或RNA結合之核酸染劑 )濃度,可於單雷射光源(488 nm)之Flow Cytometry分析時對活性污泥中的細胞進行篩選,將其從雜質顆粒區分出來,並利用FISH結合PI染色之雙重染色法進行活性污泥族群結構之分析。
以各項Flow Cytometry之最佳化操作參數對台中酒廠與台中工業區污水處理廠中的活性污泥進行菌群結構的分析,並同時進行原始FISH以顯微鏡觀察計數,其結果如下:台中酒廠中以α- Proteobacteria最具優勢、其次為β- Proteobacteria;台中工業區明顯菌相較為複雜,其中以α、β- Proteobacteria兩者較多,而且此污泥中有較多自發螢光物質的干擾。
Fluorescence in-situ hybridization (FISH) is becoming one of the powerful tools for monitoring bacteria microbial community. FISH method employs pictures-taking by epifluorescence microscope then followed by manually numerating the fluorescence signals. Not only the counting process is tedious and time-consuming but also errors occurred when the fluorescence intensity is low. On the other hand, flow cytometry is a precision instrument for rapid analysis on cell number as well as physical and chemical properties of individual cell. This study attempted to combine Flow Cytometry and FISH technique on the identification and characterization of microbial composition in activated sludge. Several parameters were investigated, including fixative agent, concentration of SDS, amount of probes, pH, and DNA dye, for establishing a proven protocol with high resolution, rapid analysis and automatic identification.
Two fixative agents (paraformaldehyde and ethanol) were studied on the fixation of pure culture of Bacillus and E.coli. When applying ethanol, most of the cells were erupted after sample dispersing and their fluorescence intensities were relatively low. Therefore, for liquid hybridization (fluorescence in-situ hybridization en suspension, FISHES), fixative paraformaldehyde is a better choice than ethanol to obtain preserved cells.
Optimized SDS concentration for performing FISH on pure culture of E. coli was found to be 0.05 %. At lower SDS concentration (0.01%), nearly no fluorescent singles were detected due to no entrance of probe into cells. Increasing SDS concentration from 0.05% to 0.5% did not increase the fluorescence intensity. Two possible reasons were concluded; First, during the hybridization process, higher SDS concentration could lyses cell wall and leach out cell substances (including RNA). Second, not only SDS acted as the detergent but also a nucleic acid denaturant. It may increase the hybridization stringency and makes the hybridization harder for probe and its targeted rRNA. The effect of SDS concentration was not significant on performing FISHES to B. subtilis. The different hybridization results between these two pure cultures (E. coli , Gram negative and B. subtilius , Gram positive) may due to the structure difference on cell wall.
The total mass of ribosomal RNA existed in cell is related to its growing status. The relation between probe concentration and hybridization fluorescence intensity was investigated. At the concentration of bacteria used in these experiments, addition of 2 ng/μL probe was not enough to hybridize all ribosomal RNA thus could not achieve maximum fluorescence intensity. Furthermore, the fluorescence responses did not increase significantly as the concentration of probe increased from 3 ng/μL to 10 ng/μL. The optimized probe concentration was between 3 ng/μL to 5 ng/μL. Finally, higher probe concentration (greater than 5 ng/μL) could enhance the nonspecific binding.
Fluorescence intensity of fluorescein dye was found to be strongly affected by pH. When the pH of hybridization suspension was controlled at 7.2, the fluorescence intensity was weaker then in pH 8.4.
There are many noncellular particles existed in activated sludge. When analyzed by flow cytometry, these impurities will result huge background noises. Applying a DNA dye, PI to the activated sludge sample analyzed by a single laser of 488-nm wavelength Flow Cytometry was found to be able to discriminate cells and noncellular particles successfully.
After optimizing the flow cytometry operating parameters, sludge samples from two wastewater treatment plants were analyzed by the proposed protocol and their community compositions were compared to the traditional FISH microscopic counts. Sludge samples from Taichung winery factory was dominated by alpha-subdivision of Proteobacteria. Similar observation was obtained on sludge sample from Taichung Industrial Park WWTP. Also beta-subdivision of Proteobacteria were identified to be the sub dominant groups in both samples.
中文摘要 I
英文摘要 III
目錄 VII
表目錄 XII
圖目錄 XIII
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 活性污泥法之原理與運用 3
2-1-1 標準活性污泥法 3
2-1-2 活性污泥中微生物的組成 3
2-2 傳統的培養分離鑑定菌種 7
2-2-1 傳統鑑定菌種的方法 7
2-2-2 傳統方法所產生的偏差 8
2-2-2-1 除磷系統(EBPR)的偏差 8
2-2-2-2 硝化系統的偏差 9
2-3 分子生物技術於環工上之運用 11
2-3-1 環工上常見的分子生物技術 11
2-3-2 螢光原位雜交(Fluorescence in-situ hybridization, FISH) 14
2-3-2-1 16S rRNA 14
2-3-3 FISH技術中各種操作條件之影響 17
2-3-3-1 固定劑所產生之效應 17
2-3-3-2 細胞打散 17
2-3-3-3 探針之濃度 18
2-3-3-4 SDS (sodium dodecyl sulfate) 濃度 18
2-3-3-5 嚴格度(stringency)對FISH實驗之影響 18
2-3-3-6 FISH結合DNA染色技術 19
2-3-4 利用FISH技術對活性泥之菌相研究 20
2-4 Flow Cytometry分析 23
2-4-1 Flow Cytometry分析之原理 23
2-4-2 Flow Cytometry之運用 27
2-4-3 Flow Cytometry於環工上之發展現況與潛力 30
2-4-3-1 生物處理系統之菌相分析 30
2-4-3-2 利用Flow Cytometry分離單一族群之研究 32
2-4-3-3 細胞中探針可及性(accessibility)分析 34
2-4-3-4 Flow Cytometry分析活性污泥之限制 35
2-4-3-5 Flow Cytometry之再現性分析 35
2-4-4 Flow Cytometry結合FISH技術之操作條件 36
2-4-4-1 探針螢光標定物質的選擇 36
2-4-4-2 Fluorescein對pH值的效應 36
2-4-4-3 Flow Cytometry之儀器設定值 37
2-4-4-4 Flow Cytometry結合DNA染色技術 39
2-5 文獻中可進一步研究的部分 40
第三章 材料與方法 41
3-1 實驗樣品 41
3-1-1 純種菌株 41
3-1-1-1 E. coli(大腸桿菌) 41
3-1-1-2 Bacillus subtilis(枯草桿菌) 41
3-1-1-3 菌種保存及預培養方法 41
3-1-2 活性污泥來源 42
3-1-2-1 台中酒廠廢水處理廠 42
3-1-2-2 台中工業區聯合污水處理廠 44
3-2 實驗流程 46
3-3 實驗藥品 47
3-3-1 化學藥品 47
3-3-2 實驗材料 47
3-3-3 實驗用水 47
3-4 實驗設備 48
3-5 實驗方法 49
3-5-1 固定( Fixation ) 49
3-5-1-1 paraformaldehyde固定 49
3-5-1-2 酒精固定 49
3-5-2 打散細胞( Dispersal ) 50
3-5-3 菌量測定 50
3-5-4 Gelatin Slides 製作方法 50
3-5-5 固定樣品(Immobilization)與脫水 51
3-5-6 螢光原位雜交(Fluorescence in situ hybridization, FISH) 51
5-5-7 DAPI染色 51
3-5-8 螢光顯微鏡之操作 52
3-5-9 以螢光顯微鏡計算菌群結構之方法 52
3-5-10 液體雜交( Fluorescence in situ hybridization en suspension, FISHES) 53
3-5-11 DNA染色 53
3-5-12 Flow Cytometry設定與操作 56
3-6 Flow Cytometry之數據分析方法 57
第四章 結果與討論 61
4-1 固定劑對固定效果之影響 61
4-1-1 固定劑對細胞結構之完整性 61
4-1-2 固定劑對探針螢光強度之影響 62
4-2 細胞打散試驗 65
4-3 探針標定螢光物質的選擇 68
4-3-1 螢光顯微鏡觀察適合之探針 68
4-3-2 Flow Cytometry分析適合之探針 68
4-4 SDS濃度測試 70
4-4-1 SDS濃度對E. coli的影響 70
4-4-2 SDS濃度對B. subtilis的影響 71
4-4-3 SDS濃度對活性污泥的影響 72
4-5 探針濃度試驗 74
4-5-1 不同探針濃度對E. coli之影響 74
4-5-2 不同探針濃度對活性污泥之影響 75
4-6 利用雙重染色法區分活性污泥之細胞與雜質顆粒 77
4-6-1 利用Universal與專一性探針進行雙重染色 77
4-6-1-1 E. coli 77
4-6-1-2 B. subtilis 78
4-6-1-3 活性污泥 78
4-6-2 利用DNA染劑與專一性探針進行雙重染色 82
4-6-2-1 E. coli 83
4-6-2-2 B. subtilis 83
4-6-2-3 活性污泥 84
4-7 pH值對fluorescein螢光的影響 90
4-7-1 E. coli 90
4-7-2 活性污泥 90
4-7-3 綜合討論 91
4-8 活性污泥之菌群結構 93
4-8-1 FISHES結合Flow Cytometry分析 93
4-8-1-1 台中酒廠 94
4-8-1-2 台中工業區 99
4-8-2 FISH結合螢光顯微鏡計數 103
4-8-2-1 台中酒廠 103
4-8-2-2 台中工業區 108
4-8-3 Flow Cytometry分析與螢光顯微鏡計數之比較 113
4-8-3-1 菌群結構結果之比較 113
4-8-3-2 實驗方法之限制 115
4-8-3-3 花費時間之比較 116
第五章 結論與建議 117
5-1 結論 117
5-2 建議 119
參考文獻 121
表目錄
表2-1 活性污泥系統中常見之微生物相 6
表2-2 文獻中常使用的寡核苷酸探針 15
表2-3 Flow Cytometry常用的螢光染劑 25
表2-4 Flow Cytometry與螢光顯微鏡之再現性比較 35
表2-5 Fluorescein對pH值之效應 37
表2-6 偵測器偵測範圍 38
表2-7 Flow Cytometry之儀器設定值 38
表3-1 台中工業區污水處理廠處理功能表 45
表3-2 各濾鏡組所適用之螢光染劑與基本資料 52
表3-3 本實驗所使用的寡核苷酸探針 54
表3-4 各緩衝液成分 55
表3-5 Formamide濃度與NaCl濃度對照表 55
表3-6 各項參數的設定值 56
表4-1 台中酒廠活性污泥各探針雜交結果所佔比例 98
表4-2 台中工業區活性污泥各探針雜交結果所佔比例 102
表4-3 台中酒廠活性污泥各探針雜交顯微鏡計數結果 107
表4-4 台中工業區活性污泥各探針雜交顯微鏡計數結果 112
表4-5 台中酒廠活性污泥由Flow Cytometry分析與螢光顯微鏡 計數之菌群結構與標準偏差比較表 114
表4-6 台中工業區活性污泥由Flow Cytometry分析與螢光 顯微鏡計數之菌群結構與標準偏差比較表 114
表4-7 Flow Cytometry分析與螢光顯微鏡計數所需時間比較表 116
圖目錄
圖2-1 標準活性污泥程序示意圖 4
圖2-2 各種探針相對於EUB探針的族群分佈結構圖 20
圖2-3 添加醋酸以增進EBPR效率之族群變化 21
圖2-4 FISH結合DAPI染色對EBPR系統中微生物菌相分析圖 22
圖2-5 液流系統 26
圖2-6 光學系統路線示意圖 26
圖2-7 細胞大小測量示意圖 28
圖2-8 FSC/SSC 偵測器示意圖 28
圖2-9 Flow Cytometry數據結果之圖形顯示方式 29
圖2-10 活性污泥之微生物族群結構 (Wallner et al., 1995) 30
圖2-11 活性污泥之微生物族群結構 (Hung, 2000) 31
圖2-12 流式分選儀之結構圖 33
圖2-13 Fluorescein 分子式 37
圖3-1 台中酒廠廢水處理流程圖 43
圖3-2 台中工業區污水處理流程圖 45
圖3-3 實驗流程圖 46
圖3-4 FISH於Flow Cytometry之結果分析圖 59
圖4-1 (A) E. coli;(B) B. subtilis;(C) 活性污泥之forward scatter對 side scater 點狀圖 63
圖4-2 (A) E. coli;(B) B. subtilis;(C) 活性污泥之forward scatter對 Fluorescein標定之EUB338探針螢光強度點狀圖 64
圖4-3 活性污泥以不同方法打散比較圖 66
圖4-4 E. coli與B. subtilis對不同標定螢光的探針EUB338 (Cy3 與Fluorescein標定) 之Histogram 69
圖4-5 SDS濃度對FISH的影響 73
圖4-6 探針濃度對雜交結果平均螢光強度之影響 75
圖4-7 探針濃度對活性污泥雜交結果分析圖 76
圖4-8 (A) Cy3標定之Univ1392探針、(B) fluorescein標定之 EUB338探針對E. coli雜交之forward scatter對螢光強 度點狀圖; (C) fluorescein標定之EUB338探針對 E. coli雜交之螢光強度histogram 79
圖4-9 (A) Cy3標定之Univ1392探針、(B) fluorescein標定之 EUB338探針對B. subtilis雜交之forward scatter對螢 光強度點狀圖;(C) fluorescein標定之EUB338探針對 B. subtilis雜交之histogram 80
圖4-10 (A) Cy3標定之Univ1392探針結合fluorescein標定之EUB338 探針對活性污泥雜交之forward scatter對螢光強度點狀圖;(B) Cy3標定之Univ1392探針、(C) fluorescein標定之EUB338探 針對活性污泥雜交之螢光強度histogram 81
圖4-11 PI濃度與平均螢光強度關係圖 85
圖4-12 (A) PI (0.1 μg/mL)染色 (B) fluorescein標定之EUB338探針對 E. coli雜交之forward scatter對螢光強度點狀圖;(C) fluorescein標定之EUB338探針對E. coli雜交之螢光強度histogram 86
圖4-13 (A) PI (0.1 μg/mL)染色 (B) fluorescein標定之EUB338探針 對B. subtilis雜交之forward scatter對螢光強度點狀圖; (C) fluorescein標定之EUB338探針對B. subtilis雜交之 螢光強度histogram 87
圖4-14 不同PI濃度對活性污泥染色forward scatter對螢光強度點狀圖 88
圖4-15 (A) PI (0.1 μg/mL)染色 (B) fluorescein標定之EUB338探針對活性污泥雜交之forward scatter對螢光強度點狀圖;(C) fluorescein標定之EUB338探針對活性污泥雜交之螢光強度histogram 89
圖4-16 不同pH值下fluorescein之histogram 91
圖4-17 不同pH值對fluorescein及PI之螢光強度變化 92
圖4-18 各探針對台中酒廠活性污泥之點狀圖 96
圖4-19 各探針對台中酒廠活性污泥之點狀圖 97
圖4-20 各探針對台中工業區活性污泥之點狀圖 100
圖4-21 各探針對台中酒廠活性污泥之點狀圖 101
圖4-22 各探針對台中酒廠活性污泥之DAPI染色(總菌數)與Cy3 探針雜交螢光(專一性雜交的菌數)影像圖 105
圖4-23 屬於γ-Proteobacteria的大型球菌 106
圖4-24 台中工業區活性污泥未經任何染色程序於螢光顯微鏡 所觀察到的自發螢光訊號 109
圖4-25 各探針對台中工業區活性污泥之DAPI染色(總菌數)與 Cy3探針雜交螢光(專一性雜交的菌數)影像圖 111
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