近年來螢光原位雜合(Fluorescence in situ hybridization, FISH)技術應用在環境土樣上，可直接觀察不同的菌群並定量菌數，對於土樣微生物社會的研究是非常有用的工具。本研究是利用甲烷氧化菌16S rRNA基因序列的專一性探針，Mγ84與Mγ705共同雜合第一型甲烷氧化菌菌群，Mα450雜合第二型甲烷氧化菌菌群。決定探針的嚴謹度(stringency)，將第一型與第二型甲烷氧化菌的標準菌株Methylococcus capsulatus與Methylosinus trichosporium OB3b，用不同甲醯胺(formamide)濃度的雜合液進行雜合後，拍攝的螢光影像用軟體測量螢光訊號的強弱，發現三支探針在20%甲醯胺濃度下，雜合的菌體螢光最強。為了得知底泥雜合探針後是否會產生螢光背景干擾，將滅菌的底泥加入菌液作為實驗組，或不加菌液作為控制組，萃取後雜合探針拍攝影像，並以軟體計數數值，結果兩者並無差異，顯然底泥的背景螢光干擾會嚴重影響菌數的計數。所以本研究在探針雜合反應前處理甲苯胺藍(toluidine blue)，發現可有效降低探針雜合的螢光背景干擾，但是雜合探針後再負染DAPI，卻使DAPI的影像出現嚴重的背景干擾，經由本研究的實驗發現，必須在雜合後處理甲苯胺藍再負染DAPI，才可抑制DAPI的背景螢光干擾。針對實驗過程中能萃取出多少底泥的目標菌，於是將甲烷氧化菌菌液加至已滅菌的南仁湖底泥，萃取後用探針雜合計數的菌數回收率約70~90%，底泥不加菌液的控制組，萃取後用探針雜合計數的數值佔加入的菌數之比例2~5%。底泥在萃取前要先分離土粒與菌體，所以經由震盪或超音波的處理並沉降之，取萃取液雜合探針Eub338並負染DAPI計數菌數，發現超音波處理後所萃取的菌數優於震盪處理。最後以20%CH4與0%CH4(控制組)馴養南仁湖底泥，並在不同時間點取樣雜合Eub338與甲烷氧化菌專一性探針，以計數第一型與第二型甲烷氧化菌佔所有細菌的比例，結果馴養至第十六天，甲烷氧化菌佔所有細菌的比例最高，第二型甲烷氧化菌所佔的比例約29%，第一型甲烷氧化菌所佔的比例約9%。

　　The fluorescence in situ hybridization (FISH) method developed recently has great potential for quantity and quality of active populations of soil bacteria. In this study, we search firstly 16S rRNA probe sequence of methanotroph in references. To determine stringency of probe , two methanotroph strain were hybridized with specific probe. Methylococcus capsulatus was targeted with probe Mγ84 and Mγ705 design to hybridize TypeⅠmethanotroph. Methylosinus trichosporium OB3b was target with probe probe Mα450 design to hybridize TypeⅡmethanotroph. Gradually increasing hybridization stringency was performed by the addition of formamide to the hybridization buffer. The optimal formamide concentration for hybridization of three probe was determined to obtain a bright fluorescent signal by software analysis of picture. After adding methanotroph to autoclaved Nanjen lake sediment, probe hybridize extraction of sediment. Autofluorescence in sediment was quenched with toluidine blue before hybridization. But DAPI autofluorescence in sediment had to be quenched with one more toluidine blue after hybridization. In order to know how many bacteria can be extract in extraction of lake sediment. Recovery of target methanotroph cells was demonstrated to be around 70~90% after hybridize extraction of autoclaved sediment which add methanotroph. Portions of sediment were added to buffer and sonicated or shoched in order to detach bacteria from the sediment. Extraction of sediment was stained with DAPI after hybridizing with Eub338 probe and counted cell number. Cells were counted by comparing images obtained for three different single fluorescence(Eub338, Mα450, Mγ84 plus 705). The number of Eub338-positive cells counted was the sum of active cells. The percentage of TypeⅠand TypeⅡ methanotroph compared respectively to the Eub338- positive cell was count 29% and 9% in the sediment cultivated to 16 days.

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