應用螢光染色法監測溫泉水微生物特性研究

Abstract

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.