李芝珊臺灣大學：環境衛生研究所陳培詩Chen, Pei-ShihPei-ShihChen2007-11-282018-06-302007-11-282018-06-302005http://ntur.lib.ntu.edu.tw//handle/246246/59772對於生物氣膠的評估來說，傳統的培養方法與顯微鏡計數方式，是相當的耗時、費力與較不精準的。因此，本研究以大腸桿菌為研究對象，在實驗室詳細評估螢光顯微鏡（EFM）、流式細胞儀（FCM）與即時定量聚合酵素連鎖反應法（real-time qPCR）等三種非培養方法相互間的關係，以及其與傳統培養方法間的關係，結果顯示，FCM所測得之總細胞濃度顯著的高於EFM所測得之總細胞濃度，且兩方法所得之總細胞濃度與活性均呈高度相關，在活性部分，以FCM所測得之活性最高，而以EFM所測得之活性最低。此外，由real-time qPCR測得的DNA濃度，也與FCM與EFM所測得之總細胞濃度成良好相關，因此，本研究指出，EFM、FCM與 real-time qPCR三種非培養方法均能對微生物濃度與活性測量提供相當快速與正確的資訊。 研究結果指出，FCM為一快速與可靠的方法，因此，針對流式細胞儀輔以螢光染色的技術（FCM/FL），再進行染劑條件、染色時間與分析條件的最佳化，結果顯示SYTO-13 與YOPRO-1的組合最能提供可靠的總濃度與活性定量，本研究也成功的將FCM /FL運用在醫院廢水處理廠中曝氣池的空氣樣本與水樣本分析，整體上， FCM/FL 被證明能夠成功的定量環境空氣與水中之細菌與真菌之濃度與活性。此外，也將FCM/FL運用於生物採樣器之採樣效率的評估，結果顯示，採樣過程對微生物活性的影響的主要因素為生物氣膠的特性（脆弱與否）與用來評估活性之採樣方式，其中以衝擊瓶為對活性影響較低之採樣方式。此外，過濾方式所產生的採樣壓力，對微生物之代謝機制的影響也較對細胞膜的影響為大，而且也跟其為細菌或真菌有關係。 最後，另一種非培養方法，real-time qPCR對空氣中致病的肺結核桿菌之分析方法，也被成功的建立與應用於肺結核病人負壓病房空氣中肺結核菌之定量。此方法的檢量線範圍可達106，而肺結核病人所在病房中，其空氣中肺結核桿菌的濃度範圍為1.43 x 10 copies/m3 到 2.06 x 105 copies/m3。此外，空氣中肺結核桿菌濃度與病人痰中肺結核桿菌濃度城中度相關。整體而言，本研究所建立之非培養方法，包括EFM、FCM與real-time qPCR的結果為環境微生物與生物氣膠的研究提供了相當有利的工具，未來也能針對各種環境微生物與生物氣膠的議題，進行更進一步的研究。Traditional culture and microscopy methods for evaluation of bioaerosols are slow, tedious, and rather imprecise. Here, using pure suspensions of E. coli, three non-culture methods, namely, flow cytometry (FCM), epifluorescence microscopy (EFM), and real-time quantitative polymerase chain reaction (real-time qPCR), were compared with a traditional culture-based method. Then, the optimal conditions of FCM with 5 different fluorescent dyes (FCM/FL) were evaluated in laboratory samples and validated to field study. In addition, FCM/FL was applied to evaluate the sampling performance of impingement and filtration with different types of fluorescent dye staining (cell membrane integrity and metabolism) and then compared with a traditional culture method (culturability). Furthermore, real-time qPCR was develop and used to measure air concentration of M. tuberculosis in a health care setting. In regard to the comparison of EFM, FCM, real-time qPCR and culture method, total cell concentrations determined using FCM were statistically higher (2.62 – 4.94 times) than those determined using EFM. In addition, EFM and FCM were highly associated for both the total cell concentration and viability. Furthermore, DNA concentrations measured by real-time qPCR with gene probe were highly associated with the total number concentrations measured by either the EFM or FCM. In summary, the three non-culture methods compared here could provide rapid and accurate information about microorganism concentrations and viabilities. For the FCM condition optimization, SYTO-13 was found to be the most suitable fluorescent dye for determining the total concentration of the bioaerosols, as well as YOPRO-1 was the most suitable for determining viability. Moreover, the established optimal FCM/FL with dyes was validated for characterizing microorganism profiles from both air and water samples from the aeration tank of hospital wastewater treatment plant. In conclusion, the FCM/FL successfully assessed the total concentration and viability for bacterial and fungal microorganisms in environmental field samples. Regarding the sampling performance of bioaerosol samplers, the bioaerosol viability during the sampling processes was highly influenced by bioaerosol characteristics (hardy or fragile), as well as by the fluorescent dyes with different physiological mechanisms. For better viability of the sampled bioaerosol, the impinger was superior to the filter. Moreover, it was found that sampling stress from filtration had more influence on the bioaerosol metabolism mechanism than cell membrane integrity. Furthermore, the differences between cell membrane integrity and the metabolism by sampling stress were found related to the bioaerosol species. By using real-time qPCR, the present study was firstly developed a quantitative assay to measure air concentration of M. tuberculosis in a health care setting. The real-time qPCR method could perform measurements of counts over 6 orders of magnitude dynamic range with a great sensitivity. The airborne M. tuberculosis concentrations were found to vary widely from 1.43 x 10 copies/m3 to 2.06 x 105 copies/m3. In addition, airborne M. tuberculosis levels, smear and culture results of sputum samples were observed to be moderately correlated. In conclusion, FCM/FL and real-time qPCR were successfully established and applied to field study. In the future, they should be powerfully tools to provide more insight in the area of bioaerosols and environmental microbiology.1. INTRODUCTION 1 1.1 Culture-based method 1 1.2 Noncultured-based methods 3 1.2.1 Microscopy 3 1.2.2 Flow cytometry (FCM) 5 1.2.3 Immunoassay 6 1.2.4 Biochemical assay 7 1.2.5 Molecular biological method 8 1.3 Chamber studies and Field Studies 11 1.3.1 Bacteria 12 1.3.2 Fungus 12 1.3.3 Interference 12 2. OBJECTIVES OF THE STUDY 15 3. REAL-TIME QUANTITATIVE PCR WITH GENE PROBE, FLUOROCHROME, AND FLOW CYTOMETRY FOR MICROORGANISM ANALYSIS 17 3.1 INTRODUCTION 17 3.2 MATERIALS AND METHOD 19 3.2.1 Test microorganism 19 3.2.2 EFM 19 3.2.3 FCM 20 3.2.4 Real-time qPCR with gene probe 20 3.2.5 Statistical methods 21 3.3 RESULTS AND DISCUSSION 22 3.3.1 Total cell concentration 22 3.3.2 Viability 25 3.4 CONCLUSION 27 3.5 REFERENCES 28 4. Bioaerosol Characterization by Flow Cytometry with Fluorochrome 34 4.1 INTRODUCTION 34 4.2. MATERIALS AND METHODS 37 4.2.1. Laboratory samples 37 4.2.2. Determination of Optimal Dyes and Staining Conditions 38 4.2.3. FCM 38 4.2.4. Viability and Culturability 40 4.2.5. Field Sample Collection and Analysis 40 4.3 RESULTS AND DISCUSSION 41 4.3.1 Laboratory Samples 41 4.3.2 Field Samples 44 4.4 CONCLUSION 46 4.5 REFERENCE 47 5. SAMPLING PERFORMANCE FOR BIOAEROSOLS BY FLOW CYTOMETRY WITH FLUOROCHROME 59 5.1 INTRODUCTION 59 5.2 MATERIALS AND METHODS 62 5.2.1 Test bioaerosols 62 5.2.2 Aerosol generation system 63 5.2.3 Bioaerosol samplers and sample processing 63 5.2.4 CFU counting 64 5.2.5 Dye and staining protocols 64 5.2.5 FCM 64 5.2.6 Indicators for sampling efficiency evaluation 65 5.3 RESULTS AND DISCUSSION 66 5.3.1 Culturability and viability in the nebulizer 66 5.3.2 Culturability and viability in bioaerosol samplers 67 5.3.3 CR and VR 68 5.4 CONCLUSION 70 5.5 REFERENCES 71 6. Concentration of Airborne Mycobacterium tuberculosis in Patient Rooms Measured using Real-Time qPCR Coupled to An Air-sampling Filter Method 75 6.1 INTRODUCTION 75 6.2 MATERIALS AND METHOD 77 6.2.1 Sampling location 77 6.2.2 Air sampling 77 6.2.3 DNA extraction method 77 6.2.4 ABI 7700 quantification 78 6.3 RESULTS AND DISCUSSION 80 6.3.1 Dynamic range and analytical sensitivity of real-time qPCR assay 80 6.3.2 Airborne M. tuberculosis in TB patient rooms 80 6.4 REFERENCES 86 LIST OF FIGURES Fig. 2.1 Study Skeleton 15 Fig. 3.3.1 Dot plots of E. coli stained with AO and PI 29 Fig. 3.3.2 Comparison of total cell concentrations measured by using EFM and FCM 30 Fig. 3.3.3 (a). Calibration curve of DNA concentrations and Ct measured by using real-time qPCR. (b). Calibration curve of cell concentrations and Ct measured by using real-time qPCR 31 Fig. 3.3.4 Comparison of total cell concentrations measured by using EFM or FCM and DNA concentration measured by using real-time qPCR 32 Fig. 3.3.5 Comparison of measured-viability and controlled-viability measured by using EFM, FCM, and culture methods 33 Fig. 4.3.1 Contour plots from AO-stained 100% controlled-viability pure suspensions 49 Fig. 4.3.2 Contour plots from SYTO-13-stained 100% controlled-viability pure suspensions 50 Fig. 4.3.3 Contour plots PI-stained 60% controlled-viability pure suspensions 51 Fig. 4.3.4 Contour plots from YOPRO-1-stained 60% controlled-viability pure suspensions 52 Fig. 4.3.5 Contour plots from CTC-stained 100% controlled-viability pure suspension 53 Fig. 4.3.6 Contour plots from AO-stained 100% controlled-viability mixed suspension 54 Fig. 4.3.7 FCM/FL analysis of water (a1) and air samples (a2) with SYTO-13 55 Fig. 4.3.8 Total concentrations, viable counts determined, and culturable counts in water and air sample) collected on two different days from the aeration tank of a hospital effluent treatment plant in Taipei 56 Fig. 6.3.1 Calibration curve of known M. tuberculosis DNA concentrations and Ct by real-time qPCR 85 Fig. 6.3.2 (a). Correlation between airborne M. tuberculosis levels and smear results of sputum samples. (b). Correlation between airborne M. tuberculosis levels and sputum culture results……………………………………………………………….86 LIST OF TABLES Table 3.3.1 Total and nonviable cell concentrations by EFM and FCM methods using AO and PI staining 33 Table 5.3.1 Culturabilities and viabilities of impinger and filter 75 Table 5.3.2 CR and VR of impinger and filter 76 Table 6.3.1 Tuberculosis patients characteristics and airborne M. tuberculosis concentrations .87en-US生物氣膠螢光顯微鏡非培養分析方法流式細胞儀及時定量PCRflow cytometrynon-culture based methodbioaerosolepifluorescence microscopyreal-time qPCR[SDGs]SDG3thesis