環境中生物氣膠之偵測 – 螢光顯微鏡、流式細胞儀與定量PCR

Abstract

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.