Applications of Molecular Biology Techniques in Monitoring Environmental Microorganisms
Date Issued
2007
Date
2007
Author(s)
Chi, Miao-Ching
DOI
en-US
Abstract
In the past decade, traditional culture-based methods are the major method for detecting microorganisms; however, culture-based methods for microorganism quantification are slow, tedious, and rather imprecise. In order to get more rapid, sensitive, and specific results for field microorganisms, non-culture-based methods for microorganism quantification should be therefore considered as an alternative. From non-culture-based methods, epifluorescence microscopy with fluorochrome (EFM/FL), flow cytometric with fluorochrome (FCM/FL), fluorescent in situ hybridization (FISH) and real-time polymerase chain reaction (real-time qPCR) for microorganisms quantification are the most promising methods to evaluate the viability, an important indicator for assessing potential health effects. To date, only limited data is available on the total cell concentration and viability of microorganisms in different environments. In this study, the optimal analysis methods of FISH and real-time qPCR for microorganism evaluation were established. The optimal conditions of FISH, and real-time qPCR were used to monitor the total cell concentration and viability of microorganisms in air and water environments. The results were then compared to those obtained using a commonly used culture-based method.
For the FISH condition optimization, four different controlled-viability samples of E.coli and yeast were made to assess the relation between FISH and culture-based method. The result showed that hybridization efficiency of FISH with varies probes was higher than 90 %. In addition, FISH and culture-based method were highly associated for viability. In summary, FISH could provide rapid and accurate information about microorganism concentrations and viability. Moreover, the established optimal FISH with probes were validated for characterizing bioaerosol profiles from air samples in swine buildings, chicken houses, and ambient air. The results were then compared to those obtained using fluorochrome with dyes and a commonly used culture-based method. The total microbial cell concentrations measured by using non-culture-based methods averaged about 9 x 10^6 cells/m^3 for swine buildings, 5 x 10^8 cells/m^3 for chicken houses, and 8 x 10^5 cells/m^3 for ambient environment. The viabilities determined by using non-culture-based methods were much higher than the culturabilities. The total microbial cell concentration and viability in the atmosphere were highly underestimated by the culture-based method in the past. Moreover, the results revealed that there was lower viability, but higher culturability in the livestock environment. The lower viability might be associated with non-viable bioaerosols accumulated on the floor resulted from longer cleaning intervals. Furthermore, the higher culturability can be explained by the sufficient nutrients form animal dander, fecal matter, and feed materials for bioaerosol growth. However, the higher viability but lower culturability was observed in the atmosphere environment. The finding might be related to that bioaerosols entered a viable but non-culturable (VBNC) state because of insufficient nutrients in a harsh environment. In conclusion, the FISH successfully assessed the total concentration and vibility for bacterial and fungal microorganisms in environmental field samples.
Regarding the microorganisms in water environments, FISH and real-time qPCR were successfully established and applied to E. coli quantification in drinking water and wastewater. Results of FISH, real-time qPCR and culture-based method indicated that viability was highly associated for culturability. By the non-culture-based methods, real-time qPCR with DNA gene probe, the total E. coli concentrations in raw sewage and final effluent averaged 5 x 10^7 cells/100 ml and 2 x 10^7 cells/100 ml, respectively. The viability determined by using non-culture-based methods was higher than those using culture-based method. In conclusion, FISH and real-time qPCR should be powerful tools to provide more insight in the area of bioaerosol and environmental microbiology.
Subjects
分子生物技術
螢光染色
即時定量聚合酶
連鎖反應
螢光原位雜交
生物氣膠
molecular biology technique
fluorochrome
real-time quantitative polymerase chain reaction
fluorescent in situ hybridization
bioaerosol
SDGs
Type
thesis
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