Epifluorescence microscope upgrade for determination of microbial abundance in natural sediments and waters

点击翻译按钮获取中文摘要

The enumeration of microbial cell abundance in environmental samples is necessary to properly evaluate the contribution of microbial biomass to biogeochemical cycles, and when coupled to phylogenetic staining methods, to differentiate between the relative distributions of archaeal, bacterial, eukaryotic and viral forms in nature. Moreover, cell abundance measures are extremely useful in normalizing quantitative PCR data targeting functional genes associated with specific metabolic pathways or taxonomic groups. One of the most basic but powerful ways to determine microbial cell abundance is by direct visualization with a DNA or RNA stain using an epifluorescence microscope under high objective magnification.  Cell counts obtained in this manner are accurate and provide a rapid and cost-effective way to obtain essential quantitative information. Over the last year I have established a laboratory of environmental genomics at the University of British Columbia to explore the genetic composition of uncultivated microbial communities in relation to the biogeochemical cycling of carbon and ammonia in marine ecosystems. I have initiated collaborative research projects focused on the anaerobic oxidation of methane in marine sediments and in the microbial community structure of the near-shore anoxic basin, Saanich Inlet, on the coast of Vancouver Island. While my laboratory is well equipped to obtain high quality genomic DNA from environmental samples we lack some of the basic tools necessary to conduct rigorous quantitative analysis. Specifically, I lack an epifluorescence microscope for accurate cell abundance measurements. I have identified an underutilized departmental Zeiss Axioskop that can be retrofitted for epifluorescence microscopy and have obtained permission to manage its use. In this proposal, I am requesting funds to obtain the necessary lamp, filters and objective to upgrade this instrument. This upgrade will greatly expand the microscopes utility and extend its research applications to include DAPI cell counts, and flourescent in situ hybridization studies. As a departmental resource it will be made available to all faculty and students on a need basis.