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

环境样品中微生物细胞丰度的计数对于正确评估微生物生物量对生物地球化学循环的贡献是必要的，并且当与系统发育染色方法相结合时，可以区分自然界中古菌、细菌、真核生物和病毒形式的相对分布。此外，细胞丰度的措施是非常有用的，在规范化定量PCR数据靶向功能基因与特定的代谢途径或分类组。确定微生物细胞丰度的最基本但最有效的方法之一是使用落射荧光显微镜在高物镜放大倍数下用DNA或RNA染色剂直接可视化。 以这种方式获得的细胞计数是准确的，并提供了一种快速和具有成本效益的方式来获得必要的定量信息。在过去的一年里，我在不列颠哥伦比亚省大学建立了一个环境基因组学实验室，以探索与海洋生态系统中碳和氨的生物地球化学循环有关的未培养微生物群落的遗传组成。我发起了合作研究项目，重点关注海洋沉积物中甲烷的厌氧氧化以及温哥华岛海岸萨尼奇湾近岸缺氧盆地的微生物群落结构。虽然我的实验室装备精良，可以从环境样品中获得高质量的基因组DNA，但我们缺乏一些进行严格定量分析所需的基本工具。具体来说，我缺少一个荧光显微镜来精确测量细胞丰度。我已经确定了一个未充分利用的部门蔡司Axioskop，可以改造为落射荧光显微镜，并已获得许可，以管理其使用。在这份提案中，我要求提供资金，以获得必要的灯泡、滤光片和物镜，以升级这台仪器。此次升级将大大扩展显微镜的实用性，并将其研究应用扩展到包括DAPI细胞计数和荧光素原位杂交研究。作为一个部门的资源，它将提供给所有教师和学生在需要的基础上。

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.