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

落射荧光显微镜升级用于测定天然沉积物和水中的微生物丰度

• 批准号: 344884-2007
• 负责人: Hallam, Steven
• 金额: $ 1.64万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2006
• 资助国家: 加拿大
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=326069
• 关键词: Epifluorescence; microscope; upgrade; determination; microbial

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.

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结合时，可以区分自然界中古细菌、细菌、真核生物和病毒形式的相对分布。此外，细胞丰度测量对于标准化定量 PCR 数据非常有用，该数据针对与特定代谢途径或分类群相关的功能基因。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直接观察 DNA 或 RNA 染色。  以这种方式获得的细胞计数是准确的，并提供了一种快速且经济有效的方式来获取重要的定量信息。去年，我在不列颠哥伦比亚大学建立了环境基因组学实验室，以探索未培养的微生物群落的遗传组成与海洋生态系统中碳和氨的生物地球化学循环的关系。我发起了一些合作研究项目，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。虽然我的实验室设备齐全，可以从环境样本中获取高质量的基因组 DNA，但我们缺乏进行严格定量分析所需的一些基本工具。具体来说，我缺乏用于精确测量细胞丰度的落射荧光显微镜。我已经确定了一台未充分利用的部门级 Zeiss Axioskop，可以改装用于落射荧光显微镜，并已获得管理其使用的许可。在此提案中，我请求资金来购买必要的灯、滤光片和物镜来升级该仪器。此次升级将极大地扩展显微镜的实用性，并将其研究应用扩展到包括 DAPI 细胞计数和荧光原位杂交研究。作为部门资源，所有教师和学生都可以根据需要使用它。