RUI: Instrumental Methods for the Analysis of Aquatic Bacteria by Flow Cytometry

9318432巴顿水生细菌控制着天然水系统的有机化学，约占海洋生物量的一半。然而，由于对常规程序的抗拒，这些被称为寡杆菌或超微细菌(UMB)的大多数生物的身份、系统发育、生物化学和营养需求尚不清楚。然而，UMB的细胞特性包括极小的尺寸和DNA含量，对有机营养的高亲和力，以及在富媒体中的生长阻力。流式细胞术提供了一个了解这些生物体的理想窗口，不仅因为这种仪器可以执行的灵活性和多样性，而且因为自然群体中的典型细胞数量非常适合这种类型的直接分析，自然细胞群体可以被保存下来供以后分析，并且分析的单细胞基础可以提供关于细胞群体结构的丰富信息。不幸的是，目前的流式细胞术分析方法仅适用于大细胞。必须开发UMB类型细胞的处理技术和标准尺寸曲线。本研究项目的目的是建立高效的流式细胞术分析UMB的方法。将完成细胞大小和每个细胞DNA的标准曲线。将为UMB RNA和蛋白质含量生成额外的标准曲线。膜电位将被用来探测细胞的生理状态，并将检验使用寡核苷酸探针作为寡杆菌分类工具的可行性。从信号邻近分析中得出混合系统中细菌的三维间距和趋化性信息的概念将在实验室和现场条件下进行测试。水生细菌，特别是寡杆菌或超微细菌(UMB)，构成了海洋中几乎一半的生物物质，因此，它们的代谢活动可以控制世界海洋的化学成分。然而，令人惊讶的是，人们对这些细胞知之甚少，除了它们很小，含有最低限度的DNA，尽管它们对有机化合物有很强的吸引力，但它们只有在浓度非常低的情况下才能生长。目前的研究计划的目标是开发利用流式细胞术直接了解海水中细胞的方法。将开发标准曲线来测量细胞大小并估计其DNA、RNA和蛋白质含量。新的方法将被用来确定哪些细胞正在代谢，以及水样本中可能存在多少种不同的UMB细胞类型。可行性将在实验室和在流式细胞仪条件下分析细胞运动的领域进行测试，以确定水生细菌是否表现出对理想营养的策略性行为。%%%

9318432 Button Aquatic bacteria control the organic chemistry of natural water systems and comprise about half the biomass of the oceans. Yet, because of recalcitrance to conventional procedures, the identity, phylogenetics, biochemistry and nutritional requirements of most of these organisms, called oligobacteria or ultramicrobacteria (UMB), are unknown. However, UMB cellular properties include extremely small size and DNA content, high affinity for organic nutrients, and resistance to growth in rich media. Flow cytometry offers an ideal window into these organisms not only because of the flexibility and the variety of measurements that can be performed by this instrumentation, but because typical cell numbers in natural populations are ideally suited for this type of direct analysis, natural cell populations can be preserved for analysis at a later time, and the single-cell basis of the analysis can provide a wealth of information on cell population structure. Unfortunately, present flow cytometry analytical methods have been developed only for large cells. Techniques for treatment and standard size curves for UMB-type cells have to be developed. The objective of the present research project is to develop methods for efficient analysis of UMB by flow cytometry. Standard curves for cell size and for DNA per cell will be completed. Additional standard curves will be generated for UMB RNA and protein content. Membrane potential will be used to probe the physiological state of cells, and the feasibility of using oligonucleotide probes as a taxonomic tool for oligobacteria will be examined. The concept of deriving three-dimensional spacing and chemotaxis information about bacteria in mixed systems from signal-proximity analysis will be tested under both laboratory and field conditions. %%% Aquatic bacteria, especially the oligobacteria or the ultramicrobacteria (UMB), comprise almost one half the biological material in the sea and, as such their metabolic activity can control the chemistry of the world's oceans. Surprisingly, however, very little is known about these cells except that they are small, they contain a minimum amount of DNA, and although they have a strong attraction to organic compounds, they only grow when the concentration is very low. The objective of the present research program is to develop methods using flow cytometry to learn about the cells directly in sea water. Standard curves will be developed to measure cell size and estimate their DNA, RNA, and protein contents. Novel methods will be used to determine which cells are metabolizing and how many different UMB-cell types may be present in a water sample. The feasibility will be tested in the laboratory and in the field of analyzing cell movement under flow cytometer conditions to determine if the aquatic bacteria exhibit tactic behavior toward desirable nutrients. %%%