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

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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. %%%