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Initial Publication Date: July 14, 2006

Methods

Created by George Rice, Montana State University


Image of sediments sampling site at Dean Creek.


The philosophy of the SIMO (link to project home page) project is that detailed descriptive studies of prokaryotic assemblages in the salt marsh are the requisite first step to a more fundamental understanding of microbial processes, but alone are insufficient. The challenge is to progress beyond descriptive studies and efforts to "link structure to function" to the point where knowledge of microbes and microbial gene expression improves understanding of ecosystem regulation. It is recognize that knowing which microorganisms are present in an environment, and even knowing which microorganisms are carrying out specific processes, does not in itself provide insight into microbial controls on biogeochemical pathways. The primary goal is then to learn what each organism is doing in complex natural assemblages in order to learn how these natural communities function.

Research Techniques:


Figure showing flow-cytometric analysis.
Flow-Cytometric Analysis (FCM): Flow cytometry is a powerful technique for analyzing large populations of single cells. The instrument consists of a pressurized hydrodynamic system, laser beam(s), flow cell/laser intercept, a series of light detectors (PMTs) and a data analysis station.

  • Cells in suspension are introduced into the machine which uses a method of hydrodynamic focusing (or laminar flow) to form the suspension into a stream of cells in single file.
  • This stream is focused through the path of one or more lasers, allowing light scatter characteristics to be ascertained (related to size and density characteristics). Fluorochrome labeled target(s) of interest are excited and detected by one of a series of photomultiplier tubes (PMTs).
  • The signals are sent to a computer where the characteristics of large populations (usually 10-15,000 cells or events but is fully user-definable) are ascertained and tabulated. At this point it is also possible to physically sort the cells. Once the cell has passed through the laser and has been determined to possess particular user-defined traits (i.e. to exhibit high GFP fluorescence) it is possible to send the desired cell into a collection vial by means of electrostatic charging and subsequent charge mediated deflection.
  • The results are typically displayed in the form of distribution histograms (single parameter studies) or 2D dot plots (multiple parameter studies). Closer looks at the characteristics of sub populations are possible by carefully selecting or 'gating' on those cell subsets of interest.

(from "What is Flow Cytometry?" - Queen's University, Ontario, Canada)

Flow cytometry (FCM) discriminates between populations within complex bacterial communities based on fluorescence and size differences among the cells. In conjunction with a sorting unit, defined FCM populations can be physically separated and then subjected to further taxonomic analysis (2, 3, 23, 28, 32, 36). However, the combined methods are challenging to use, primarily because of the low bacterioplankton densities typical of seawater and the large number of sorted cells needed for subsequent DNA-based analysis.

For use of FCM in this study see- Mou et al., Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations. Applied and Environmental Microbiology, March 2005, p. 1405-1416.


Copyright on all images, and material by Mary Ann Moran, 2006.