New method to characterize microbial diversity using flow cytometry

Abstract

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

“…Flow cytometry was used to get quantitative information on the community dynamics and for the identification of active populations. Flow cytometry is a well-established method for depicting bacterial consortia in natural environments (15,22,55,65,68) and was recently combined with cell sorting as a prerequisite for phylogenetic analyses of subcommunities (49,53). We used narrow ranges of DNA contents and cell sizes as the criteria for cell sorting.…”

“…Multiparametric flow cytometry has the potential to identify the active community members without requiring knowledge of metabolic genes. It provides information on the physiological states and activities of individuals and populations within communities, e.g., by indicating proliferation activity, while raising the possibility of identifying individuals by specific fluorescence labeling (50) or by rRNA-based methods after cell sorting (49,53).…”

Quantifying inorganic sources of geochemical energy in hydrothermal ecosystems, Yellowstone National Park, USA

“…Complementary work on phylogeny of environmental samples from Yellowstone, based on sequencing of subunits of ribosomal RNA, adds scores of genetically distinct, although typically uncultured organisms to the list of microorganisms in Table 1 (Ward et al, 1990(Ward et al, , 1998Barns et al, 1994Barns et al, , 1996Reysenbach et al, 1994Reysenbach et al, , 2000Reysenbach et al, , 2005Nold and Ward, 1995;Santegoeds et al, 1996;Hugenholtz et al, 1998;Graber et al, 2001;Blank et al, 2002;Boomer et al, 2002;Ferris et al, 2003a;Fouke et al, 2003;Botero et al, 2005;Hamamura et al, 2005;Meyer-Dombard et al, 2005;Park et al, 2005;Sheehan et al, 2005;Spear et al, 2005a,b;Walker et al, 2005;Hall et al, 2008;Toplin et al, 2008). In all of these cases, the metabolic strategies pursued by these organisms are unknown, as the metabolic function of uncultured organisms is not conveyed by 16S rRNA data.…”

Genomics, metagenomics, and microbial oceanography—A sea of opportunities

“…Integrating cultivation with other technologies (e.g., flow cytometry, molecular genetics) to develop new methodologies is a new direction (e.g., the FACS approach (florescence activated cell sorting); ref. [73]). Directed cultivation and phenotypic characterization of microorganisms and coupling metagenomics and culture-based techniques is another new direction in microbial oceanography [43].…”

“…These cultivation devices should be able to mimic the natural environmental conditions in the lab and can be deployed in the ocean as an in situ microcosm/mesocosm (e.g., refs. [71][72][73]). Integrating cultivation with other technologies (e.g., flow cytometry, molecular genetics) to develop new methodologies is a new direction (e.g., the FACS approach (florescence activated cell sorting); ref.…”