An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting

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doi: 10.1111/1462-2920.12153
Author(s): Morono, Yuki; Terada, Takeshi; Kallmeyer, Jens; Inagaki, Fumio
Author Affiliation(s): Primary:
Japan Agency for Marine-Earth Science and Technology, Geomicrobiology Group, Japan
Marine Works Japan, Japan
Deutsches GeoForschungsZentrum, Germany
Volume Title: Environmental Microbiology
Source: Environmental Microbiology, 15(10), p.2841-2849. Publisher: Blackwell Science, Oxford, United Kingdom. ISSN: 1462-2912
Note: In English. 24 refs.; illus.
Summary: Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼50% of cells were recovered from deep samples (100-365?m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104-108cellscm3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.
Year of Publication: 2013
Research Program: IODP Integrated Ocean Drilling Program
Key Words: 07 Marine Geology and Oceanography; East Pacific; Expedition 316; Expedition 329; Flow cytometry; Integrated Ocean Drilling Program; Marine sediments; Microorganisms; NanTroSEIZE; North Pacific; Northwest Pacific; Pacific Ocean; Sample preparation; Sediments; South Pacific; Southeast Pacific; Techniques; West Pacific
Coordinates: N330100 N331400 E1364800 E1364300
S455800 S235100 W1230900 W1660000
Record ID: 2019044686
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