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Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4'6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS)

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Title: Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4'6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS)
Authors: Terashima, Mia Browse this author
Kamagata, Yoichi Browse this author
Kato, Souichiro Browse this author
Keywords: Polyphosphate-accumulating organisms
flow cytometry
wastewater sludge
bacteria isolation
DAPI
Issue Date: 30-Apr-2020
Publisher: Frontiers Media
Journal Title: Frontiers in microbiology
Volume: 11
Start Page: 793
Publisher DOI: 10.3389/fmicb.2020.00793
Abstract: Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4'6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 mu g/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.
Rights: https://creativecommons.org/licenses/by/4.0/
Type: article
URI: http://hdl.handle.net/2115/78758
Appears in Collections:低温科学研究所 (Institute of Low Temperature Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 寺島 美亜

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