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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)
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Submitter: 寺島 美亜
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