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

Frontiers in Microbiology

Yoichi Kamagata²  Souichiro Kato³  Mia Terashima³ 

[1] Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan;Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan;Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan;

关键词: Polyphosphate-accumulating organisms;    flow cytometry;    wastewater sludge;    bacteria isolation;    DAPI;   

DOI  :  10.3389/fmicb.2020.00793

来源: DOAJ

【 摘 要 】

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 μg/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.

【 授权许可】

Unknown