Frontiers in Microbiology (Apr 2020)
Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4’6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS)
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 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.
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