Technical note: Flow cytometry assays for the detection, counting and cell-sorting of polyphosphate-accumulating bacteria

Abstract. In the context of the ecological sustainability of phosphorus (P), the emerging evidence of the ubiquitous presence of polyphosphate-accumulating bacteria (PAB) in natural environments invites efforts to reveal their unknown functions and roles in the biogeochemical cycle of P. This requires high-throughput methods to characterise PAB structure and dynamics in the environment. A promising strategy is to combine efficient staining of intracellular polyphosphate (polyP) granules in PAB and their subsequent detection by flow cytometry, enabling rapid data acquisition and multiparametric analysis. In this study, we provide a generic protocol for the detection, quantification, and cell sorting of PAB by flow cytometry using the dye 4’6-diamidino-2-phenylindole (DAPI). The assays were performed using Tetrasphaera elongata, which represent a large part of the microbial biomass in enhanced biological phosphate removal systems for wastewater treatment. We also included, as a negative control, a bacterial strain characterized by very low quantities of cellular polyP and carried out tests on water and lake sediment samples. We also show that the synthetic fluorochrome JC-D7, a new selective fluorescent dye used for the specific labeling of endogenous polyP in living cells, is promising for achieving these purposes, particularly in complex environmental samples.