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, emerging evidence for the ubiquitous presence of polyphosphate-accumulating bacteria in natural environments invites efforts to reveal their roles in the biogeochemical cycle of phosphorus. This requires high-throughput methods to characterize their structure and dynamics in ecosystems. A promising strategy is to combine the staining of intracellular polyphosphate granules and their subsequent detection by flow cytometry, enabling rapid data acquisition. In this study, we evaluated the potential of this approach by testing various factors that could affect the efficiency and specificity of polyphosphate labeling. Most of our experiments were performed using the 4′,6-diamidino-2-phenylindole dye (DAPI). However, we also carried out a preliminary study using the synthetic fluorochrome JC-D7, a new selective fluorescent dye used for the specific labeling of endogenous polyphosphate in living cells. The assays were performed on Tetrasphaera elongata, a Gram-positive bacterium known to accumulate large amounts of intracellular polyphosphates. We also used six bacterial strains belonging to different phyla, in particular a Gram-negative bacterial strain belonging to the genus Pseudomonas, which is characterized by low levels of cellular polyphosphate. The potential of flow cytometry to quantify and sort polyphosphate-accumulating bacteria in complex environmental samples, including soil, freshwater and sediments, was also examined. Our tests provide useful information for the design of future experiments and highlight the potential pitfalls and limitations of detecting polyphosphate-accumulating bacteria using the cytometric approach. We also show that JC-D7 is a promising dye for achieving these objectives, particularly for enumerating polyphosphate-accumulating bacteria from environmental samples.