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https://doi.org/10.5194/egusphere-2024-1085
https://doi.org/10.5194/egusphere-2024-1085
02 May 2024
 | 02 May 2024
Status: this preprint is open for discussion.

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

Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

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.

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Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

Status: open (until 13 Jun 2024)

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  • RC1: 'Comment on egusphere-2024-1085', Anonymous Referee #1, 03 May 2024 reply
  • RC2: 'Comment on egusphere-2024-1085', Anonymous Referee #2, 06 May 2024 reply
Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours
Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

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Short summary
In the context of the ecological sustainability of phosphorus (P), the ubiquitous presence of polyphosphate-accumulating bacteria (PAB) in natural environments invites efforts to reveal their unknown roles in the biogeochemical cycle of P. In this study, we provide a generic protocol for the detection, quantification, and cell sorting of PAB by combining efficient staining of intracellular polyphosphate (polyP) granules in PAB and their subsequent detection by flow cytometry.