title: Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).
abstract: Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.
authors.affiliation: School of Biological Sciences and QUESTOR Centre, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Rd., Belfast, BT9 7BL, Northern Ireland.
authors.forename: Anna N
authors.initials: AN
authors.lastname: Kulakova
authors.forename: Darragh
authors.initials: D
authors.lastname: Hobbs
authors.forename: Matthew
authors.initials: M
authors.lastname: Smithen
authors.forename: Evgeny
authors.initials: E
authors.lastname: Pavlov
authors.forename: Jack A
authors.initials: JA
authors.lastname: Gilbert
authors.forename: John P
authors.initials: JP
authors.lastname: Quinn
authors.forename: John W
authors.initials: JW
authors.lastname: McGrath
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes; D007211:Indoles; D008856:Microscopy, Fluorescence; D010758:Phosphorus / Q000032:analysis; D011122:Polyphosphates / Q000302:isolation & purification*; D016958:Pseudomonas putida / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D011122:Polyphosphates; D010758:Phosphorus; C007293:DAPI