title: A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.
abstract: The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: L P S
authors.initials: LP
authors.lastname: Alves
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: A T
authors.initials: AT
authors.lastname: Almeida
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: L M
authors.initials: LM
authors.lastname: Cruz
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: F O
authors.initials: FO
authors.lastname: Pedrosa
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: E M
authors.initials: EM
authors.lastname: de Souza
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: L S
authors.initials: LS
authors.lastname: Chubatsu
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: M
authors.initials: M
authors.lastname: Müller-Santos
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.|Departamento de Análises Clínicas, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: G
authors.initials: G
authors.lastname: Valdameri
mesh_terms: D015806:Azospirillum brasilense / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D042803:Herbaspirillum / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018517:Plant Roots / Q000382:microbiology*; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates