A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

• Published in: Brazilian journal of medical and biological research Vol. 50; no. 1; p. e5492
• Main Authors: Alves, L P S, Almeida, A T, Cruz, L M, Pedrosa, F O, de Souza, E M, Chubatsu, L S, Müller-Santos, M, Valdameri, G
• Format: Journal Article
• Language: English
• Published: Brazil Associacao Brasileira de Divulgacao Cientifica (ABDC) 01-01-2017; Associação Brasileira de Divulgação Científica
• Subjects: Azospirillum brasilense; Azospirillum brasilense - metabolism; BIOLOGY; Biomedical Sciences; Flow cytometry; Flow Cytometry - methods; Herbaspirillum - metabolism; Herbaspirillum seropedicae; Hydroxybutyrates - metabolism; MEDICINE, RESEARCH & EXPERIMENTAL; Microscopy, Fluorescence; Nile red; Nitrogen-fixing microorganisms; Plant Roots - microbiology; Poly-3-hydroxybutyrate; Poly-beta-hydroxybutyrate; Polyesters - metabolism; Azospirillum brasilense; Flow cytometry; Nile red; Herbaspirillum seropedicae; Poly-3-hydroxybutyrate
• ISSN: 0100-879X; 1414-431X; 1414-431X
• DOI: 10.1590/1414-431X20165492

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.