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

By L.P.S. Alves, A.T. Almeida, L.M. Cruz, F.O. Pedrosa, E.M. de Souza, L.S. Chubatsu, M. Müller-Santos and G. Valdameri

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

Topics: Flow cytometry, Nile red, Poly-3-hydroxybutyrate, Herbaspirillum seropedicae, Azospirillum brasilense, Medicine (General), R5-920, Biology (General), QH301-705.5
Publisher: Associação Brasileira de Divulgação Científica
DOI identifier: 10.1590/1414-431x20165492
OAI identifier: oai:doaj.org/article:144181400c4e4221be51b9e89cc70a16
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