Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas, 50, e5492
January, 2017

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

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

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.

Digital object identifier (DOI): 10.1590/1414-431X20165492

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