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

• Autor(a) principal: Alves,L.P.S.
• Data de Publicação: 2017
• Outros Autores: Almeida,A.T., Cruz,L.M., Pedrosa,F.O., de Souza,E.M., Chubatsu,L.S., Müller-Santos,M., Valdameri,G.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Brazilian Journal of Medical and Biological Research
• Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2017000100606
• topic: Flow cytometry; Nile red; Poly-3-hydroxybutyrate; Herbaspirillum seropedicae; Azospirillum brasilense

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.