Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining

H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granule...

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Bibliographic Details
Published inBio-protocol Vol. 8; no. 5; p. e2748
Main Authors Juengert, Janina, Bresan, Stephanie, Jendrossek, Dieter
Format Journal Article
LanguageEnglish
Published United States Bio-Protocol 05.03.2018
Bio-protocol LLC
Subjects
Biology
Methods
Gas chromatography
Nile red
Ralstonia eutropha
Polyhydroxybutyrate (PHB)
Acidic methanolysis
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Summary:H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence microscopy qualitatively. Our second approach enables the conversion of PHB to volatile hydroxycarboxylic acid methyl esters by acidic methanolysis and their quantification by gas chromatography. Through this method, it is possible to obtain an absolute quantification of PHB, ., per cell dry weight.
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Contributed equally to this work
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.2748