rapid quantification of intracellular bacterial polyhydroxyalkanoates via nile red staining
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Date
2014
Authors
Randeran, Zuriani
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Abstract
This current study presents the improvements of Nile red staining method for rapid quantification of accumulated polyhydroxyalkanoate (PHA) produced by a locally isolated Gram negative Cupriavidus sp. USMAA1020. A 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the stained cells containing PHA. Prior to fluorescence measurement, the optimal ranges of excitation and emission wavelengths were determined using various types of PHA which consist of different co-monomers and compositions. Despite of the differences, all tested PHAs produced maximum fluorescence at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. Based on a constructed calibration curve, two standard equations were used to determine both PHA content (wt%) and concentration (g/L). The improved staining method had demonstrated similar correlation between the amounts of accumulated PHA determined from the fluorescence measurement and that from gas chromatography analysis to estimate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV-co-4HB)] produced by Cupriavidus sp. USMAA1020. More importantly, a similar correlation was also observed in the monitoring of P(3HB) synthesis by Chromobacterium sp. USM2 and Bacillus megaterium KB-1, deducing the potential of Nile red method in rapid quantification of PHA produced by other microorganisms.