A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds

Abstract

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 microgram/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Production and characterization of poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending with other polymers

“…The N-limit media showed more number of PHB granules than the P-limit media. The reports says that this viable-colony staining method was in particular applicable to gram-negative bacteria and it was less suitable for discriminating between PHAnegative and PHA-positive strains of grampositive bacteria such as Bacillus megaterium or Rhodococcus ruber (Spiekermann et al, 1999). But our studies show the presence of PHB in all the five positive strains and it was confirmed by FTIR and NMR analysis.…”

“…Bright orange fluorescing PHB granules were more prominent in Bacillus sphaericus NII 0838. The Nile blue stain binds specifically to PHB granules and they fluoresce with orange color under excitation wavelength of 390 nm (Spiekermann et al, 1999). The N-limit media showed more number of PHB granules than the P-limit media.…”

Biosynthesis of biodegradable polyhydroxyalkanotes biopolymers in genetically modified yeasts

“…Positive yeast colonies containing the designated plasmid pBK/PHB were screened for PHB expression using the Nile-Red staining assay (Spiekermann, et al, 1999). It is known that the Nile-red stain emitted strongly positive red fluorescence signals only with hydrophobic compounds like PHAs and lipids.…”

“…A cassette including the three genes of the PHA biosynthesis pathway of Ralstonia eutropha {beta-ketothiolases (phbA); NADPH-linked-acetoacetyl-CoA reductase (phbB) and PHA synthase (phbC)} was obtained from the plasmid pBHR68 (pBluscriptSK -) that contains a 5.2 Kb fragment from chromosomal DNA of Ralstonia eutropha (Spiekermann, et al, 1999). Subsequently, the cassette was digested with endonucleases enzymes Xba1 and BamHI and subcloned by ligation into the pBK-CMV phagemid vector (Stratagene, La Jolla, CA, USA).…”

“…Saccharomyces cerevisiae INVSc1 and Kloeckera spp KY1 were transformed with the constructed plasmid designated pBK/PHB. Yeast colonies containing plasmid pBK/PHB were then screened for PHB expression using the Nile-Red staining assay described by Spiekermann et al, (1999). To confirm results of the Nile-Red assay, positive clones were screened for the presence of inserts by restriction mapping (Sambrook, et al, 1989).…”