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

The oxazine dye Nile blue A and its fluorescent osazone form, Nile red, wer e used to develop a simple and highly sensitive staining method to detect p oly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) direc tly in growing bacterial colonies. In contrast to previously described meth ods, these dyes were directly included in the medium at concentrations of o nly 0.5 mu g/ml, and growth of the cells occurred in the presence of the dy es. This allowed an estimation of the presence of PHAs in viable colonies a t any time during the growth experiment and a powerful discrimination betwe en 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 m ethod was in particular applicable to gram-negative bacteria such as Azotob acter vinelandii,Escherichia coli, Pseudomonas putida, and Ralstonia eutrop ha. It was less suitable for discriminating between PHA-negative and PHA-po sitive strains of gram-positive bacteria such as Bacillus megaterium or Rho dococcus ruber, but it could also be used to discriminate between wax-ester - and triacylglycerol-negative and -positive strains of Acinetobacter calco aceticus or Rhodococcus opacus. The potential of this new method and its ap plication to further investigations of PHA synthases and PHA biosynthesis p athways are discussed.