Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produc ed polyhydroxyalkanoic acids were investigated as film castings and in livi ng cells respectively after staining with Nile red. All these polyesters sh ow a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wave length between 570 nm and 605 nm. This could be shown by the use of two-dim ensional fluorescence spectroscopy and flow cytometry. The examination of n ative poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 mu g Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspension s at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a hi gh potential for the quantitative determination of hydrophobic bacterial po lyhydroxyalkanoic acids.