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Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining
V Gorenflo1, A Steinbüchel, S Marose
1Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Germany.
Applied Microbiology and Biotechnology
|July 28, 1999
Summary
Nile red effectively quantifies bacterial polyhydroxyalkanoic acids. This hydrophobic polyester can be accurately determined using Nile red staining, with fluorescence intensity correlating to concentration in both isolated granules and living cells.
Area of Science:
- Biochemistry
- Microbiology
- Analytical Chemistry
Background:
- Polyhydroxyalkanoic acids are bacterial polyesters with diverse applications.
- Accurate quantification of these hydrophobic compounds is crucial for research and industrial use.
- Nile red is a lipophilic stain with potential for detecting such polymers.
Purpose of the Study:
- To investigate the fluorescence properties of chemically and biologically produced polyhydroxyalkanoic acids.
- To evaluate Nile red as a quantitative staining agent for polyhydroxyalkanoic acids in film castings and living cells.
- To determine the optimal conditions for Nile red staining and quantification.
Main Methods:
- Staining of polyhydroxyalkanoic acid film castings and living bacterial cells with Nile red.
- Two-dimensional fluorescence spectroscopy to determine excitation and emission maxima.
- Flow cytometry and spectrophotometry for quantitative analysis of stained samples.
Main Results:
- All investigated polyhydroxyalkanoic acids exhibited similar fluorescence behavior with Nile red, showing maxima at specific excitation (540-560 nm) and emission (570-605 nm) wavelengths.
- Optimal Nile red concentration for staining poly(3-hydroxybutyric acid) granules was determined.
- High correlation was observed between fluorescence intensity and poly(3HB) concentration in granule suspensions and cell cultures.
Conclusions:
- Nile red is a suitable fluorescent stain for polyhydroxyalkanoic acids.
- The fluorescence properties are consistent across different polyhydroxyalkanoic acid types.
- Nile red demonstrates high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids in various biological matrices.