Modified rapid determination of lipid content in Nannochloropsis by Nile Red fluorescence staining

Abstract:

[Objective] This study was aimed to investigate the optimal Nile Red fluorescence staining method to quickly determine the quantification of total lipid in Nannochloropsis. [Methods] We systematically adjusted the excitation wavelength, emission wavelength and optimized the dimethyl sulfoxide (DMSO) concentration, Nile Red concentration, staining time and cell density range. Meanwhile, we evaluated the relationship between the florescence intensity of Nile Red staining and the lipid contents of gravimetric method and Triolein standard method, respectively. [Results] The relationship between the Nile Red fluorescence intensity and the lipid content of green algae Nannochloropsis was obtained. The results showed that the optimal dyeing conditions are as follows: DSMO concentration was 5%, the final concentration of Nile Red was 1 mg/L, staining time was 6 min and the cell number ranged from 0.5×106 to 3.0×106 cells/mL, under the excitation and emission wavelengths was 515 nm and 570 nm. The correlation between the lipid content from gravimetric method and Triolein standard indicated that the Nile Red fluorescence staining method can be used to rapidly and exactly detect the lipid content in Nannochloropsis. The lipid content was positively correlated with the fluorescence intensity, and the correlation coefficient R2 was 0.997 3. The detection limit of lipid content reached 2 μg after the optimization of Nile Red staining, which greatly reducing the amount of cells required for the determination of lipid content. [Conclusion] These results indicated that the Nile Red fluorescence staining method can be used to determine the lipid content rapidly and exactly in the Nannochloropsis with a microdose cell, and be applied in screening Nannochloropsis mutants with higher lipids content in large scale, conveniently.

Key words:Nile Red, Fluorescence intensity, Lipid content, Nannochloropsis