Nile Red Staining: A Technique to Detect and Quantify Intracellular Neutral Lipids in Algae

Algae are aquatic photosynthetic organisms that produce a variety of lipids, of which neutral lipids are essential for biodiesel production.

To detect and quantify neutral lipids, mix an optimum volume of algal suspension and ethanol in a black microtiter plate well. The algal cell wall and membrane, which prevent most stains from penetrating, become permeable when exposed to ethanol. Next, combine the ethanol with a lipid-soluble fluorescent dye - Nile red. Add this mixture to the algae-containing well.

Place the microtiter plate in a spectrophotometer. Set a program that ensures incubation with intermittent shaking to facilitate interaction between the dye and the algae.

As Nile red is hydrophobic, it cannot easily traverse the cytoplasm. Solvents such as ethanol, when coupled with Nile red, mobilize the dye and help it reach the lipid bodies dispersed in the cytoplasm. Nile red then penetrates the lipid bodies and solubilizes there, thus staining the lipids.

Lipid-deficient cells appear as fluorescent rings with dark bodies, while lipid-rich cells show a bright orange-red glow where the lipid bodies are accumulated.

After staining the cells, expose them to a wavelength that maximally excites the neutral lipids. Record the resulting fluorescence and correlate it against a calibration curve to quantify the neutral lipids in the test samples.