This article discusses the detection and quantification of neutral lipids in algae, which are crucial for biodiesel production. The method involves using Nile red dye in conjunction with ethanol to stain lipid bodies within algal cells, allowing for fluorescence measurement.
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.
For fluorometric quantification, prepare all algal samples at the same biomass concentration, and in the same manner, as the standards used in the measurement. Do this by suspending pre-dried samples in the appropriate amount of phosphate buffer. If necessary, use a homogenizer to facilitate resuspension of dried algal cells.
For each sample, mix 80 microliters of a prepared 30% ethanol solution, 10 microliters of a prepared Nile Red solution, and 10 microliters of algae suspension, in a single well of a 96-well plate. In order to properly account for the variability of the fluorescence measurement, perform five replicates of each sample. Next, run a two-point calibration curve with standards prepared previously, in order to account for day-to-day variations in the instrument and preparation.
To perform the fluorescence measurements in a multi-well plate reader spectrophotometer, set the parameters to shake at 1,200 rpm and orbit 3 millimeters for 30 seconds, followed by incubation at 40 degrees Celsius for 10 minutes. Another shake at 1,200 rpm, and orbit 3 millimeters for 30 seconds, and to record fluorescence, with excitation at 530 nanometers and emission at 604 nanometers. Then, start the run. As a final step, convert the fluorescence measurements to oil content using the results from the internal standards.
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