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

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.