Development of an indirect method of microalgal lipid quantification using a lysochrome dye, Nile red

Earlier studies showed that the lipophilic dye, Nile red (9-diethylamino 5H-benzo[α]phenoxazine-5-one) can be used to measure the lipid content of microalgae by cellular in vivo fluorescence. It was observed that a higher amount of lipid present in lipid droplets of microalgal cells would result in higher degree of emitted fluorescent light. In this present study, the feasibility of using Nile red, a fluorescent dye specific for intracellular lipid droplets, as an indirect method of lipid quantifications was investigated. Following cellular staining of different microalgal species with nile red, the in vivo fluorescence of the whole cell was visualized by fluorescence microscopy (excitation: 450 to 590 nm and emission: 520 nm). Intensity of the relative in vivo fluorescence was measured using a fluorescence spectrophotometer at excitation and emission wavelengths of 485 and 590 nm, respectively. Lipid content was determined gravimetrically and the fluorescence of the extract was measured using the microemulsion method at emission and excitation wavelengths of 540 and 617 nm. The equivalent oil content of the extracted lipid was correlated to the fluorescence of pure olive oil using the microemulsion method. Cellular in vivo fluorescence of stained cells (ex: 485 nm and em: 590 nm), fluorescence of extracted lipid (ex: 540 nm and em: 617 nm) and gravimetrically determined lipid were linearly correlated. This suggests that Nile red can serve as a vital stain which allows a relatively rapid method of determining the lipid content of microalgal samples and is as good as the gravimetric method used for lipid determination, eliminating the requirement for the toxic solvents and timeconsuming manipulations. Keywords: Nile red, microalgae, lipid, fluorescence