The fluorescent stain Nile Red has been used extensively for the quantification of lipids in phytoplankton, including microalgae, because it preferentially stains neutral lipids and it is economical and sensitive to use for screening purposes. Although its basic application has not changed for several decades, recent improvements have been made to improve its utility across applications. Here we describe additional refinements in its application and interpretation as a high-throughput method for the rapid quantification of neutral lipids in liquid cultures of marine phytoplankton. Specifically we address (1) interspecies comparisons, (2) fluorescence excitation and emission wavelengths, and (3) the time course of the Nile Red signal in the context of using bulk or cell-specific fluorescence to quantify neutral lipids of live or preserved cells. We show that with proper caution in its interpretation across species and physiological states the quantity of lipid in hundreds of small volume samples can be reliably assessed daily using a refined Nile Red protocol.
Johnson ZI, Bidigare RR, Blinebry SK, Brown SL, Cullen JJ, Loftus SE, Redalje DG, Swink C, Van Mooy BAS. Screening for lipids from marine microalgae using Nile Red (2017) in Handbook of Hydrocarbon and Lipid Microbiology Series. Consequences of Microbial Interactions with Hydrocarbons, Oils and Lipids: Production of Fuels and Chemicals Springer. DOI: 10.1007/978-3-319-31421-1_382-1