Monitoring Microalgal Neutral Lipid Accumulation with Nile Red.

The widely applied Nile red (NR) method allows near real-time monitoring of microalgal neutral lipid accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for monitoring of microalgal neutral lipid accumulation.

Monitoring cell-specific neutral lipid accumulation in Phaeodactylum tricornutum (Bacillariophyceae) with Nile Red staining - a new method for FlowCAM.

With the fluorescent stain Nile Red (NR), phytoplankton lipid accumulation can be monitored quickly and in situ. In the light of recent results in phytoplankton diversity research, there is also a need for cell- and species-specific lipid measurement techniques. The objective of this work was to investigate whether cell-specific phytoplankton lipid accumulation could be monitored with the image-based particle analyzer FlowCAM™ and NR staining. Applying Phaeodactylum tricornutum as a model species, we compared the FlowCAM method to two established lipid quantification methods: spectrofluorometric NR fluorescence measurement and total lipid analysis by gas chromatography. The experiment was carried out in batch cultures under nitrogen limitation to induce lipid accumulation. We showed significant correlation between the three different lipid quantification methods confirming the applicability of the novel FlowCAM method in cell-specific and near real-time lipid quantification. Furthermore, with the method described here, the lipid content of taxonomically distinguished cells can eventually be measured from multispecies cultures, opening several new possibilities to study species-specific responses to stress conditions and the complementarity effect.

Nile Red staining of phytoplankton neutral lipids: species-specific fluorescence kinetics in various solvents.

Nile Red (NR) staining potentially offers a simple method for monitoring lipid accumulation in microalgal cultivation. However, variable staining efficiencies and methods have been reported. The effect of dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol on NR penetration with four different phytoplankton species representing different taxonomical groups was studied. Treatment with the solvents enhanced the NR fluorescence of the diatom Phaeodactylum tricornutum during kinetic fluorescence measurements, but high concentrations of solvents were needed. None of the solvents improved NR staining of the green alga Chlorella pyrenoidosa and Scenedesmus obliquus, which are known to be difficult to stain due to their thick and rigid cell walls. The naked Isochrysis sp. cells stained best without solvents. The results confirm that NR staining protocol needs to be optimized for each species.