Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

Abstract

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

Yeast Cells in Microencapsulation. General Features and Controlling Factors of the Encapsulation Process

“…It is worth mentioning that a non-destructive and rapid quantification of hydrophobe encapsulation can be achieved via Nile Red lipid staining [31,130]. This method has some significant limitations: it requires an equilibration of at least 30 min [131] and a ), but permeabilized cells behave similarly. The most striking result is the large increase in the number and dimensions of bodies that can be colored by the lipid stain Nile Red (in the insets, confocal images where the cell wall is stained by Concanavalin A, and Nile Red fluorescence highlights the lipidic bodies).…”

“…It is worth mentioning that a non-destructive and rapid quantification of hydrophobe encapsulation can be achieved via Nile Red lipid staining [ 31 , 130 ]. This method has some significant limitations: it requires an equilibration of at least 30 min [ 131 ] and a thorough calibration [ 31 ], which is critically necessary using different yeast strains (or other forms of microorganisms such as algae [ 132 ]) or operational conditions, since the results are quite dependent on cell morphology [ 133 ] and on whether an organic solvent was used as a carrier [ 134 ], leaving the fluorescence-free test as the most accurate approach [ 133 ].…”

Extracts from Argentinian native plants reverse fluconazole resistance in Candida species by inhibiting the efflux transporters Mdr1 and Cdr1

“…1 and 2). At the emission and excitation wavelengths used in this study (488 nm and a 574/26 nm, respectively), Nile Red labels neutral lipids within intracellular lipid droplets [51,52]. The differences in the MFI values obtained, suggest that S. cerevisiae strains contain more neutral lipids than Candida strains.…”

Key role of fluorescence quantum yield in Nile Red staining method for determining intracellular lipids in yeast strains

“…Thus, the feasibility of this methodology was confirmed for lipid determination from different carbon sources. Many researchers have tried to optimize the fluorometric method by investigating the effect of biomass concentration, use of carriers for dyes penetration or the use of different staining dyes, concentration or incubation time [ 13 , 28 , 29 ]. Results are always displayed in terms of relative units, while the solution to avoid inconsistent measurement would be the use of fluorimeter absolute values, as the ф fl provides.…”

“…As shown in Fig. 4 , lipid content obtained with I E , which is the most common method used when conducting this type of yeast screenings [ 23 , 29 ], presented the highest error bars. Furthermore, significant differences ( p < 0.05) were observed in the lipids content obtained when using the gravimetric method and I E .…”