The guidelines would improve the robustness of the staining method across the different species and facilitate a more reliable diagnostic tool for the quantification of biodiesel-convertible lipid in microalgal cells. The second objective of the study was to develop a simple set of guidelines for the optimization of Nile red staining across various microalgal species. To generate the model, response surface methodology was applied on the results of factorial experiments. The model would enable the identification of significant variables and elucidate some of the fundamental mechanisms governing lipid/dye interaction. The first one was to create a mathematical model that described microalgal Nile red fluorescence as a function of variables of the staining procedure (cell concentration, ultrasonication power, incubation temperature, and Nile red dye concentration). Through the review process, we were able to develop a simple framework that provided a set of guidelines for the standardization of the Nile red staining procedure across different microalgal species. In its second phase, the study reviewed findings from previous Nile red studies to elucidate some of the fundamental mechanism underlying the diffusion of Nile red dye molecules into the microalgal cells and their subsequent interaction with intracellular lipids. Up to a certain threshold, fluorescence intensity was shown to increase with Nile red dye concentration.
Cell concentration was shown to have a significant effect on the fluorescence intensity. A quadratic surface model that successfully described the Nile red fluorescence intensity as a composite function of its variables was generated ().
In its first phase, this study examined the dependence of microalgal Nile red fluorescence ( Tetraselmis suecica) in terms of its most pertinent staining variables. There is a need for the development of a more uniform staining procedure. Nile red staining acts as a rapid diagnostic tool to measure the amount of biodiesel-convertible lipid that the cells accumulate. In the wake of global warming and rapid fossil fuel depletion, microalgae emerge as promising feedstocks for sustainable biofuel production. 'Near-infrared solid-state fluorescent naphthooxazine dyes attached with bulky dibutylamino and perfluoroalkenyloxy groups at 6- and 9-positions'. Journal of Histochemistry and Cytochemistry. 'Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O'. 'Synthetic Polymer Contamination in Bottled Water' (PDF). Welch, Victoria Neratko, Joseph (15 March 2018).
'Nile Red, A Selective Fluorescent Stain for Intracellular Lipid Droplets'. 'Highly-branched poly(N-isopropyl acrylamide)s with core–shell morphology below the lower critical solution temperature'. ^ R Plenderleith T Swift & S Rimmer (2014).This strain grows partly as cell chains, so a membrane dye may be useful to distinguish internal cell boundaries. Bacillus subtilis stained with Nile red as a membrane dye (shown in red).
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