Lipid determination in qualitative and quantitative analysis is crucial for identification of suitable strains for biodiesel production. Conventional methods such as solvent extraction or gravimetric methods have been used by Bligh and Dyer [30]. Separation and profiling of lipid components re­quire elaborate techniques in order to satisfy criteria of biodiesel qual­ity and includes thin layer chromatography (TLC), gas chromatography — mass spectroscopy (GC/MS) and/or high pressure liquid chromatography (HPLC) [31]. These methods are time-consuming for lipid extraction and analysis, especially for a large number of samples. Thus, a rapid screening for lipid content in organisms or cells is necessary and important for high — throughput screening. Nile red (9-diethylamino-5-benzo[a] phenoxazi — none), a lipophilic stain, maybe used for this purpose. It was first synthe­sized by Thorpe in 1907 by boiling Nile blue with sulfuric acid, and in the same year, Smith reported the use of Nile red for detecting lipids in human cells [32]. The application of Nile red for lipid staining in microorganisms such as bacteria, yeasts and microalgae is now a common practice that allows a rapid qualitative determination of lipids in cells (Figure 1) [33].

Although Nile red can be applied for rapid lipid screening, this meth­od has not been successful in some particular microalgae species due to variables such as staining time, temperature, rigid cell walls, etc. [34]. Thus, Nile red dye concentrations applied for lipid staining are different for particular microalgae species. To improve staining efficiency some factors can be considered. For instance, microwaves applied for staining were first introduced by Leong and Milios and then improved by Chiu et al. in 1987 [34-36]. Microwave exposure time was optimized for pro­cesses of pretreatment and staining. Results of this research showed that microwave-assisted staining increased remarkably fluorescence intensity

using a spectrofluorometer from 476 to 820 arbitrary units (a. u.) for Pseu- dochlorococcum sp. and from 662 to 869 a. u. for Chlorella zofingiensis after 50 s of microwave exposure in a pretreatment process and after 60 s of staining. Dimethyl sulfoxide (DMSO) has also been used for enhancing lipid staining effectiveness [33,34,37] and maybe used in low quantities instead of acetone as a solvent to allow viability of cells after staining. This means that Nile red staining can not only be used as a preliminary quantitative fluorometric assay for relative comparisons among closely — related strains, but potentially also for mutant screening and selection of high lipid-yielding strains.