Abstract：A quick, simple, in situ, cheap and accurate method for lipid content determination in microalgae is important to microalgae biodiesel research. In this study, Chlorella vulgaris was cultivated in nitrogen depleted media, and total lipids were extracted by Soxhlet extraction at different growth stages, and the result showed lipids content increased with the increase of cultivation time. Lipid content of the biomass was highest (12%~13%) at the 4th days of cultivation. At the same time, we used three staining methods (Nile red, Sudan Black B and Oil Red O dye methods) for quantification the relative lipid levels in various stage strains of C. vulgaris. The results showed that there was a linear correlation between the lipids level and the light absorb value of stained microalgal cells. The content of microalgal cell lipids (y) was related to the absorbance(x) of cells and can be expressed with a regression equation of the form y =kx+b. The regression equation of Nile red method was y=0.009543x+3.087(fluorescence value at 580 nm), and the correlation coefficient (R2) was 0.9577(P＜0.001). For Sudan Black B method, the equation was y=30.06x+3.705(optical density at 645 nm), and R2 is 0.9603(P＜0.001). In Oil Red O dye manner, the regression equation was y=72.83x-27.87(optical density at 600 nm), and R2 was 0.8878(P＜0.005), but optical density at 600 nm ranged only within 0.4~0.6. These results indicated that three staining strategies were suitable for estimating the lipid content of C. vulgaris cells. The Nile red method involved incubating live cells in the dye and then reading fluorescence with a spectrophotometer, and application of this method was simple, high sensitivity, rapid but high cost. Sudan black B method had relatively complicated steps, but this strategy had lower cost and equipment. Oil Red O dye manner also could be used to test lipids content, but its most significant disadvantage is its low sensitivity and large error. The research results provide guidance for testing lipids content according to experimental conditions.
