尼罗红-荧光光谱法测定布朗葡萄藻油脂含量的方法研究

Determination of lipid content in Botryococcus braunii by Nile red-fluorescence spectrometry

布朗葡萄藻因胞外分泌物多、细胞壁厚、聚集生长等原因,不能用传统的尼罗红染色方法进行油脂含量测定。通过探索染色前处理和染色条件,优化了布朗葡萄藻的染色方法:藻液摇匀后超声波处理3 min(超声波参数为温度20℃、发射功率100 W、频率40 k Hz),3 000×g离心去除培养基,用体积分数8%的DMSO重悬细胞,调整悬液OD750到0.16,每1 m L样品加入10#L 120μg/m L的尼罗红丙酮溶液(尼罗红质量浓度1.2#g/m L),40℃水浴锅中染色10 min,荧光激发光波长520nm。改进的方法促进了尼罗红与布朗葡萄藻的结合,提高了测定的稳定性和准确度。

The traditional Nile red fluorescence dyeing method could not be applied to determine the lipid content in Botryococcus braunii because of its extracellular excretion,thick cell wall and dense cell colonies. By studying the pretreatment and staining conditions,the detection method was optimized as follows： treating algal cells by ultrasound for 3 min at 20 ℃,100 W and 40 k Hz,centrifugation at 3 000 ×g to remove the culture medium,suspending the cells with 8% dimethylsulfoxide to OD7501. 6,every 1m L sample added by 10 μL Nile red acetone solution of 120 μg / m L（ mass concentration of Nile red 1. 2μg /m L）,staining at 40 ℃ for 10 min. Then the sample was measured by fluorescence spectrometer excited at 520 nm. The interaction between Nile red and microalgal cells,and the determination stability and accuracy were enhanced after optimization.