DAPI荧光染色计数法的感潮河段沉积物细菌数量测量影响因素研究

Factors Affecting the DAPI Fluorescence Direct Count in the Tidal River Sediment

采集珠江广州河段沉积物样品,分析沉积物质地与有机质含量,同时分析了改良DAPI染色操作方法并讨论各因素对DAPI染色剂染色效果的影响,研究DAPI(4′,6-diamidino-2-phenylidole)荧光染色剂在具有感潮性质的沉积物样品中的染色影响因素.结果表明,沉积物稀释2 000倍,超声浴10 min,DAPI浓度为10μg.mL-1,染色时间30 min以上时,可以得到最佳细菌计数数量.细菌数量与褪色比例有良好的相关性(r=0.587,p=0.004).粒径组成与有机质含量有较强的相关性,它是通过影响有机质含量来影响褪色比例的,因此有机质也与褪色比例有一定的相关性.将褪色比例与有机质含量、细菌数量及黏粒比例做回归分析,得到与实测值相关性较好(r=0.694)的拟合值.这些结果表明,在使用DAPI荧光染色法研究感潮河段沉积物细菌数量的过程中,应充分考虑上述影响因素.

The factors affecting the DAPI（4′,6-diamidino-2-phenylidole） fluorescence direct count in the tidal river sediment were examined.Sediment samples were collected from the Guangzhou section of the Pearl River.Besides sediment texture and organic matter,an improved staining procedure and the involved parameters were analyzed.Results showed that the procedure with the sediment with 2 000 fold dilution and ultrasonic water bath for 10 min,and with a final DAPI concentration of 10 μg.mL-1 and staining time for more than 30 min produced the optimum results of DAPI direct count in the sediment.The total bacterial number was correlated to the proportion of the non-nucleoid-containing cells to the total bacterial number（r = 0.587,p = 0.004）.The organic matter content also correlated to the ration.The clay content had a strong correlation with the organic matter,through which the clay content also affected the ratio.A multiple regression analysis between the ration versus the organic matter,the total bacterial number,and the clay content showed that the regression equation fit the measure values satisfactorily（r = 0.694）.These results indicated that the above factors needed to be considered in the applications of the DAPI fluorescence direct counting method to the tidal river sediment.