荧光示踪技术对土壤中诱变菌的生长研究

Investigation of growth in a UV-mutant species in soil using fluorescent tracing technique

采用荧光染料DAPI(4',6-Diamidino-2-Phenylindole)对外源微生物Bacillus subtilis 38(B38)的DNA分子进行标记,利用荧光示踪技术对外源微生物的生长繁殖进行有效监测.通过筛选DAPI工作液的使用浓度与菌悬液的稀释比例,得出采用0.1μg/mL浓度的DAPI对稀释10~6倍的菌悬液进行染色,荧光信号强度适宜,菌体分散度良好,可实现准确计数.采用上述染色条件,对60d内土壤B38菌体数量的变化进行阶段性监测.结果发现,接种后菌体浓度显著增加,平衡后菌体浓度达到(1.05±0.01)×10^(10)cells/mL,与初期施加量相比,增长量达到3个数量级.以上结果表明,采用DAPI染料对外源微生物进行荧光示踪计数,为监测外源微生物的生长繁殖状态提供了一种简便、有效的方法.

The DNA molecular of the exotic microorganism Bacillus subtilis38(B38,a mutant species of Bacillus subtilis)was used labelled by a fluorescent dye,DAPI(4’,6-Diamidino-2-Phenylindole),to monitor the microorganism growthduring the process of soil remediation by bioaugmentation technique.By optimizing the DAPI concentration and thedilution ratio of the bacterial suspension,an ideal DAPI concentrations of0.1μg/mL and a bacterial suspension dilutionfold of106were applied,and the appropriate fluorescence intensity and cell dispersity for the accurate fluorescent dotcounting were obtained.The quantitative changes of B38in60days soil remediation was monitored under the optimizedconditions.The results showed that the numbers of B38in soil increased obviously,and then reached an equilibriumconcentration of(1.05±0.01)×1010cells/mL.The equilibrium concentration was as high as3orders of magnitude of theinitial dosage.Thus,DAPI labelled fluorescent tracing technique was a convenient and effective approach to monitor thegrowth and reproduction of exotic microorganism.