Department of Chemical Engineering, McGill University , Montreal, Quebec H3A 2B2, Canada.
Environ Sci Technol. 2011 Oct 1;45(19):8345-51. doi: 10.1021/es201496q. Epub 2011 Sep 2.
The risk of groundwater contamination by microbial pathogens is linked to their survival in the subsurface. Although there is a large body of literature on the inactivation behavior of suspended (planktonic) microorganisms, little is known about the inactivation of bacteria when attached to sand grain surfaces in groundwater aquifers. The main goal of this study was to develop a fluorescence-based experimental technique for evaluating the extent of inactivation over time of bacteria adhered onto a surface in an aqueous environment. Key features of the developed technique are as follows: (i) attached cells do not need to be removed from the surface of interest for quantification, (ii) bacterial inactivation can be examined in real-time for prolonged time periods, and (iii) the system remains undisturbed (i.e., the aqueous environment is unchanged) during the assay. A negatively or positively charged substrate (i.e., bare or coated glass slide) was mounted in a parallel-plate flow cell, bacteria were allowed to attach onto the substrate, and the loss of bacterial membrane integrity and respiratory activity were investigated as a function of time by fluorescence microscopy using Live/Dead BacLight and BacLight RedoxSensor CTC (5-cyano-2,3-ditolyl tetrazolium chloride) viability assays. These two different measures of bacterial inactivation result in comparable trends in bacterial inactivation, confirming the validity of the experimental technique. The results of this work show that the developed technique is sensitive enough to distinguish between the inactivation kinetics of different representative bacteria attached to either a negatively charged (bare glass) surface or a positively charged (coated glass) surface. Hence, the technique can be used to characterize bacterial inactivation kinetics when attached to environmentally relevant surfaces over a broad range of groundwater chemistries.
微生物病原体通过地下水造成污染的风险与它们在地下环境中的存活有关。尽管关于悬浮（浮游）微生物失活动力学已有大量文献，但对于地下含水层中砂粒表面附着细菌的失活动力学却知之甚少。本研究的主要目的是开发一种基于荧光的实验技术，用于评估随时间推移附着在水相环境中表面上的细菌失活动力的程度。所开发技术的主要特点如下：（i）无需从感兴趣的表面去除附着细胞即可进行定量，（ii）可以实时长时间检查细菌失活动力学，以及（iii）在测定过程中系统保持未被干扰（即水相环境不变）。带负电荷或带正电荷的基底（即裸玻璃或涂覆玻璃载玻片）安装在平行板流动池中，使细菌附着在基底上，然后通过使用 Live/Dead BacLight 和 BacLight RedoxSensor CTC（5-氰基-2,3-二甲苯四唑氯化物）活力测定法通过荧光显微镜研究细菌细胞膜完整性和呼吸活性随时间的损失。这两种不同的细菌失活动力学测量方法导致细菌失活动力学的趋势相似，证实了实验技术的有效性。这项工作的结果表明，所开发的技术足够灵敏，可以区分附着在带负电荷（裸玻璃）表面或带正电荷（涂覆玻璃）表面的不同代表性细菌的失活动力学。因此，该技术可用于在广泛的地下水化学范围内表征附着在环境相关表面上的细菌失活动力学。
