Banning N, Toze S, Mee B J
CSIRO Land and Water, Floreat, Western Australia, Nedlands, Western Australia.
J Appl Microbiol. 2002;93(1):69-76. doi: 10.1046/j.1365-2672.2002.01670.x.
The aim of this study was to deterimine the survival of an enteric bacterium in anaerobic groundwater and effluent microcosms using the green fluorescent protein (GFP) marker gene in combination with the viability indicator propidium iodide (PI).
The pEGFP vector (Clontech) was transformed into Escherichia coli DH5alpha and was stable for at least 100 generations of growth in nonselective medium at 28 degrees C and 37 degrees C. Using an epifluorescent microscope, GFP cells could be detected under blue light (450-490 nm) and the numbers of PI-positive GFPs could be detected under green light (530-560 nm). GFP-tagged E. coli could be detected for at least 132 d in sterilized water microcosms. GFP fluorescence was not lost from the culturable cell population for the duration of the experiment. However, a slow decline in the number of GFP-fluorescent cells in sterilized groundwater was observed. Escherichia coli die-off and loss of green fluorescence was more rapid in nonsterilized waters than in sterilized. Viable numbers of the GFP-tagged E. coli determined by PI counterstaining were compatible with numbers of colony-forming units.
The long-term survival of E. coli and maintainance of GFP-conferred fluorescence in these cells was demonstrated in both groundwater and effluent, under sterilized conditions. However, severe starvation and/or the presence of indigenous microorganisms were found to be factors affecting the maintenance of fluorescence in dead or dying cells.
This study demonstrates the successful application of PI with GFP-tagging to monitor long-term bacterial survival in nutrient-limited conditions and mixed microbial populations.
本研究的目的是利用绿色荧光蛋白（GFP）标记基因结合活力指示剂碘化丙啶（PI），确定一种肠道细菌在厌氧地下水和废水微观世界中的存活情况。
将pEGFP载体（Clontech公司）转化到大肠杆菌DH5α中，在28℃和37℃的非选择性培养基中至少传代100次仍保持稳定。使用落射荧光显微镜，在蓝光（450 - 490nm）下可检测到GFP细胞，在绿光（530 - 560nm）下可检测到PI阳性的GFP数量。在灭菌水微观世界中，GFP标记的大肠杆菌至少可检测到132天。在实验期间，可培养细胞群体中的GFP荧光没有消失。然而，在灭菌地下水中观察到GFP荧光细胞数量缓慢下降。在未灭菌的水中，大肠杆菌的死亡和绿色荧光的丧失比在灭菌水中更快。通过PI复染确定的GFP标记大肠杆菌的活菌数与菌落形成单位数相符。
在灭菌条件下，在地下水和废水中均证明了大肠杆菌的长期存活以及这些细胞中GFP赋予的荧光的维持。然而，发现严重饥饿和/或本地微生物的存在是影响死亡或濒死细胞中荧光维持的因素。
本研究证明了PI与GFP标记相结合在监测营养受限条件和混合微生物群体中细菌长期存活方面的成功应用。
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