Fuller M E, Streger S H, Rothmel R K, Mailloux B J, Hall J A, Onstott T C, Fredrickson J K, Balkwill D L, DeFlaun M F
Envirogen, Inc., Princeton Research Center, Lawrenceville, New Jersey 08648, USA.
Appl Environ Microbiol. 2000 Oct;66(10):4486-96. doi: 10.1128/AEM.66.10.4486-4496.2000.
Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.
以往的细菌运输研究使用的荧光团已被证明会对染色细胞的生理产生不利影响。本研究旨在确定不会对细菌运输或活力产生不利影响的替代荧光染料。最初的工作是用一种地下水分离菌——食酸菌属菌株DA001进行的。首先对潜在化合物进行筛选，以确定染色效率和不良副作用。5-（和6-）-羧基荧光素二乙酸琥珀酰亚胺酯（CFDA/SE）能有效染色DA001，且不会对细胞黏附或活力产生不良影响。许多其他革兰氏阴性和革兰氏阳性细菌属的成员也能用CFDA/SE有效染色。通过落射荧光显微镜和流式细胞术测定，在人工地下水中（无生长条件下）孵育的CFDA/SE染色的食酸菌属菌株DA001细胞中，超过95%至少28天保持荧光。在地下水或饱和沉积物微观世界中，未检测到CFDA/SE染色和未染色的DA001细胞在存活率和可培养性上的差异。通过落射荧光显微镜，明亮的黄绿色细胞很容易与自发荧光的沉积物颗粒区分开来。开发了一种使用微孔板荧光分光光度法的高通量方法，其检测限为10⁵CFDA染色细胞/毫升左右；流式细胞术的检测限约为1000细胞/毫升。用完整沉积物岩心和不分裂的DA001细胞进行的实验室规模细菌运输实验结果表明，微孔板测定法测定的水样细胞浓度与其他计数方法测定的结果吻合良好。本研究表明，CFDA/SE在细菌运输实验中标记细胞非常有效，并且可能对其他微生物生态学研究也有用。
