Environmental Biotechnology Institute, Environmental Science Program, University of Idaho, Moscow, ID 83844, USA.
J Microbiol Methods. 2010 Apr;81(1):26-32. doi: 10.1016/j.mimet.2010.01.019. Epub 2010 Jan 28.
Low concentrations (microg/L) of the perchlorate anion, ClO(4)(-), have been measured in surface and ground water supplies in many locations throughout the United States. Perchlorate is known to affect the function of the thyroid gland in mammals and its toxicity primarily results from its inhibition of thyroid hormone output. The major sources of perchlorate contamination in surface and ground waters are defense contractors, military installations, propellant manufacturers and agriculture. The currently accepted method of perchlorate analysis, recommended by the US EPA, is neither fast nor easy to use and requires purchase of an expensive high performance ion chromatograph (IC). The novel method described here uses dye resazurin to measure perchlorate reduction by bacterial cultures and bacterial consortia in a high-throughput, multi-well, culture plate format. The method is based on the observation that perchlorate reduction and the decrease of resazurin fluorescence occur simultaneously in perchlorate degrading cultures. The bioassays were performed in anaerobic serum bottles or 96-well plates with constant shaking, using a minimal ATCC medium with 10 mM acetate as electron donor/carbon source and 200 ppm perchlorate as an electron acceptor. Fluorescence measurements with excitation at 570 nm and emission at 590 nm were taken in 20 min intervals. Changes in perchlorate concentration were confirmed using IC. Based on the experimental data, a simple model showing the correlation between perchlorate concentration in microbial culture and resazurin fluorescence level was proposed. Other dyes including redox indicators, reactive azo dyes and electron shuttle chemicals were also tested for comparison and were found less useful.
在美国许多地方的地表水和地下水中都检测到低浓度（微克/升）的高氯酸盐阴离子（ClO4-）。高氯酸盐已知会影响哺乳动物甲状腺的功能，其毒性主要来自对甲状腺激素分泌的抑制。地表水和地下水中高氯酸盐污染的主要来源是国防承包商、军事设施、推进剂制造商和农业。美国环保署推荐的目前用于高氯酸盐分析的方法既不快也不容易使用，并且需要购买昂贵的高性能离子色谱仪（IC）。本文描述的新方法使用染料 Resazurin 来测量细菌培养物和细菌群落在高通量、多孔、培养板格式中对高氯酸盐的还原。该方法基于这样的观察结果，即在高氯酸盐降解培养物中，高氯酸盐还原和 Resazurin 荧光的减少同时发生。生物测定在厌氧血清瓶或 96 孔板中进行，使用最小 ATCC 培养基，其中 10 mM 醋酸盐作为电子供体/碳源，200 ppm 高氯酸盐作为电子受体。使用 570nm 激发和 590nm 发射进行荧光测量，间隔 20 分钟进行一次。使用 IC 确认高氯酸盐浓度的变化。根据实验数据，提出了一个简单的模型，该模型显示了微生物培养物中的高氯酸盐浓度与 Resazurin 荧光水平之间的相关性。还测试了其他染料，包括氧化还原指示剂、反应性偶氮染料和电子穿梭化学品，用于比较，发现它们不太有用。
