Manganese oxide minerals (Mn(III/IV)O_x) are ubiquitous in natural environments and interactions between Mn(III/IV)O_x and microbes play important roles in biogeochemical cycles. Current techniques for determining the spatial distribution of microbes with Mn(III/IV)O_x include electron microscopy and synchrotron radiation analyses. However, these techniques may not be readily available in most laboratories or may be cost prohibitive. Here we present a rapid, cost‐effective “simul‐staining” method for imaging particulate Mn(III/IV)O_x and cells on the same filter using epifluorescence microscopy with differential interference contrast (DIC) capability as a prescreening tool before higher resolution and/or more time‐intensive analyses. This method uses leucoberbelin blue (LBB) dye, which turns blue when oxidized by particulate Mn(III/IV)O_x on filters, and the fluorescent nucleic acid stain, SYBR Green, which fluoresces when bound to nucleic acids. First, the DIC configuration is used to locate blue “haloes” of oxidized LBB around Mn(III/IV)O_x particles. Second, a filter set that excites dyes with blue light and emits green fluorescence is used to image SYBR Green bound to nucleic acids in cells. Third, ImageJ is used for image analysis to associate Mn(III/IV)O_x particles and microbes. We demonstrate that this simul‐staining is suitable for laboratory cultures of manganese‐oxidizing bacteria as well as environmental samples from a marine oxycline.

中文翻译：

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同时染色的锰氧化物和微生物细胞

氧化锰矿物（Mn（III / IV）O _x）在自然环境中无处不在，并且Mn（III / IV）O _x与微生物之间的相互作用在生物地球化学循环中起着重要作用。用于确定具有Mn（III / IV）O _x的微生物的空间分布的当前技术包括电子显微镜和同步加速器辐射分析。但是，这些技术在大多数实验室中可能不容易获得，或者可能过于昂贵。在这里，我们提出了一种快速，经济高效的“模拟染色”方法来对颗粒Mn（III / IV）O _x进行成像在具有更高的分辨率和/或更耗时的分析之前，使用具有微分干涉对比（DIC）功能的落射荧光显微镜将细胞和同一滤膜上的细胞用作预筛选工具。该方法使用的是白带蓝色（LBB）染料，当在过滤器上被颗粒Mn（III / IV）O _x氧化时会变成蓝色，而荧光核酸染料SYBR Green会在与核酸结合时发出荧光。首先，DIC配置用于在Mn（III / IV）O _x颗粒周围定位氧化的LBB的蓝色“晕圈” 。其次，使用激发蓝光染料并发出绿色荧光的滤光片组对与细胞核酸结合的SYBR Green成像。第三，ImageJ用于图像分析以关联Mn（III / IV）O _x颗粒和微生物。我们证明该模拟染色适用于锰氧化细菌的实验室培养以及来自海洋奥克西林的环境样品。