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Simul‐staining manganese oxides and microbial cells
Author(s) -
Cavazos Amanda R.,
Glass Jennifer B.
Publication year - 2020
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.1002/lom3.10362
Subject(s) - manganese , nucleic acid , fluorescence , staining , fluorescence microscope , chemistry , microscopy , oxidizing agent , nuclear chemistry , analytical chemistry (journal) , environmental chemistry , biology , optics , biochemistry , physics , organic chemistry , genetics
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.