Simul‐staining manganese oxides and microbial cells

Abstract

Manganese oxide minerals (Mn(III/IV)Ox) are ubiquitous in natural environments and interactions between Mn(III/IV)Ox and microbes play important roles in biogeochemical cycles. Current techniques for determining the spatial distribution of microbes with Mn(III/IV)Ox 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)Ox 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)Ox 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)Ox 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)Ox 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.

Challenges of Measuring Soluble Mn(III) Species in Natural Samples

“…In spite of this limitation, the LBB method remains a very valuable assay, particularly in contexts such as the study of biological Mn oxidation processes [ 20 , 21 , 22 ] or the detection of Mn oxides in unknown materials [ 23 , 33 , 37 ]. When using LBB to detect soluble species, it must be understood that, since LBB can only access a subset of Mn(III)–L complexes, an additional LBB-invisible pool of Mn(III) may exist.…”