Bacterial and Mineral Elements in an Arctic Biofilm: A Correlative Study Using Fluorescence and Electron Microscopy

Abstract

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

Epifluorescence, SEM, TEM and nanoSIMS image analysis of the cold phenotype ofClostridium psychrophilumat subzero temperatures

“…Only a few psychrophilic organisms, for example, Psychromonas arctica (Groudieva et al ., ), Psychrobacter arcticus (Hinsa‐Leasure et al ., ) and Thiomicrospira species (Niederberger et al ., ) have been described so far as capable of producing biofilms at low temperatures (4 °C). In frozen environments, ‘biofilms’ may help bacterial cells to attach onto the surface of soil minerals and organic particles and gain access to the surrounding thin film of liquid water (Clarke et al ., ; Hinsa‐Leasure et al ., ). Moreover, biofilms are particularly important in perennially cold habitats to facilitate lateral gene transfer as such to compensate for the very low rates of generation time of the indigenous organisms as demonstrated to occur for the haloarchaea populating an isolated Antarctic lake (DeMaere et al ., ).…”

Analysis of engineered nanomaterials in complex matrices (environment and biota): General considerations and conceptual case studies

“…The aggregation state of nanomaterials in association with cells is, for example, resolved by ESEM but also by cryogenic SEM [41]. Environmental scanning electron microscopy, EDS, and fluorescence microscopy have been used together to resolve CdSe QDs and their integrity, in part from Cd and Se atomic ratios by EDS but also by QD fluorescence [42]. Both ESEM-EDS and fluorescence microscopy, like confocal scanning laser microscopy, are relatively easy methods with potentially little preparation and thus minimal sample disturbance.…”

“…Biological samples, such as biofilms, may be imaged intact [42] or subsampled and then subsamples analyzed, using a variety of techniques. For example, after a subsample is homogenized, one may use approaches that avoid inducing nanoparticle dissolution during sample handling, then divided again for dry mass and ICP-MS analyses.…”

“…1, pp. [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]2012 # 2011 SETAC Printed in the USA DOI: 10.1002/etc.723 This paper evolved from discussions held at a SETAC-endorsed Technical Workshop held at Clemson University in August, 2010. The workshop was sponsored by the United States Environmental Protection Agency (U.S. EPA), Arcadis-US, and the Clemson University Institute of Environmental Toxicology.…”

Mineralogy of saline perennial cold springs on Axel Heiberg Island, Nunavut, Canada and implications for spring deposits on Mars

“…Elemental sulfur is found in the sediments inside the vent at WS and in sediments downstream from the WS and CP outlets, sometimes forming nodules, forming channels at GH, and in almost all cases is in association with gypsum. Elemental sulfur is associated with sulfur oxidizing bacteria in ''streamer'' biofilms (Clarke et al, 2010), but the mechanism is not yet understood. It is possible that H 2 S oxidizing bacteria deposit S°inside the cell as an energy reserve (Madigan and Martinko, 2006), in case the system becomes depleted in H 2 S (e.g., in the summer, when the snow cover has melted and no longer ''locks Typically present only in trace amounts, spread throughout all three sites in'' H 2 S), and S°is needed as an electron donor for oxidation to sulfate.…”