Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy

Abstract

Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment.Electron microscopy (EM)-based imaging and analyses provide excellent high-resolution tools for studying the structural and compositional features of microorganisms and their immediate surroundings. However, the conventional sample processing that includes dehydration as a prerequisite for imaging by high-resolution vacuum instruments can cause substantial changes in microbial cell ultrastructure. Chemical fixation with aldehydes and treatment with heavy metals help to preserve cell morphology and enhance contrast but can also result in substantial shrinkage (41). Dehydration with organic solvents can extract cell constituents, cause cell membrane discontinuities (23), induce distortion of delicate structures such as membrane-associated components (4), and have other deleterious effects on morphology that can consequently lead to inaccurate interpretations of cellular features.

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime

“…Therefore, the volume of the periplasm of a single cell was calculated to later estimate the average concentration of c-type cytochromes. The average length and width of S. oneidensis cells were measured using environmental scanning electron microscopy images of logarithmically growing ferric iron-reducing cells, and the width of the periplasm was obtained from the work of Dohnalkova et al (2011). Using equation 2, these values led to an average periplasmic volume of 0.153 fl per cell (Supplementary Figure S1).…”

Mineralogical Diversity in Lake Pavin: Connections with Water Column Chemistry and Biomineralization Processes

“…(some species being known to form electrically-conductive nanowires under some conditions), were not detected. Alternatively, these filaments could also result from the collapse of extracellular organic polymers during sample preparation [80,81]. Indeed, previous STXM analyses at the C K-edge of samples collected in Lake Pavin at similar depths revealed the presence of extracellular polymeric substances surrounding bacteria and nucleating Fe phosphates [20].…”

Membrane Vesicles as a Novel Strategy for Shedding Encrusted Cell Surfaces

“…This finding is probably due to the use of cryo-TEM, which allows fixation by flash freezing, preserving the samples intact in a frozen hydrated state. It is well established that cryo fixation is the most effective sample preparation to avoid artifacts originating from chemical fixation and dehydration [45]. We also avoided centrifugation, which has been suggested to artificially generate MVs.…”