Advanced biofilm staining techniques for TEM and SEM in geomicrobiology: Implications for visualizing EPS architecture, mineral nucleation, and microfossil generation

Microbial biofilms and mats have long been studied for their role in mineral precipitation reactions in natural environments. Scanning electron microscopy (SEM) is often used to characterize biofilms and their associated precipitates, however, conventional SEM sample preparation methods do not typically preserve the structure of the extracellular polymeric substances (EPS), which account for a large portion of biofilm material and play crucial roles in biofilm function and mineral nucleation. In the present investigation, EPS preservation and visualization using transmission electron microscopy (TEM) was explored using three biofilm fixation and staining protocols. Although aspects of these protocols were developed for preserving complex eukaryotic tissue samples, the heterogeneous, three-dimensional nature of biofilms make them suitable candidates for these sample processing techniques. The results suggest that cryofixation provides the best preservation of cyanobacteria-dominated biofilm structures. A staining protocol including six different pre-embedding stains allowed for TEM visualization of the EPS matrix that encompasses biofilm cells and precipitates. Of the stains used, uranyl acetate appears to be important in avoiding biofilm deformation during sample processing. Using these staining protocols, cell-EPS-mineral relationships were observed, including the precipitation of hydromagnesite [Mg₅(CO₃)₄(OH)₂·4H₂O] on the EPS adjacent to the exterior of cyanobacteria filaments. Beachrock-associated biofilms were characterized using both TEM of ultrathin sections, as well as SEM of resin embedded osmium stained biofilms prepared as petrographic thin sections. Combining these two approaches enabled characterization of both the micrometer-scale cell-carbonate mineral contacts, as well as the larger scale microbial colony-mineral cement relationships. These results suggest that sample preparation techniques developed for rapid preservation of eukaryotic tissue samples can be used to preserve and characterize biofilm architecture. These findings have applications to understanding mineral nucleation in biofilms, and the preservation of biofilms as microfossils in the rock record.