EFFECT OF SEM PRESERVATION TECHNIQUES ON BIOFILM APPEARANCE, DISTRIBUTION, AND HETEROGENEITY

Biofilms (aggregations of bacteria and extracellular polymer secretions) within samples from the Carter Sandstone of Alabama were prepared for scanning electron microscopy using different dehydration techniques, which resulted in organic material with visibly different textures and distributions. In order to assess whether this variation was attributable to SEM preservation or to inherent biofilm heterogeneity, five preservation techniques were tested on small (1 cm) pieces of sandstone containing either a strain of bacteria cultured from and re-introduced into the rock, or an in situ biofilm grown by injection of nutrients through core samples. The techniques tested were 1) air drying alone, 2) fixation in 10% glutaraldehyde with air drying, 3) ethanol dehydration with hexamethyldisilazane (HMDS) drying [2.5% glutaraldehyde, ethanol dehydration, and HMDS]; 4) ethanol dehydration with critical point drying; and 5) ethanol and acetone dehydration with critical point drying. Unpreserved control samples were either imaged wet in an environmental scanning electron microscope (ESEM) or vacuum-dried for SEM. Bacteria were best preserved by ethanol dehydration with HMDS drying, ethanol dehydration with critical point drying, or ethanol-acetone dehydration with critical point drying. Critical point drying preserved bacteria but stripped away mucilagenous material, revealing filamentous structures within the biofilm. These filaments, along with masses of microspheres (nannobacteria) and the smooth mucilagenous outer layer of the biofilm, are present in unpreserved ESEM samples and are not dehydration artifacts. Cultured bacteria produced a biofilm that had a different surface texture and was more susceptible to preservation artifacts than the in situ biofilm. Different preservation techniques accentuated different components of the heterogeneous biofilm, thus resulting in strikingly different textures from the same sample. We conclude that use of more than one preservation technique, for example fixation in 10% glutaraldehyde with air drying and ethanol dehydration with critical point drying, on the same sample, may result in a better understanding of biofilms in lithologic samples.