COMPARISON OF ORGANIC MATTER PRESERVATION TECHNIQUES FOR SEM STUDY OF GEOLOGIC SAMPLES

Rock samples have been inoculated with bacteria, fed, then prepared for SEM analysis using several widely accepted methods purported to preserve organic textures. The protocols tested are: A) Air dry; B) Gluteraldehyde fixation (Fix sample in 10% gluteraldehyde for 2 hrs); C) Gluteraldehyde fixation and ethanol dehydration (Step 1. Fix specimens in 2.5% gluteraldehyde for 2 hrs; Step 2. Dehydrate in ethanol, 10-15 min per step: 35%, 50%, 70%, and 95% ethanol; Step 3. Continue dehydration: 100% ethanol (4 changes) for one hr; Step 4. Change to HMDS (hexamethyldisilazane), 2 changes, 10 min each; Step 5. Air dry sample.); D) Gluteraldehyde fixation, ethanol dehydration, and critical-point drying (Steps 1-3 from C; Step 4. Five CO₂ flushes in critical-point drier); E) Gluteraldehyde fixation, ethanol dehydration, acetone dehydration, and critical-point drying (Steps 1-3 from C; Step 4. Dehydrate in acetone, 10-15 min per. step: 35%, 50%, 70%, and 95% acetone; Step 5. Five CO₂ flushes in critical-point drier).

No tested protocol correctly maintains the in situ textures of both the bacteria and the biofilms. Biofilm slime occurs as a more or less smooth continuous coating over both rock and bacteria in air-dried or gluteraldehyde fixed samples (procedures A and B). Slight to moderate curling of the edges of the biofilm is sometimes observed, especially in air-dried samples. In these samples, it is uncommon to observe bacteria completely uncovered by biofilm. Individual or colonies of bacteria most commonly occur as lumps and bumps beneath the slime or as partially emergent bodies. The bacterial bodies themselves are deformed to varying degree in both procedures.

Bacterial shape is well preserved in the dehydration and drying procedures (C, D, and E), however, these techniques greatly change the morphology of the biofilm from a continuous coat to curled, desiccated, and tattered remnant sheets and strings. Tangled nests of vermicelli-shaped bacteria are commonly observed and are frequently completely devoid of slime. Biofilm fragments also occur as small (<100 nm) spheres, ovoids, and beaded chains. Similar small features have been interpreted as nannobacteria or ultramicrobacteria in terrestrial and extraterrestrial geologic samples.