A comparison of conventional SEM techniques, low temperature SEM and the electroscan wet scanning electron microscope to study the structure of a biofilm of Streptococcus crista CR3

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Abstract
Biofilms of Streptococcus crista CR3 were generated on hydroxyapatite (HA) discs for 20 h in a continuous flow system with brain heart infusion broth dripped over the disc at a rate of 6 ml h-1. This study compares the conventional scanning electron microscope (SEM) preparation techniques, of critical point drying and freeze-drying, with low temperature SEM (LTSEM) and Electroscan generated images of hydrated biofilms, which preserve the integrity of hydrated polymers. Critical point drying and freeze-drying caused almost complete disappearance of the matrix of extracellular polymeric substances (EPS). Critical point drying, however, showed evenly spaced single or paired cocci remaining on the HA disc whereas freeze-drying caused the biofilm to detach from the HA leaving only patchy clumps of cells visible. By comparison LTSEM preserved the EPS better than critical point drying and freeze-drying, but holes were seen in the top and side of the biofilm and the EPS did show some shrinkage artefacts. An untreated wet biofilm viewed in the Electroscan showed an intact, hydrated, smooth matrix of EPS with cell shapes only visible indistinctly in a canopy of moist EPS. No holes were visible and no shrinkage artefacts were evident. Therefore, Electroscan imaging of the biofilm was the only method that preserved the integrity of the matrix with no apparent shrinkage artefacts.