Evaluating Adherent Bacteria and Biofilm Using Electron Microscopy

Abstract

The secreted exopolysaccharide that constitutes the bacterial glycocalyx allows bacterial proliferation in a well-protected environment.^2,19 Bacterial adherence, proliferation and biofilm production on a biomedical device can have catastrophic consequences for the patient, resulting in infection or failure of the device. Therefore, laboratory studies of the pathogenesis of these latent device-related infections is critical.³ However, attempts to study the bacterial biofilm by electron microscopy employing only conventional fixation techniques suffer from inadequately preserved or stained glycocalyx. This is largely due to the characteristics of the glycocalyx as a highly hydrated, polymerized anionic matrix of variably substituted polysaccharides. Polysaccharides are not well stabilized by the conventional fixatives, namely the aldehydes, glutaraldehyde and paraformaldehyde, or osmium tetroxide. Graded dehydration with alcohol to gradually remove water from this highly hydrated structure may further distort delicate features resulting in observation of condensed or collapsed structure.^2,15,16,18 Not naturally electron-dense, the conventional means for adding contrast with the poststains uranyl acetate and lead citrate is usually not sufficient to enable exopolysaccharide constituents to appear dark or electron-dense on the phosphorescent screen of the transmission electron microscope. Too electron-translucent on its own, and unable to gain contrast from conventional poststains, the glycocalyx is often indistinguishable from the embedding resin background.¹⁶