A new in-block staining method to visualize biofilms

Objectives: In the past, the visualization of the extracellular matrix of biofilms on an ultrastructural level was hampered by shrinkage artefacts. In addition, the reproducible contrasting of extracelluar polysaccharides (EPS) has not been satisfactorily solved. The aim of the present paper was to overcome these difficulties and produce artefact-free transmission electronmicroscopic images of oral biofilms. Methods: To avoid shrinkage, the classical alcohol/acetone dehydration had to be replaced. This was achieved by substituting water by a number of polar organic molecules. The second difficulty, to stain the EPS matrix, was overcome by using an osmophilic base instead of the Schiff's reagent which thereupon was reacted with OsO₄. These experiments were carried out using biofilms created in the Zurich feast/famine biofilm model. Results: Applying this method provided images of biofilms with an intact matrix in which differentially contrasted bacteria were embedded. All five members of the biofilm consortium could be distinguished. This distinction was based on shape and characteristic species-specific contrasting of microbial ultrastructures. Within the matrix, structural differences of EPS probably due to different proportions of α 1,3 and 1,6 linkages were apparent. This was evident around micro-colonies of S. sobrinus where fibrilar and S. oralis where fluffy structured EPS were regularly detected. Conclusions: The ultrastructure of biofilms prepared for transmission electron microscopy by this method allows the imaging of undistorted EPS together with a differentiated contrasting of microbial species. This is a major step forward to determine the spatial arrangement of microorganisms in biofilms on an ultrastructural level.