A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms

In the past, the visualization of the extracellular matrix of biofilms on an ultrastructural level was hampered by shrinkage artifacts. In addition, the reproducible contrasting of extracellular polysaccharides (EPS) has not satisfactorily been solved. Here we describe a method overcoming these difficulties, which produces artifact-free transmission electron microscopic (TEM) images using multispecies biofilms grown in vitro. Sufficient contrast was achieved by replacing Schiff's reagent with the osmiophilic amino acid methionine. In addition, shrinkage was avoided by replacing the classical dehydration agents with ethylene glycol and 1,2-pentanediol. Applying this method provided images of biofilms with an intact matrix in which differentially contrasted bacteria were embedded. All six members of the biofilm consortium (Streptococcus sobrinus, Streptococcus oralis, Veillonella dispar, Fusobacterium nucleatum, Actinomyces naeslundii, and Candida albicans) could be distinguished. Within the matrix, structural differences of EPS, probably due to different proportions of alpha-1,3 and alpha-1,6 linkages, were apparent. Fibrilar polysaccharides were evident around microcolonies of S. sobrinus, and fluffy polysaccharides were detected in the vicinity of S. oralis microcolonies. The ultrastructure of biofilms prepared for TEM using this method allows the imaging of undistorted EPS as well as the differentiated contrasting of the six microbial species of the in vitro biofilm model. This is a major step forward in determining the spatial arrangement of microorganisms in biofilms on an ultrastructural level.