Biofilm structural heterogeneity visualized by three microscopic methods

Abstract

The structural heterogeneity of a microbial biofilm was demonstrated by scanning electron microscopy, confocal scanning laser microscopy (CSLM), and cryoembedding followed by sectioning and microscopic examination. Biofilm was composed of a binary population of Pseudomonas aeruginosa and Klebsiella pneumoniae grown in a continuous flow annular reactor. The three microscopic methods provided a consistent picture of the biofilm as a non-uniform structure characterized by variable thickness and variable local cell and polymer densities. Significant changes in these parameters occurred in the biofilm over distances of 10 μm or less. Though the biofilm was several hundred microns thick in places, areas of bare substratum were also observed on the same sample coupon. Cell-free pores and channels in the biofilm interior were evident. Specific staining of cellular nucleic acids with ethidium bromide and extracellular polymeric substances (EPS) with calcofluor showed that cell and EPS distributions did not always overlap. The ethidium bromide-stained region was contained within the larger region of calcofluor staining; thus, some cell-free areas actually were filled with EPS. CSLM and cryoembedding approaches are superior to SEM in their ability to image the biofilm interior and in their potential to provide quantitative information.