BIOFILM STRUCTURAL HETEROGENEITY VISUALIZED BY 3 MICROSCOPIC METHODS

The structural heterogeneity of a microbial biofilm was demonstrated b y scanning electron microscopy, confocal scanning laser microscopy (CS LM), and cryoembedding followed by sectioning and microscopic examinat ion. Biofilm was composed of a binary population of Pseudomonas nerugi nosa and Klebsiella pneumoniae grown in a continuous flow annular reac tor. The three microscopic methods provided a consistent picture of th e biofilm as a non-uniform structure characterized by variable thickne ss and variable local cell and polymer densities. Significant changes in these parameters occurred in the biofilm over distances of 10 mu m or less. Though the biofilm was several hundred microns thick in place s, areas of bare substratum were also observed on the same sample coup on. 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 bromid e-stained region was contained within the larger region of calcofluor staining; thus, some cell-free areas actually were filled with EPS. CS LM and cryoembedding approaches are superior to SEM in their ability t o image the biofilm interior and in their potential to provide quantit ative information.