QUANTITATIVE-ANALYSIS OF BIOFILM THICKNESS VARIABILITY

The thickness variability of biofilms of Pseudomanas aeruginosa, Klebs iella pneumoniae, and the binary population combination of these two s pecies was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and app lying image analysis to construct thickness profiles along linear tran sects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell att ached to the surface (<5 mu m) and as thick as 1000 mu m. Week-old bio films of three different species compositions displayed distinct struc tural features as indicated by their mean thicknesses and by a roughne ss coefficient. Monopopulation biofilms of P. aeruginosa (29 mu m mean thickness) or K. pneumoniae (100 mu m mean thickness) were thinner th an the binary population biofilm (400 mu m mean thickness). A roughnes s coefficient developed in this investigation corroborated the qualita tive visual characterization of P. aeruginosa biofilms as relatively u niformly thick (mean roughness coefficient 0.15), K. pneumoniae biofil ms as patchy (mean roughness coefficient 1.14), and the binary populat ion biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum c ompletely, significant areas of essentially bare substratum were appar ent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilm s may be due to the fact that this organism is nonmotile. A spatial co rrelation analysis of the thickness data indicated that thickness meas urements were still correlated even when separated by distances that e xceeded the mean biofilm thickness. Cell aggregates, some of them hund reds of microns in size, were observed in the effluent of K. pneumonia e and binary population biofilm reactors. Measurements of thickness va riability and other observations reported in this article provide a qu antitative basis for analysis of microscale structural heterogeneity o f biofilms. (C) 1995 John Wiley and Sons, Inc.