MEASUREMENT OF LOCAL DIFFUSION-COEFFICIENTS IN BIOFILMS BY MICROINJECTION AND CONFOCAL MICROSCOPY

A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluores cent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluores cein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) wer e measured in the cell clusters and interstitial voids of a heterogene ous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capa city decreased and the diffusion coefficient could be measured. The di ffusivity of phycoerythrin in cell clusters was impeded by 41%, compar ed to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteri stics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. ( C) 1997 John Wiley & Sons, Inc.