Small colonies of P. aeruginosa cells are dispersed homogeneously and thinly throughout the biofilm matrix on Etafilcon A after 72 h growth. The non-confocal transmitted light micrograph and the acridine orange stained micrograph are x-y projections of a slice of the z-stack (z = 12 μm) of the biofilm matrix. Bacterial cells were stained with the dye acridine orange to observe the total amount of bacterial cells (viable and dead). The three-dimensional representation of the biofilm stained with acridine orange illustrates the distribution of the bacterial cells throughout the biofilm matrix and the thickness of the biofilm matrix (~ 30 μm). Furthermore, the fluorescent dye acridine orange intercalates not only into nucleic acids but also into the contact lens hydrogel polymer matrix.