Properties of a novel, synthetic biofilm were examined by using confocal scanning laser microscopy (CSLM) in combination with fluorescent probes and by investigating total protein content and specific beta-galactosidase activity during various steps of the biofilm preparation. Viable, but nongrowing Escherichia coli were entrapped in 10- to 80-mu m-thick multilayer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. Cell viability and distribution within the films were evaluated by developing a protocol to stain the bacteria with fluorescein isothiocyanate and propidium iodide, thereby labeling all cells green and dead cells red, respectively. Confocal microscopy facilitated viewing samples in the XY and XZ planes, and image analysis enabled counting of the cells. These experiments showed that the initial viability of the entrapped bacteria was 85% to 90%, cell distribution was uniform in the XY plane and cell number increased with increasing depth into the film. Specific beta-galactosidase assays developed here allowed comparison of the induction of lacZ in suspended and immobilized cells, These experiments demonstrated that rehydration was an important step in biofilm preparation, and E. coli cast into synthetic biofilms with cell layers of at least 20 to 35 mu m in thickness had gene induction characteristics similar to suspended cells.