This article presents a study on the influence of the protocol used for immobilization of bacterial cells onto surfaces by mechanically trapping them into a filter. In this sense, the surface and structure of trapped cells are analyzed. Bacteria can be present solely or with extracellular polymeric substances (EPS). To test the behavior of the EPS layer duing the filtering process, different strains of a well-known EPS-producer bacteria (Staphylococcus epidermidis), which produce an extracellular matrix clearly visible in AFM images, have been used. Results show that this immobilization method can cause severe structural and mechanical deformation to the cell membrane. This altered mechanical state may possibly influence the parameters derived from AFM force curves (which are micro/nano-mechanical tests). Also, our results suggest that the EPS layer might move during the filtering process and could accumulate at the upper part of the cell, thus favoring distorted data of adhesion/pull-off forces as measured by an AFM tip, especially in the case of submicron-sized microbial cells such as bacteria.