The main goal of this paper is to probe mechanical properties of living and dead bacteria via atomic force microscopy (AFM) indentation experimentations. Nevertheless, the prerequisite for bioAFM study is the adhesion of the biological sample on a surface. Although AFM has now been used in microbiology for 20 years, the immobilization of micro-organisms is still challenging. Immobilizing a single cell, without the need for chemical fixation has therefore constituted our second purpose. Highly ordered arrays of single living bacteria were generated over the millimeter scale by selective adsorption of bacteria onto micrometric chemical patterns. The chemically engineered template surfaces were prepared with a microcontact printing process, and different functionalizations of the patterns by incubation were investigated. Thanks to this original immobilization strategy, the Young moduli of the same cell were measured using force spectroscopy before and after heating (45 °C, 20 min). The cells with a damaged membrane (after heating) present a Young modulus twice as high as that of healthy bacteria.