We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.

We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc.