Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.During recent years, atomic force microscopy (AFM) has been increasingly used in the biosciences (5, 16). Theoretically, it combines the two most important aspects of studying structure-function relationships of biological specimens: it performs high-resolution imaging with a high signal-to-noise ratio on a molecular or submolecular scale and has the ability to operate in aqueous environments, allowing the observation of dynamic molecular events in real time and under physiological conditions. The AFM is surprisingly simple in its concept. A sharp tip located at the free end of a flexible cantilever scans over a surface. Interaction forces between the tip and the sample surface subsequently cause the cantilever to deflect. The deflection signal is acquired and digitized to provide a threedimensional image of the surface.Several biological specimens have been imaged, with lateral and vertical resolution on a nanometer and a subnanometer scale, respectively (9, 14, 23). However, when living microbial cell surfaces are imaged, the softness of the cell surface together with the high pressure over the contact area between the tip and the cell can prevent high-resolution imaging. Image contrast is indeed influenced by the probe's geometry, the imaging parameters, the surface topography, and the viscoelastic and physicochemical properties of the cell surface. Additional problems arise from friction and from lateral displacement of the organism under study, which makes immobilization strategies critical.Beyond being an imaging device, the AFM has evolved as an instrument for measuring molecular interaction forces (21,22). Bio...

Transition from reversible to irreversible bacterial adhesion is a highly relevant but poorly understood step in initial biofilm formation. We hypothesize that in oral biofilm formation, irreversible adhesion is caused by bond strengthening due to specific bacterial interactions with salivary conditioning films. Here, we compared the initial adhesion of six oral bacterial strains to salivary conditioning films with their adhesion to a bovine serum albumin (BSA) coating and related their adhesion to the strengthening of the binding forces measured with bacteria-coated atomic force microscopy cantilevers. All strains adhered in higher numbers to salivary conditioning films than to BSA coatings, and specific bacterial interactions with salivary conditioning films were accompanied by stronger initial adhesion forces. Bond strengthening occurred on a time scale of several tens of seconds and was slower for actinomyces than for streptococci. Nonspecific interactions between bacteria and BSA coatings strengthened twofold faster than their specific interactions with salivary conditioning films, likely because specific interactions require a closer approach of interacting surfaces with the removal of interfacial water and a more extensive rearrangement of surface structures. After bond strengthening, bacterial adhesion forces with a salivary conditioning film remained stronger than those with BSA coatings.Oral biofilm ("dental plaque") formation proceeds according to a well-known sequence of events (7,18,19). The first step in this sequence is the adsorption of conditioning film components or, specified to the oral cavity, the adsorption of salivary components that form the acquired enamel pellicle, followed by bacterial transport to the substratum surface. Subsequently, bacteria initially (co)adhere (19) reversibly, after which a transition to an irreversible state sets in, and eventually the adhering bacteria start to grow and form a mature biofilm. The transition from reversible to irreversible adhesion is intriguing as it is largely unknown what actually happens during this transition. The transition is partly due to active bacterial processes such as anchoring through the excretion of extracellular polymeric substances (9). However, also inert polystyrene particles adhering to surfaces have demonstrated a transition from a reversible to a nearly irreversible state which has been attributed to (i) the progressive removal of interfacial water from in between the interacting surfaces, (ii) the reorientation of an adhering particle in order to face a substratum surface with its most favorable site, and (iii) conformational changes of protruding polymer chains (2,3,14). Using image sequence analysis (14, 15), it was found that the desorption probabilities of bacteria and polystyrene particles adhering to inert substrata decrease after their arrival at a surface within 30 to 60 s by a factor of 200 for bacteria and within 100 to 1,000 s by a factor of 100 for polystyrene particles.The change from a reversible to irreversi...

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In this paper, direct measurement by atomic force microscopy (AFM) of the cell surface softness of a fibrillated oral streptococcal strain Streptococcus salivarius HB and of a nonfibrillated strain S. salivarius HBC12 is presented, and the data interpretation is validated by comparison with results from independent techniques. Upon approach of the fibrillated strain in water, the AFM tip experienced a long-range repulsion force, starting at approximately 100 nm, attributed to the compression of the soft layer of fibrils present at the cell surface. In 0.1 M KCl, repulsion was only experienced when the tip was closer than approximately 10 nm, reflecting a stiffer cell surface due to collapse of the fibrillar mass. Force-distance curves indicated that the nonfibrillated strain, probed both in water and in 0.1 M KCl, was much stiffer than the fibrillated strain in water, and a repulsion force was experienced by the tip at close approach only (20 nm in water and 10 nm in 0.1 M KCl). Differences in cell surface softness were further supported by differences in cell surface morphology, the fibrillated strain imaged in water being the only specimen that showed characteristic topographical features attributable to fibrils. These results are in excellent agreement with previous indirect measurements of cell surface softness by dynamic light scattering and particulate microelectrophoresis and demonstrate the potential of AFM to directly probe the softness of microbial cell surfaces.