Bacteria attachment to surfaces--AFM force spectroscopy and physicochemical analyses

Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterial-surface interactions. Biofilm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterial-metal adhesion force was quantified at different surface delay time from 0 to 60s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53±1.40 nN and 7.88±0.94 nN) when compared to Gram-positive bacteria (1.44±0.21 nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and surface charges than B. subtilis along with the capability of producing extracellular polymeric substances (EPS). The higher hydrophobicity, surface charges, and greater propensity to form EPS by M. timonae and P. aeruginosa led to high adhesive force on the metal surface.     

Quantitative changes in the elasticity and adhesive properties of Escherichia coli ZK1056 prey cells during predation by bdellovibrio bacteriovorus 109J

Atomic force microscopy (AFM) was used to explore the changes that occur in Escherichia coli ZK1056 prey cells while they are being consumed by the bacterial predator Bdellovibrio bacteriovorus 109J. Invaded prey cells, called bdelloplasts, undergo substantial chemical and physical changes that can be directly probed by AFM. In this work, we probe the elasticity and adhesive properties of uninvaded prey cells and bdelloplasts in a completely native state in dilute aqueous buffer without chemical fixation. Under these conditions, the rounded bdelloplasts were shown to be shorter than uninvaded prey cells. More interestingly, the extension portions of force curves taken on both kinds of cells clearly demonstrate that bdelloplasts are softer than uninvaded prey cells, reflecting a decrease in bdelloplast elasticity after invasion by Bdellovibrio predators. On average, the spring constant of uninvaded E. coli cells (0.23 +/- 0.02 N/m) was 3 times stiffer than that of the bdelloplast (0.064 +/- 0.001 N/m) when measured in a HEPES-metals buffer. The retraction portions of the force curves indicate that compared to uninvaded E. coli cells bdelloplasts adhere to the AFM tip with much larger pull-off forces but over comparable retraction distances. The strength of these adhesion forces decreases with increasing ionic strength, indicating that there is an electrostatic component to the adhesion events.     

Analysis of bacterial adhesion using a gradient force analysis method and colloid probe atomic force microscopy

The atomic force microscope (AFM) has been used to examine the stickiness of bacteria on the basis of the analysis of approach and retraction force curves between the AFM tip and the bacterial surface. One difficulty in analyzing approach curve data is that the distance between the AFM tip and the surface of the bacterium is difficult to define. The exact distances are difficult to determine because the surface of the bacterium deforms during force imaging, producing a highly nonlinear region in the approach curve. In this study, AFM approach and retraction curves were obtained using a colloid probe AFM for three strains of Escherichia coli (D21, D21f2, and JM109). These strains differed in their relative adhesion to glass surfaces, on the basis of measurements of sticking coefficients in packed bed flow through column tests. A gradient force curve analysis method was developed to model the interactions between the colloid probe and a surface. Gradient analysis of the approach curve revealed four different regions of colloid-surface interactions during the approach and contact of the probe with the bacterial surface: a noninteraction region, a noncontact phase, a contact phase, and a constant compliance region. The noncontact phase, which ranged from 28 to 59 nm for the three bacterial strains, was hypothesized to arise primarily from steric repulsion of the colloid by extracellular polymers on the bacterial surface. The contact phase, spanning 59-113 nm, was believed to arise from the initial pressure of the colloid on the outer membrane of the cell. The constant compliance region likely reflected the response of the colloid probe to the stiff peptidoglycan layer that confers strength and rigidity to gram negative bacteria. It was shown that the sticking coefficients reported for the three E. coli strains were correlated with the length of the noncontact phase but not the properties of the other phases. Sticking coefficients were also not correlated with any parameters determined from retraction force curves such as pull-off distances or separation energies. These results show that gradient analysis is useful for studying the contribution of the length of the exopolymers on the cell surface to bacterial adhesion to glass surfaces.     

Probing surface adhesion forces of Enterococcus faecalis to medical-grade polymers using atomic force microscopy

The aim of this study was to compare the initial adhesion forces of the uropathogen Enterococcus faecalis with the medical-grade polymers polyurethane (PU), polyamide (PA), and poly(tetrafluoroethylene) (PTFE). To quantify the cell-substrate adhesion forces, a method was developed using atomic force microscopy (AFM) in liquid that allows for the detachment of individual live cells from a polymeric surface through the application of increasing force using unmodified cantilever tips. Results show that the lateral force required to detach E. faecalis cells from a substrate differed depending on the nature of the polymeric surface: a force of 19 +/- 4 nN was required to detach cells from PU, 6 +/- 4 nN from PA, and 0.7 +/- 0.3 nN from PTFE. Among the unfluorinated polymers (PU and PA), surface wettability was inversely proportional to the strength of adhesion. AFM images also demonstrated qualitative differences in bacterial adhesion; PU was covered by clusters of cells with few cell singlets present, whereas PA was predominantly covered by individual cells. Moreover, extracellular material could be observed on some clusters of PU-adhered cells as well as in the adjacent region surrounding cells adhered on PA. E. faecalis adhesion to the fluorinated polymer (PTFE) showed different characteristics; only a few individual cells were found, and bacteria were easily damaged, and thus detached, by the tip. This work demonstrates the utility of AFM for measurement of cell-substrate lateral adhesion forces and the contribution these forces make toward understanding the initial stages of bacterial adhesion. Further, it suggests that initial adhesion can be controlled, through appropriate biomaterial design, to prevent subsequent formation of aggregates and biofilms.     

Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength

The S-layer present at the outermost cell surface of some lactobacillus species is known to convey hydrophobicity to the lactobacillus cell surface. Yet, it is commonly found that adhesion of lactobacilli to solid substrata does not proceed according to expectations based on cell surface hydrophobicity. In this paper, the role of cell surface hydrophobicity of two lactobacillus strains with and without a surface layer protein (SLP) layer has been investigated with regard to their adhesion to hydrophobically or hydrophilically functionalized glass surfaces under well-defined flow conditions and in low and high ionic strength suspensions. Similarly, the interaction of the lactobacilli with similarly functionalized atomic force microscope (AFM) tips was measured. In a low ionic strength suspension, both lactobacillus strains show higher initial deposition rates to hydrophobic glass than to hydrophilic glass, whereas in a high ionic strength suspension no clear influence of cell surface hydrophobicity on adhesion is observed. Independent of ionic strength, however, AFM detects stronger interaction forces when both bacteria and tip are hydrophobic or hydrophilic than when bacteria and tip have opposite hydrophobicities. This suggest that the interaction develops in a different way when a bacterium is forced into contact with the tip surface, like in AFM, as compared with contacts developing between a cell surface and a macroscopic substratum under flow. In addition, the distance dependence of the total Gibbs energy of interaction could only be qualitatively correlated with bacterial deposition and desorption in the parallel plate flow chamber.     

The effect of solvent polarity on the molecular surface properties and adhesion of Escherichia coli

The elasticity and molecular surface characteristics of Escherichia coli JM109 were investigated via atomic force microscopy (AFM) in solvents expressing different polarities. The nature of bacterial adhesion and surface characteristics was probed in formamide, water, and methanol, with dielectric constants of 111, 80, and 33, respectively. Solvent polarity affected the elasticity of the bacterium, the conformation of the cell surface biopolymers, the height of the surface biopolymers, and measured adhesion forces between the bacterium and silicon nitride. By applying the Hertz model to force-indentation data, we determined that the Young's modulus was greatest in the least polar solvent, with values of 182 +/- 34.6, 12.8 +/- 0.1, and 0.8 +/- 0.3 MPa in methanol, water, and formamide, respectively. The thickness of the biopolymer brush layer on the bacterial surface was quantified using a steric model, and these values increased as polarity increased, with values of 27, 93, and 257 nm in methanol, water, and formamide, respectively. The latter results suggest that highly polar conditions favor extension of the biopolymer brush layer. Cross-sectional analysis performed on tapping mode images of the bacterial cells in methanol, water, and formamide further supported this hypothesis. The image height values are larger, since the image analysis measures the height of the bacterium and the polymer layer, but the trend with respect to solvent polarity was the same as was obtained from the steric model of the brush length. Measured adhesion forces scaled inversely with solvent polarity, with greatest adhesion observed in the least polar solvent, methanol. The combined conformational changes to the bacterial surface and biopolymer layer result in different presentations of macromolecules to a substrate surface, and therefore affect the adhesion forces between the bacterial molecules and the substrate. These results suggest that polarity of the solvent environment can be manipulated as a design parameter to control or modify the bacterial adhesion process.     

Microscopic origin of the humidity dependence of the adhesion force in atomic force microscopy

Water condenses between an atomic force microscope (AFM) tip and a surface to form a nanoscale bridge that produces a significant adhesion force on the tip. As humidity increases, the water bridge always becomes wider but the adhesion force sometimes decreases. The authors show that the humidity dependence of the adhesion force is intimately related to the structural properties of the underlying water bridge. A wide bridge whose width does not vary much with tip-surface distance can increase its volume as distance is increased. In this case, the adhesion force decreases as humidity rises. Narrow bridges whose width decreases rapidly with increasing tip-surface distance give the opposite result. This connection between humidity dependence of the adhesion force and the structural susceptibility of the water bridge is illustrated by performing Monte Carlo simulations for AFM tips with various hydrophilicities.     

Atomic force microscopy investigation of phage infection of bacteria

Atomic force microscopy (AFM) was used to study the process of infection of bacterial cells by bacteriophages, for which purpose experimental protocols were elaborated. Three types of bacteriophages were characterized with AFM and transmission electron microscopy (TEM). Bacteriophage interaction with cells was studied for three bacterial hosts: Gram-negative Escherichia coli 057 and Salmonella enteritidis 89 and Gram-positive Bacillus thuringiensis 393. Depending on the phase of lytic cycle, different cell surface changes are observed in AFM images of infected cells in comparison with intact cells: from phage adsorption on the cells and flagella to complete lysis of the cells, accompanied by the release of a large number of newly formed phages. Control experiments (cells without phages and cells with nonspecific phages) did not reveal any surface changes. Penetration of phages inside obligate aerobe Bacillus thuringiensis was shown to be oxygen-dependent and required aeration in laboratory conditions. Our results show great potential of using AFM for numerous fundamental and applied tasks connected with pathogen-host interaction.     

Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.     

Measurement of polyamide and polystyrene adhesion with coated-tip atomic force microscopy

This work presents atomic force microscopy (AFM) measurements of adhesion forces between polyamides, polystyrene and AFM tips coated with the same materials. The polymers employed were polyamide 6 (PA6), PA66, PA12 and polystyrene (PS). All adhesion forces between the various unmodified or modified AFM tips and the polymer surfaces were in the range -1.5 to -8 nN. The weakest force was observed for an unmodified AFM tip with a PS surface and the strongest was between a PS-coated tip and PS surface. The results point to both the benefits and drawbacks of coated-tip AFM force-distance measurements. Adhesion forces between the two most dissimilar (PA6-PS and PA66-PS) materials were significantly asymmetric, e.g., the forces were different depending on the relative placement of each polymer on the AFM tip or substrate. Materials with similar chemistry and intermolecular interactions yielded forces in close agreement regardless of placement on tip or substrate. Using experimental forces, we calculated the contact radii via four models: Derjaguin, Muller, and Toporov; Johnson, Kendall, and Roberts; parametric tip-force-distance relation; and a square pyramid-flat surface (SPFS) model developed herein. The SPFS model gave the most reasonable contact tip radius estimate. Hamaker constants calculated from the SPFS model using this radius agreed in both magnitude and trends with experiment and Lifshitz theory.     

AFM probing in aqueous environment of Staphylococcus epidermidis cells naturally immobilised on glass: physico-chemistry behind the successful immobilisation

AFM probing of microbial cells in liquid environments usually requires them to be physically or chemically attached to a solid surface. The fixation mechanisms may influence the nanomechanical characterization done by force curve mapping using an AFM. To study the response of a microbial cell surface to this kind of local measurement this study attempts to overcome the problem associated to the uncertainties introduced by the different fixation treatments by analysing the surface of Staphylococcus epidermidis cells naturally (non-artificially mediated) immobilised on a glass support surface. The particularities of this natural bacterial fixation process for AFM surface analysis are discussed in terms of theoretical predictions of the XDLVO model applied to the systems bacteria/support substratum and bacteria/AFM tip immersed in water. In this sense, in the first part of this study the conditions for adequate natural fixation of three S. epidermidis strains have been analyzed by taking into account the geometries of the bacterium, substrate and tip. In the second part, bacteria are probed without the risk of any possible artefacts due to the mechanical or chemical fixation procedures. Forces measured over the successfully adhered cells have (directly) shown that the untreated bacterial surface suffers from a combination of both reversible and non-reversible deformations during acquisition of force curves all taken under the same operational conditions. This is revealed directly through high-resolution tapping-mode imaging of the bacterial surface immediately following force curve mapping. The results agree with the two different types of force curves that were repeatedly obtained. Interestingly, one type of these force curves suggests that the AFM tip is breaking (rather than pushing) the cell surface during acquisition of the force curve. In this case, adhesive peaks were always observed, suggesting a mechanical origin of the measured pull-off forces. The other type of force curves shows no adhesive peaks and exhibits juxtaposing of approaching and retraction curves, reflecting elastic deformations.     

Reduced hydrophobic interaction of polystyrene surfaces by spontaneous segregation of block copolymers with oligo (ethylene glycol) methyl ether methacrylate blocks: force measurements in water using atomic force microscope with hydrophobic probes

Reduction of hydrophobic interaction in water is important in biological interfaces. In our previous work, we have found that poly(styrene- b-triethylene glycol methyl ether methacrylate) (PS-PME3MA) segregates the PME3MA block to the surface in hydrophobic environment, such as in air or in a vacuum, and shows remarkable resistance against adsorption or adhesion of proteins, platelets, and cells in water. In this paper, we report that atomic force microscopy (AFM) with hydrophobic probes can directly monitor the reduced hydrophobic interaction of the PS surfaces modified by poly(styrene- b-origoethylene glycol methyl ether methacrylate) (PS-PME NMA), where N is the number of ethylene glycol units. The pull-off forces between the hydrophobic probes that are coated with octyltrichlorosilane (OLTS) and the PS-PME NMA modified polystyrene (PS) surfaces in water were measured. The absolute spring constants and tip-curvatures of the AFM cantilevers were measured to compute the work of adhesion by the Johnson, Kendall, and Roberts (JKR) theory, which relates the pull-off force at which the separation occurs between a hemisphere and a plane to the work of adhesion. The hydrophobic interactions between the hydrophobic tip and polymer surfaces in water were greatly reduced with the segregated PME NMA blocks. The hydrophobic interactions decrease with increasing N of the series of PS-PME NMA and show a correlation with the amount of protein adsorbed.     

Interaction forces measured using AFM between colloids and surfaces coated with both dextran and protein

Both proteins and polysaccharides are biopolymers present on a bacterial surface that can simultaneously affect bacterial adhesion. To better understand how the combined presence of proteins and polysaccharides might influence bacterial attachment, adhesion forces were examined using atomic force microscopy (AFM) between colloids (COOH- or protein-coated) and polymer-coated surfaces (BSA, lysozyme, dextran, BSA+dextran and lysozyme+dextran) as a function of residence time and ionic strength. Protein and dextran were competitively covalently bonded onto glass surfaces, forming a coating that was 22-33% protein and 68-77% dextran. Topographic and phase images of polymer-coated surfaces obtained with tapping mode AFM indicated that proteins at short residence times (<1 s) were shielded by dextran. Adhesion forces measured between colloid and polymer-coated surfaces at short residence times increased in the order protein+dextran < or = protein < dextran. However, the adhesion forces for protein+dextran-coated surface substantially increased with longer residence times, producing the largest adhesion forces between polymer coated surfaces and the colloid over the longest residence times (50-100 s). It was speculated that with longer interaction times the proteins extended out from beneath the dextran and interacted with the colloid, leading to a molecular rearrangement that increased the overall adhesion force. These results show the importance of examining the effect of the combined adhesion force with two different types of biopolymers present and how the time of interaction affects the magnitude of the force obtained with two-polymer-coated surfaces.     

Using the adhesive interaction between atomic force microscopy tips and polymer surfaces to measure the elastic modulus of compliant samples

An atomic force microscope (AFM) method for measuring surface elasticity based on the adhesive interactions between an AFM tip and sample surfaces is introduced. The method is particularly useful when there is a large adhesion between the tip and soft samples, when the indentation method would be less accurate. For thin and soft samples, this method will have much less interference from the substrate than is found using the indentation method because there is only passive indentation induced by tip-sample adhesion; in contrast, a large indentation with a sharp tip in the sample may break its stress-strain linearity, or even make it fracture. For the case where it is difficult to accurately locate the tip-sample contact point, which is problematic for the indentation method, the method based on adhesive interactions is helpful because it does not require locating the tip-sample contact point when fitting the whole retraction force curve. The model is tested on PDMS polymers with different degrees of cross-linking.     

Rupture kinetics of liquid bridges during a pulling process: a kinetic density functional theory study

Capillary bridge is a common phenomenon in nature and can significantly contribute to the adhesion of biological and artificial micro- and nanoscale objects. Especially, it plays a crucial role in the operation of atomic force microscopy (AFM) and influences in the measured force. In the present work, we study the rupture kinetics and transition pathways of liquid bridges connecting an AFM tip and a flat substrate during a process of pulling the tip off. Depending on thermodynamic conditions and the tip velocity, two regimes corresponding to different transition pathways are identified. In the single-bridge regime, the initial equilibrium bridge persists as a single one during the pulling process until the liquid bridge breaks. While, in the multibridge regime the stretched liquid bridge transforms into an intermediate state with a collection of slender liquid bridges, which then break gradually during the pulling process. Moreover, the critical rupture distance at which the bridges break changes with the tip velocity and thermodynamic conditions, and its maximum value occurs near the boundary between the single-bridge regime and the multibridge regime, where the longest range capillary force is produced. In this work, the effects of tip velocity, tip size, tip-fluid interaction, and humidity on rupture kinetics and transition pathways are also systematically studied.     

Atomic force microscopy of mechanically trapped bacterial cells.

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.     

The role of few-asperity contacts in adhesion

The surface roughness of a few asperities and their influence on the work of adhesion is of scientific interest. Macroscale and nanoscale adhesion data have seemingly given inconsistent results. Despite the importance of bridging the gap between the two regimes, little experimental work has been done, presumably due to the difficulty of the experiment needed to determine how small amounts of surface roughness might influence adhesion data lying in between the two scales. To investigate the role of few-asperity contacts in adhesion, the pull-off force was measured between different sized atomic-force microscope (AFM) tips (with different roughnesses) and sample surfaces that had well-controlled material properties. There were seventeen tips of four different types, with radii from 200 nm to 60 microm. The samples were unpatterned single crystal silicon with a chemical silicon dioxide surface resulting from a standard silicon wafer clean. Some of the samples were treated with a few angstroms of vapor deposited diphenylsiloxane. We observed that the uncorrected (for surface roughness) pull-off force was independent of the radius of the AFM tip, which was contrary to all continuum-mechanics model predictions. To explain this behavior, we assumed that the interactions between the AFM tip and sample were additive, material properties were constant, and that the AFM tip, asperities, and sample surfaces were of uniform density. Based on these assumptions, we calculated a simple correction due to the measured root mean square (RMS) surface roughness of the AFM tips. The simple correction for the RMS surface roughness resulted in the expected dependence of the pull-off force on radius, but the magnitudes were higher than expected. Commercial and heat-treated AFM tips have minimal surface roughness and result in magnitudes that are more reliable. The relative uncertainty for the pull-off force was estimated to be 10%. In this paper, we derive how the cantilever and tip parameters contribute to the measured pull-off force and show how the corrected results compare with theory. Although much work is still needed, the work presented here should advance the understanding of adhesion between the macroscale and nanoscale regimes.     

Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy

Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes.     

Analysis of different approaches for evaluation of surface energy of microbial cells by contact angle goniometry

Microbial adhesion on solid substrate is important in various fields of science. Mineral-microbe interactions alter the surface chemistry of the minerals and the adhesion of the bacterial cells to mineral surface is a prerequisite in several biobeneficiation processes. Apart from the surface charge and hydrophobic or hydrophilic character of the bacterial cells, the surface energy is a very important parameter influencing their adhesion on solid surfaces. There were many thermodynamic approaches in the literature to evaluate the cells surface energy. Although contact angle measurements with different liquids with known surface tension forms the basis in the calculation of the value of surface energy of solids, the results are different depending on the approach followed. In the present study, the surface energy of 140 bacterial and seven yeast cell surfaces has been studied following Fowkes, Equation of state, Geometric mean and Lifshitz-van der Waals acid-base (LW-AB) approaches. Two independent issues were addressed separately in our analysis. At first, the surface energy and the different components of the surface energy for microbial cells surface are examined. Secondly, the different approaches are evaluated for their internal consistency, similarities and dissimilarities. The Lifshitz-van der Waals component of surface energy for most of the microbial cells is realised to be approximately 40 mJ/m2 +/-10%. Equation of state and Geometric mean approaches do not possess any internal consistency and yield different results. The internal consistency of the LW-AB approach could be checked only by varying the apolar liquid and it evaluates coherent surface energy parameters by doing so. The electron-donor surface energy component remains exactly the same with the change of apolar liquid. This parameter could differentiate between the Gram-positive and Gram-negative bacterial cells. Gram-negative bacterial cells having higher electron-donor parameter had lower nitrogen, oxygen and phosphorous content on their cell surfaces. Among the four approaches, LW-AB was found to give the most consistent results. This approach provides more detailed information about the microbial cell surface and the electron-donor parameter differentiates different type of cell surfaces.     

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.