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or as two- or three-dimensional images with color variations (different colors or shading). A line plot of a single P. stutzeri KC bacterium is shown in Figure 16.1a. The image was taken using tapping mode in air of a bacterium originally prepared in MOPS buffer; the cell was covalently bound to the surface to prevent it from moving during imaging. The tapping of the tip was driven by a piezo at a set frequency, but as the tip contacted the surface, it shifted out of phase with the driving frequency. This phase shift was monitored and graphed as shown in Figure 16.1b (phase image) for the same bacterium. Changes in the phase shift indicate differences in the chemical and physical properties of the surface, making it easier to distinguish the boundaries of the bacterium (a soft surface) from the hard glass surface. The phase image also provides evidence of heterogeneity of structure of the bacterial surface itself.

The height and elasticity of bacteria can introduce artifacts in the images produced with the AFM.19 Shown in Figure 16.2 is a false-color image of a single E. coli cell (strain D21, fixed with 2.5% glutaraldehyde) obtained using tapping mode (Figure 16.2a) along with the corresponding phase image (Figure 16.2b). The cells in this image were anchored to the surface with PEI and imaged in 100 mM NaCl and 1 mM Tris buffer. It looks as if there is material that is adjacent to the bacterium (upper right of Figure 16.2a) that could represent material pulled off the cell or attached to the cell. However, the apparent presence of that material is an imaging artifact. The

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FIGURE 16.2 (Color Figure 16.2 appears following page 236.) A single E. coli (strainD21) imaged in water that apparently shows some "material" off to the right side of the bacterium: (a) false color height image and (b) phase image. (c) Imaging the bacterium under air reduces the height of the cell, and the absence of any "material" off to the right side of the cell indicates that the "material" is an imaging artifact. (Adapted from figure 2 and figure 4 in Velegol et al., Langmuir, 19, 851-857, 2003. With permission.)

height of the bacterium is large enough that the side of the pyramid-shaped tip, which is angled ~10° from the surface, hits the side of the cell making it appear as if there is some material that the bottom of the tip is hitting. The key factors in identifying the material as an artifact are that the material appears on only one side of the cell (it has a ~30° offset to the horizontal line during rastering of the tip across the surface), and that the location of the material relative to the cell and tip orientation is always on the same side.18 When the water was removed for imaging, the cell dried and the height of the cell decreased due to desiccation of the bacterium, and the artifact disappeared (Figure 16.2c).

16.3.2 Surface Roughness

When particles attach to surfaces, the properties of both the particles and the surfaces will be important. Chemical factors such as surface hydrophobicity and energy are known to be important, but physical factors such as roughness can also be important. Shellenberger and Logan20 examined the attachment of inorganic colloids (latex microspheres) and bacteria to two surfaces (glass beads) having different average roughnesses. One surface had an average roughness of 15 ± 2 nm, as measured by contact mode AFM, while the other surface had a roughness of 38 ± 4 nm. The rougher surface, shown in Figure 16.3, has many large asperities that served to interact with the surface of the bacteria and other particles. These asperities reached heights

Digital Instruments ManoScope Scan size 30.00 ^m

Scan rate 1.001 Hz

Number of samples 256

Image date Height

Data scale 500.0 nm

Digital Instruments ManoScope Scan size 30.00 ^m

Scan rate 1.001 Hz

Number of samples 256

Image date Height

Data scale 500.0 nm

FIGURE 16.3 The surface of a glass bead roughened using chromic acid shows asperities on the glass surface that average 38 ± 4 nm and can reach over 50 nm. (From Shellenberger and Logan, Environ. Sci. Technol., 36, 184-189, 2002. With permission.)

of over 50 nm and therefore extended well outside the conventional electrostatic repulsion layer of colloids and larger particles. The researchers found that attachment of bacteria and latex microspheres was greater to the rougher surface than to the smoother surface. The extent that surface roughness is important to adhesion efficiencies between different types of biological and inorganic particles in marine systems is an issue that should be further explored.

16.3.3 Interaction Forces

One of the most interesting aspects of probing surfaces with AFM is the ability to measure interaction forces between the AFM tip (or a colloid on the tip) and a surface or particle. Camesano and Logan21 examined the interaction between a silicon nitride (SiN4) tip and individual cells of P. putida KT2442. During the approach of the tip to the surface, they observed repulsion of the tip by the bacterial surface at pHs ranging from 2.2 to 8.7 (Figure 16.4a). The magnitude of the force measured at

Tip-to-sample distance (nm)

Tip-to-sample distance (nm)

FIGURE 16.4 Force curves obtained on individual bacteria of P. putida strain KT2442 in water at different pH values: (a) approach curves and (b) retraction curves. (Adapted from Camesano and Logan, Environ. Sci. Technol., 34, 3354-3362, 2000. With permission.)

Tip-to-sample distance (nm)

FIGURE 16.4 Force curves obtained on individual bacteria of P. putida strain KT2442 in water at different pH values: (a) approach curves and (b) retraction curves. (Adapted from Camesano and Logan, Environ. Sci. Technol., 34, 3354-3362, 2000. With permission.)

the "origin" decreased with pH, suggesting that electrostatic effects were important because decreasing the pH decreased the repulsive layer thickness of the bacterial surface. That is, a reduction in pH brought the net surface charge of the bacterium closer to its isoelectric point (2.3 for strain KT2442; typically around a pH of 2 to 3 for bacteria). The "origin" shown in Figure 16.4a was not a fixed point during force curve imaging. During the approach of the tip to the surface, both the tip and surface can be deflected by electrostatic repulsion between the tip and molecules as well as by small numbers of molecules on the surface.18 Thus, the surface deflected downward during imaging and was lower than its original point by the time the tip and surface were in complete contact.

During the retraction of the tip from the bacterial surface, there were adhesion forces measured between the tip and the surface at the two lowest pH values (Figure 16.4b). However, there were repulsive forces measured between the tip and surface at the two higher pH values (7.0 and 8.7). Attractive forces could be due to covalent and electrostatic bonds between the tip and surface, but capillary forces could be important in this system as well.

Solution ionic strength, as well as pH, can be an important effect in determining bacterial interactions with surfaces. As solution ionic strength is decreased, it is expected from DLVO theory that the electrostatic repulsion layer thickness will increase. It has been observed that the repulsion between an AFM tip and a bacterial surface increases as ionic strength is decreased in accordance with this theory (Fig. 16.5). However, the interaction distances (100s of nanometers) are much larger than those predicted from electrostatic interactions alone (10s of nanometers). Thus, it is likely that these interactions result from changes in orientation of large molecules on the surface of the bacteria in response to changes in ionic strength as well as to changes in pH.

The finding that interaction distances between bacteria and AFM tips are large has prompted extensive studies into measurements of molecular interactions at bacterial surfaces. Razatos and coworkers8,9 first worked with two strains of E. coli that had different lengths of LPS (strains D21 and D21f2). In their experiments, the AFM tip was coated with bacteria and this coated tip brought down near a glass surface. They found that the AFM tip coated with D21f2 was repelled by the glass surface but that coated with strain D21 was attracted to the surface. They hypothesized that the extra polysaccharide on outer layer of D21 shielded the charge of the highly negatively charged keto group (that was exposed on D21f2) because this outer polysaccharide layer was attracted to the surface. They therefore concluded that adhesion increased with an increase in the length of the LPS.

The conclusion that the presence or absence of an O-antigen affects adhesion has been postulated in other studies.22 However, findings by Razatos8,9 that the LPS layer length determines the adhesion properties of these E. coli strains has not been confirmed by others.16 AFM measurements were made between AFM tips and individual bacteria for the two strains used by the Razatos group, and a third strain of E. coli that contained a full LPS layer (keto group, core polysaccharide, and O-antigen). For all three strains, AFM force curves between a bare silicon nitride tip and the bacterial surface were found to be identical (Figure 16.6). In addition, sticking coefficients measured in column tests between the bacteria and glass beads showed that the adhesion of D21 was larger than that of D21f2 only for conditions of low ionic strength

FIGURE 16.5 Approach force curves obtained on individual bacteria of P. putida strain KT2442 in different ionic strength solutions. (Adapted from Camesano and Logan, Environ. Sci. Technol., 34, 3354-3362, 2000. With permission.)

Tip-to-sample distance (nm)

FIGURE 16.5 Approach force curves obtained on individual bacteria of P. putida strain KT2442 in different ionic strength solutions. (Adapted from Camesano and Logan, Environ. Sci. Technol., 34, 3354-3362, 2000. With permission.)

FIGURE 16.6 Approach force curves as a function of the relative piezo movement when silicon nitride tip interacts with E. coli strains D21(0), D21f2 (-), and JM109 (I). Cells were prepared in 1 mM Tris and attached to glass slides using PEI. (From Burks et al., Langmuir, 19(6), 2366-2371. With permission.)

Relative piezo position (nm)

FIGURE 16.6 Approach force curves as a function of the relative piezo movement when silicon nitride tip interacts with E. coli strains D21(0), D21f2 (-), and JM109 (I). Cells were prepared in 1 mM Tris and attached to glass slides using PEI. (From Burks et al., Langmuir, 19(6), 2366-2371. With permission.)

and for non-glutaraldehyde-treated cells.16 When bacteria were placed in a solution of higher ionic strength, or were treated with glutaraldehyde (all bacteria used by Razatos were fixed with glutaraldehyde), the sticking coefficient was larger for D21f2 than D21. Thus, trends between cell adhesion and the molecular composition of the bacteria observed with AFM techniques were not always correlated to conditions observed in macroscopic adhesion tests. Additional research is needed to understand how these chemical treatment and solution changes affect the adhesion of these bacteria to glass surfaces.

16.3.4 Outlook for Using AFM to Study Attachment Processes

There have been many successes using AFM to study the topography of colloids, flocs, and surfaces, and forces between molecules, colloids, and surfaces. The AFM remains one of the most useful tools to study such systems, but additional work will be needed to understand how molecular interactions affect the binding of bacteria, and other particles, to surfaces. The flexibility of the AFM in terms of different methods of operation (contact, tapping, phase, and forces imaging), as well as the ability to chemically modify tips or colloids on the AFM cantilever, make AFM an exceptional tool for further investigations of molecular and nanoscale interactions that affect particle adhesion.

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