Single Bacterial Cell Surfaces
Figure 3: AFM 3-D height image of the S. meliloti wild-type-strain Rm2011, fixed, dried, and imaged in air. The whole bacterial surface is covered with protrusions that show an elongated and wrinkled structure closely connected. Scan size = 4.5 µm × 4.5 µm.
Switching from SEM to AFM, similar surface patterns
became visible using AFM under ambient conditions. In this nanomechanical probing technique, a tiny tip scans the topology of the surface. Tis provides surface imaging of fixed and dried bacterial cells without metal coating or vacuum. In the AFM image shown in Figure 3, we found that the bacterial surface is completely decorated with a dense network of surface protrusions (12.1 ± 4.9 nm heights, 26 ± 6 nm lateral diameters and 72 ± 28 nm lengths). A quantitative comparison of the observed patterns
from XHR SEM and AFM is difficult because SEM suffers from beam penetration and interaction volume effects, and AFM can suffer from tip-convolution effects. However, the estimated values determined by AFM are in good agreement with the XHR SEM data. So the AFM results confirm that the structures are actually present on the cell surface and not part of sub-surface structures. In order to evaluate pos-
sible drying artifacts, AFM images were taken of fixed S. meliloti bacteria in PBS buffer (see Figure 4). Under these conditions the protru- sions appear mostly round- shaped with a height of 4.7 ± 2.7 nm and a lateral diameter of 33 ± 8 nm. Next, the fixation was
omitted to analyze living bac- teria. Te bacteria exhibited protrusions of a height of 3.9 ± 1.7 nm and a width of 28 ± 6 nm (see Figures 5 and 6). Te dimensions are similar
to chemically fixed
bacteria. In consequence, the observed structures are not artifacts caused by the fixation
24 Figure 5: AFM 3-D height image of the S. meliloti wild-type-strain Rm2011, living bacterium in culture medium. Protrusions can be recognized, positioned closely together on the bacterial surface. Scan size = 1 µm × 2 µm.
www.microscopy-today.com • 2011 September
procedure but naturally occurring features on the outer bacterial envelope.
Discussion No obvious change in surface morphology could be
observed comparing bacteria imaged with AFM in liquid with and without fixation (Figures 4 and 5). Drying, however, changes the morphology, although no patterns are generated de novo. On dried bacteria, the observed patterns are more wave-like instead of round protrusions found on cell surfaces imaged by AFM in liquid (see AFM in air, Figure 3; XHR SEM, Figure 2; and SEM Figure 1). Tere are several possible mechanisms for wrinkle formation. One possibility is the wrinkling of the outer membrane during drying. A second possibility is
the formation of elongated wrinkles by the
Figure 4: AFM 3-D images of the topography of the S. meliloti wild-type-strain Rm2011, fixed and imaged in PBS buffer. The bacterial surface is completely decorated with round protrusions. Scan size = 2.5 µm × 2.5 µm.
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