MICROBIAL CELL IMAGING USING ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 51

using glutaraldehyde ( Figs. 3.1e,f ) . 33 This method has been used for imaging
Gram-negative B. cepacia , Pseudomonas stutzeri, E. coli, Pseudomonas putida

and Gram-positive . 56,57 Immobilization

by covalent attachment depends on favourable microbe-to-substrate contact,
and any repulsion forces that might prevent this contact must be overcome.
This immobilization technique has been used successfully for AFM imaging
with various buffers. 21,23,24,48,49

Bacillus subtilis and Micrococcus luteus

3.3 GENERAL CONSIDERATIONS FOR AFM IMAGING OF
MICROBIAL CELLS

In addition to cell mounting procedures, other considerations related to
microbial cell imaging should be considered. In general, imaging bacteria in
liquid shows that hydrated cells have a smooth surface with greater heights
compared with bacteria imaged in air. However, the imaging mode used,
either contact or non-contact, can also affect the image. For example, the
morphology of treated with the antimicrobial peptide colistin

appears remarkably different when imaged in MACmode when compared
with images taken in contact mode. 30 After 3 hours of colistin treatment, the
cell surface changes and appears rough in MACmode images. In contrast,
contact mode images result in a wavy morphology ( Fig. 3.2 ) . Even though
both imaging modes indicate that colistin strongly affects the bacterial
envelope, the actual morphology of the bacterial surface after treatment with
colistin is dificult to ascertain. Contact mode images of bacterial spheroplasts
reinforce the importance of selecting the appropriate imaging mode for the
probed sample. When untreated spheroplasts were imaged in contact mode
using a cantilever with a relatively low spring constant (0.01 nN/nm), they
conformed to the shape of the tip ( Fig. 3.3 ) . Intermittent contact imaging (e.g.
MACmode, acoustic, tapping), which applies a lower force, prevents these tip
artifacts and allows for imaging soft bacterial cell surfaces where the cell wall
has been removed.
Although liquid imaging is preferred for the reasons mentioned earlier,
imaging in air generally results in better resolution of ine structures ( Fig. 3.4 ) .
Presumably, the luidity of the microbial surface and its various appendages
are reduced as the surface is dried and appendages become immobilized on
the surface. This allows for routine resolution of bacterial lagella, pili and
changes to surface ultrastructure. Nevertheless, imaging and interpreting
dried samples will have to account for artifacts that result from the drying
process.

P. aeruginosa