Biology Reference
In-Depth Information
or cell wall deicient forms of bacteria, at least one report applied the technique
for imaging rod-shaped mycobacteria.
25
To accommodate other bacterial
shapes, lithographic patterning of silicon wafers to purposefully deine
pores suitable for microbial immobilization has been described.
26
However,
entrapment within pores is not without risk of mounting artifacts. Mendez-
Vilas
strains
and found that the friction between the spherical bacterial cell and the sides
of the ilter pore can lead to accumulation of extracellular polymeric material
deposited on the exposed surface of the cell.
22
et al.
evaluated mechanically trapped
Staphylococcus epidermidis
3.2.2
Chemical Aachment
Chemically attaching the cells to the surface is yet another approach to
immobilizing microbial cells for AFM imaging in liquid. One variation is to
modify the substrate in such a way that it facilitates adsorption of the cells
to the surface. This approach typically takes advantages of the negatively
charged surface of most bacteria. Hence, cationic substrate modiications are
effective at immobilizing a wide variety of cells.
17,27-34
is suitable for immobilizing both Gram-negative and Gram-positive bacteria
and involves briely incubating a bacterial suspension with a gelatin-coated
mica surface.
The bacteria are typically suspended in water and allowed
to stand on the gelatin-coated surface. Afterwards, the sample is rinsed for
several minutes with water. The immobilized bacteria on the surface can
be imaged in liquid without further alteration. The technique results in
isolated bacteria, distributed throughout the sample, thereby reducing the
time required to ind a region of interest. Apparently, not all commercially
available gelatins can be effectively used in this technique. Several bovine
gelatins purchased from Sigma did not immobilize the bacteria while two
porcine gelatins purchased from Sigma (G-2624, G6144) did.
17,35
Bacterial
adhesion to gelatin is believed to occur in two stages: the initial, reversible
attachment and the more durable, irreversible attachment which follows.
17
36,37
Rinsing has been shown to displace bacteria in the earlier stages of adhesion
while irreversible attachment occurs within minutes.
Bacteria immobilized
on gelatin are stably attached and can withstand rinsing under a stream
of liquid for several minutes. Gelatin is denatured collagen, and several
bacterial species have been shown to bind collagen via speciic binding sites
on the bacterial surface.
38
It is likely that these binding sites, along with
electrostatic and hydrophobic interactions, contribute to retaining bacteria
on gelatin-coated substrates. Gelatin-coated mica has been effective for
immobilizing a number of different bacteria including
39,40
Escherichia coli, P.
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