Biology Reference
In-Depth Information
In other reports, AFM was used to examine the development and
structure of various bacterial spores (see also
Chapter 4
).
9,13,14,52,76,77
Images
of the dynamic process of germination of
spores revealed
germination-induced changes in spore coat topography and structure.
31
These imaging studies began in water (non-inducing conditions) and later
conducted at 37
B. atrophaeus
o
C and continued for several hours. Ultimately, the authors
found that outer rodlet structures were disrupted by small defects that formed
perpendicular to the rodlet array. These defects expanded and coalesced over
time until vegetative cells emerged. A previously unrecognized ordered layer
was also revealed as a result of the impressive images in this report. The same
group also followed spore germination of
in liquid.
14
AFM has been used to visualize other dynamic processes of microbes.
For example, the enzymatic digestion of a bacterial cell wall has been
documented by time-series images of a single bacterial cell during enzyme
exposure. When the cell wall of
Clostridium novyi
was treated with the enzyme
lysostaphin, the cell surface roughness increased with time.
21
An example
from virology illustrates the beneit of using AFM for simulating the dynamic
environment encountered by biological systems
S. aureus
. used
MACmode® to monitor the release of RNA from a type of human rhinovirus
(HRV2).
54
High-resolution images of the immobilized viruses yielded height
measurements around 30 nm, which correlated well with those previously
reported in literature. It was known before their study that RNA is released
from the HRV2 capsid in low pH conditions
in vivo
. Kienberger
et al
. The authors were able
to simulate this condition by reducing the pH of the imaging buffer. After
a period of time in the low pH buffer, the extrusion of RNA molecules was
visualized, illustrating the resolving power of AFM in dynamic environments.
The presence of RNA was conirmed by comparing these images with those
taken in buffer supplemented with RNase A. In the latter case, the ibres
believed to be RNA were not present. On the basis of the presence of fork-like
structures at the end of fully released RNA, the authors were able to speculate
about the initial orientation of the RNA during extrusion. The ability to alter
buffer conditions during imaging permits visualization of dynamic processes
and is an important advantage of AFM.
in vivo
3.4.3 AFM Studies of Microbial Cell Substructures
Although microbes do not have organelles in the classical sense, some have
subcellular macromolecular structures that are important to their physiology.
Such components are impossible to visualize using optical microscopy
without luorescent constructs. Extracellular structures of microbes can be
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