Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 164

electron probe microimaging augments biochemical analyses by providing
structural and spatial information at resolution 2-3 orders of magnitude
greater than that achievable by standard far-field optical microscopy. Together
with optical imaging of fixed and live preparations, TEM and SEM provide
numerous techniques for investigating and analyzing chronological and struc-
tural relationships of interactions between bacteria and their hosts and

environments.
Protocols for conducting such investigations vary considerably according to
particular aspects of experimental design and desired outcome (1,2,3,4,5,6,7) .
Examples include imaging techniques, such as negative staining for struc-
tural or immunological analyses of bacteria, rapid freezing and freeze-
substitution for preservation and immobilization of fine structures and aqueous
solutes, microwave processing for rapid diffusion and infiltration of fixatives
and embedding resins, variable pressure SEM and containment chambers
for examining hydrated samples, and enzymatic or immunological labeling
for locating and identifying specific target molecules. Such techniques are
commonly used to assess bacterial morphology and phenotypic expression
of surface and extracellular components such as flagella, pili, fimbrae,
and capsules. Furthermore, TEM and SEM can reveal the chronology of
microbial cell cycles, phase changes, sporulation, and cytopathic effects on
host cells and tissues, such as cytoplasmic and cytoskeletal rearrangements,
plasma membrane ruffling and pedestal formation, organelle trafficking and
vesicle fusion, and cell lysis, necrosis, apoptosis, and autophagy (1,2,3,4) .
Despite the diversity of TEM and SEM applications and approaches to
microbial investigations, most protocols share common processes to address
properties and requirements inherent with electron probe analyses of biological
material, such as a high vacuum environment and both the damaging
effects and exploitable attributes of focused cathode-ray irradiation. This
chapter provides descriptions of conventional preparative and immunological-
labeling techniques with which many questions can be addressed and
modifications adapted. For in-depth review of theoretical and practical appli-
cations of electron microscopy, readers are directed to any of several excellent
volumes (1,2,3,4) .
Because of the broad nature of microbial samples prepared for EM, this
chapter cannot describe methods to address all types of ultrastructural studies.
Rather, this chapter provides methods for preparing samples that are either
cultured on or subsequently attached to a rigid or semi-rigid substrate. Examples
of such substrates are silicon chips, Thermanox® cover slips, Aclar® film,
filter membranes, or agar-based culture medium. Adherence to a firm substrate
is advantageous for positioning TEM samples for sectioning along planes
of interest, for maintaining the integrity of biofilms and host cell layers,