Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 165

and for interpreting structures and events that may be polarized with respect
to basal, lateral, or apical cell surfaces. In many instances, such layers
are appropriate and useful in vitro models for the native associations of
bacteria and host cells. Microbial and cell adherence to such substrates can
be promoted, if necessary, by pre-coating the substrate with a biologically
relevant substance, such as fibronectin, vitronectin, collagen, or poly-l-lysine.
It should be noted, however, that many situations exist in which adherence to a
surface is not advantageous and use of centrifugal pellets, for example, may be
preferable.
During all steps of sample preparation, care should be taken to assure
that the samples, reagents, containers, and tools are kept clean and free
of particulates and volatile contaminants. Dust, fingerprints, trace salts, and
other materials in solution degrade image quality and can damage or destroy
samples and instrumentation. To minimize problems with contamination,
several preventative steps are advisable, including (1) acquiring high quality
water and reagents, (2) using pre-cleaned or sterile plasticware and thoroughly
cleaning and rinsing all glassware, (3) filtering buffers, stains, and sample
rinse water with 20-200 nm filters prior to use, (4) handling samples, tools,
and sample holders with protective gloves, and (5) reducing dust by filtering
incoming ventilation and controlling foot traffic into preparative and instrument
areas. Furthermore, several reagents and resins used in sample preparation
are highly toxic, carcinogenic, and/or radioactive. Therefore, material safety
data and recommendations from the suppliers should be obtained, under-
stood, and followed for safe handling and disposal of all potentially hazardous
reagents.
1.1. Conventional TEM
This section provides preparative methods for examining thin sections of
microbial material. The examples shown in Fig. 1 depict mixtures of bacteria
with human host cells prepared by these methods. These techniques are
suitable for samples that may or may not include specific staining or labeling
steps for identifying or highlighting structures or molecules of interest (see
Subheading 1.3 ). It is presumed that certain equipment including a fume hood,
ultramicrotome and diamond or glass knife, and transmission electron micro-
scope are available for use.
Preparative steps involve fixing with cross-linking agents, contrasting with
electron-dense compounds, dehydrating, embedding, and sectioning. Typically,
primary fixation involves immersion in buffered glutaraldehyde or a mixture
of glutaraldehyde and paraformaldehyde. These reagents react with and can
cross-link free amino groups. If needed, the primary fixative can also be used