Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 172

Samples for SEM examination are often cells, cell layers, or bulk tissues
mounted on rigid or semi-rigid substrates ( see Note 7 ). Several preparative steps
are similar to those listed above for TEM, in Subheading 3.1 . The methods
diverge to prepare the samples for viewing exposed surfaces rather than internal
structures.
3.2.1. Fixation
1. Apply suspended cells to substrate if necessary. Place the desired number of
silicon chips into wells of a 24-well tissue-culture plate, shiny side up. Pipette
30 μL of cell and/or bacterial suspensions that have been washed and resus-
pended at 10 6 or 10 7 /mL of HBSS, respectively, onto the shiny surface of
silicon chips. Cover and place in a hydrated CO 2 incubator. Allow to settle for
10-15 min.
2. Gently add 1 mL of fixative containing 2.5% glutaraldehyde, 4%
paraformaldehyde, and 0.1 M sodium cacodylate buffer, pH 7.2, to each well.
Allow 30-60 min for primary fixation. Mammalian cells, cell monolayers, and
Gram-negative bacteria fix relatively rapidly. Bulk tissue samples, Gram-positive
bacteria (particularly spore-forming bacteria), and hydrophobic mycobacteria may
require extended fixation time periods.
3. Wash the samples twice for 15-30 min with 1-2 mL of cacodylate buffer.
4. Post-fix the samples with 1 mL of 1% OsO 4 in cacodylate for 30-60 min.
5. Wash the samples once with cacodylate buffer and twice with water.
3.2.2. Dehydration, Coating, and Mounting
1. Dehydrate the samples by replacing the water with hour-long series of 70, 100,
and 100% ethanol.
2. Dry the samples using a critical point dryer and carbon dioxide.
3. Mount the samples, oriented with the material of interest facing upward, onto
sample stubs manufactured for the available scanning EM, using conductive
material such as silver, gold, or graphite paint, or double-adhesive carbon
disks.
4. (Optional) Sputter-coat the samples with suitable material such as chromium,
iridium, gold-palladium, or carbon. The samples are ready for examination.
3.3. Immune Labeling with Quantum Dots
The methods described in this section pertain to labeling antigens located on
the surface of cells or thin sections for examination by either transmission or
scanning electron microscopy (SEM). Despite being similar to colloidal gold
for detection by electron microscopy, quantum dots offer the ability to observe
labeling and associated biological processes by fluorescence microscopy prior
to fixation for electron microscopy. Although use of quantum dots for biological