Symbiotic Fungi: Principles and Practice (Soil Biology)

232 K. Vogel-Mikusˇ et al.
such specimens ensure the best possible level of preservation of tissue morphology,
and thus the most reliable results. In addition, to control the quality of the sections,
a dissecting binocular should be provided with the cryo-microtome. The specimens
should be sectioned using disposable stainless steel cryo-microtome blades, which
are superior to the standard steel or diamond knives (Schneider et al. 2002). The
sections of the specimens are then put on pre-cooled filter paper into specially
designed pre-cooled aluminium beakers with a cover, and kept in liquid nitrogen
for freeze-drying.
14.2.3.4 Freeze-Drying
Freeze-drying is a very sensitive step in the whole procedure, since the samples can
wilt and shrink drastically due to the large amounts of water in plant tissues. The
samples should therefore be freeze-dried at the lowest temperature possible (they
are best put in the freeze-dryer in liquid nitrogen) and at low pressure (10 5 bar).
To ensure the flatness of the sections, they should be laid between two layers
of precooled filter paper and fixed with a pre-cooled heavy object (e.g., a part of
microscope object glass).
14.2.3.5 Mounting of the Samples into Holders
The freeze-dried samples are mounted into aluminium holders that are covered with
a thin foil (cca. 300 nm thick) of Pioloform (SPI Chem) (Fig. 14.3). The foil is
prepared by dissolving 1 g of Pioloform in 75 ml chloroform (Vogel-Mikusˇ et al.
2007), which can be kept in a dark flask for cca. 6 months, with the dissolved
solution then poured into a beaker. The easiest way of making the foils is to dip a
clean microscope object glass into the foil solution for 2 s and then let it dry for
3 min. Then the edges of the foil are scraped with another clean object glass, to
enable the detachment of the foil from the object-glass. The foil is then floated from
the object glass by dipping it into bidistilled water and picking out the foil on a
specially designed aluminium section holder, as schematically represented in
Fig. 14.3. The sections should be carefully put on a holder with forceps and covered
with another holder covered with Pioloform foil, to fix the section into a sandwich
(Vogel-Mikusˇ et al. 2007, 2008b). The sections can also be mounted on foil with
special two-component glue (e.g., Araldite), being sure that the areas of interest for
scanning remain clean.
14.2.4 Micro-PIXE Analysis
Micro-PIXE permits quantitative studies of element distributions, with lateral
resolution of the order of 1 mm for elements from Na to U. Relatively few nuclear
microprobe set-ups for the analysis of biological material exist worldwide because