Symbiotic Fungi: Principles and Practice (Soil Biology)

230 K. Vogel-Mikusˇ et al.
Fig. 14.1 Schematic representation of root sample preparation and rapid freezing
additionally isolated with a polystyrene bed to avoid rapid evaporation of the liquid
nitrogen. Great care needs to be taken when handling propane and liquid nitrogen: a
mixture of propane and air can be explosive, and handling liquid nitrogen in a
closed room can cause asphyxiation. Make sure that this is carried out in a wellventilated
room (door and windows always open) so that the concentrations of both
gases in the air are kept to a minimum.
After freezing, the specimens in the propane (middle thermo-block chamber) are
rapidly transferred to a liquid nitrogen (bigger thermo-block chamber) (Fig. 14.1).
Then all of the prepared specimens together with the thermo-block are transferred
directly into the cryo-microtome chamber to avoid the thawing of the samples.
In general, root-specimen preparation using polished needles (Schneider 2002;
Scheloske et al. 2004; Vogel-Mikusˇ et al. 2008b) enables more rapid freezing,
due to the direct contact of the specimen with the cryogen, and thus the ice
crystals formed are smaller, providing superior preservation of cell morphology.