Symbiotic Fungi: Principles and Practice (Soil Biology)

8 Use of the Autofluorescence Properties of AM Fungi 127
8.3.4 Spores
The most frequently used techniques for inoculum production in most of laboratories
working with mycorrhiza are the propagation of AM fungi in pot cultures
with trap plants, or in monoaxenic cultures with transformed carrot roots, according
to Bécard and Fortin (1988). Consistently, only these two methods are considered
here. The methods for producing AM fungus inocula in pot cultures in association
with trap plants have been extensively described (Menge 1984; Jarstfer and Sylvia
1993; Brundrett et al. 1996), while several reviews are devoted to inoculum production
by means of root organ cultures (Bécard and Piché 1992; Fortinetal.2002;
Declerck et al. 2005).
In the case of pot cultures, spores are isolated by wet sieving and decanting
(Gerdemann and Nicolson 1963). In the case of monoaxenic cultures, the method of
Doner and Bécard (1991) is followed.
After these isolation steps, the spores are transferred to microtubes and subjected
to several washing steps in sterile deionized water to eliminate as much debris as
possible. Some spore samples are mounted on slides with the help of a Pasteur
pipette and examined immediately under the epifluorescence microscope. They can
then be subjected to staining directly afterwards.
Other spore solutions are prepared for flow cytometry. It is recommended that
the mean spore number be determined under the stereomicroscope when setting up
this experiment. Additionally, it is important to determine the mean diameter of the
spores of the AM fungus species to be studied. The spore solutions of species
with spore sizes bigger than 200 mm should be sieved through a 200 mm sieve to fit
the particle size that can be handled by the flow cytometer to prevent the tubes of
the fluid system from obstruction. This is not necessary, for example, for Glomus
intraradices since the spores of this species range between 40 and 150 mm in size,
while Glomus clarum spore diameters range between 100 and 260 mm, making
sieving advisable.
8.4 Bright-Field Microscopy, Epifluorescence
Microscopy and Lambda-Scan
All the root sections, isolated intra-radical structures and isolated spores prepared as
stated above can be observed by bright-field and epifluorescence microscopy. In our
laboratory, we use a Leica Leitz DMRD epifluorescence microscope fitted with a
mercury lamp (Leica, Wetzlar, Germany), which permits easy switching between
the different settings and filter cubes.
Unstained fresh material should always be observed under bright-field settings
before subjection to illumination.
The autofluorescence of AM fungal structures is observed with our microscope
under epifluorescence settings with the filter cubes IR (excitation filter BP 450–490,