Symbiotic Fungi: Principles and Practice (Soil Biology)

4 In Vivo Model Systems for Visualisation, Quantification and Experimental Studies 53
a b
c
Fig. 4.1 Light micrographs showing: (a) a germinated spore of G. mosseae grown on a cellulose
ester membrane, (b) G. mosseae anastomosed hyphae after staining for SDH activity, (c)
G. mosseae anastomosed hyphae after staining for SDH activity and mounting in Trypan blue,
and (d) G. mosseae hyphae showing incompatible responses, after staining for SDH activity and
mounting in Trypan blue
4.2.2 Dynamics of Anastomosis Formation in Living Hyphae
Hyphal growth and anastomoses formation can be monitored in living hyphae by
means of microchambers (Logi et al. 1998; Giovannetti et al. 2000) (Fig. 4.2).
Surface-sterilised spores are germinated on 20 mm 20 mm sterile cellophane
membranes placed on a thin layer of 1% water agar (WA) in 5.5 cm diameter Petri
dishes, for direct observations of spore germination and hyphal growth under the
dissecting microscope. Cellophane membranes bearing germinated spores are
placed on microscope slides in SDW. The coverslip is sealed with 1% WA, and
periodically wetted with SDW. Alternatively, Petrislide plates, bearing the cellophane
membrane placed on a 2-mm WA layer poured on the lid, may be used both
to germinate spores and as imaging chambers. The microchambers obtained are
then transferred to the light microscope and contacting hyphae can be observed for
periods of several hours, in order to monitor the entire process of anastomoses
formation, during which organelle flow between anastomosing hyphae can be
monitored and images can be captured with a video camera and then recorded.
d