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Three Alternative Methods for Long-Term Storage of S. sclerotiorum
Submitted by Linda Kohn, Fungal Systematics, University of Toronto
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| Sclerotia of S. sclerotiorum in a soybean stem. Photo credit: Craig Grau. |
Storage of dry sclerotia at room temperature
Harvest mature sclerotia ( when no traces of exudate are observed on the rind surface and sclerotia are easily detached from the mycelial mat — about 3 to 4 weeks after inoculation). Store in paper envelopes.
Viability estimate: usually at least one year, possibly longer. Requires surface sterilization prior to germination to produce axenic cultures.
Storage of sclerotia in water blanks at -20°C
Harvest mature sclerotia ( when no traces of exudate are observed on the rind surface and sclerotia are easily detached from the mycelial mat — about 3 to 4 weeks after inoculation). Freeze in sterile, distilled water . The 2.0 ml microfuge tubes with o-ring sealed caps or plastic centrifuge tubes are good containers.
Viability estimate: usually at least one year, often longer. Does not require surface sterilization to germinate to produce axenic cultures.
Storage of mycelium (NOT sclerotia) at -70 to -80C.
Grow isolates on solid medium for 24 to 48 hrs. Transfer four or five 1.5-2 mm pieces of mycelium taken from hyphal tips at the actively growing periphery of the colony to 10% glycerol in sterile, distilled water. The 2.0 ml microfuge tubes with o-ring sealed caps are excellent containers.
Freeze in two steps: first put tubes at -20°C for 24 hrs, then move to -70 to -80°C for storage. To thaw, place tube in water at 37°C (a water bath works well). Take care not to contaminate the mouth of the storage tube.
Viability estimate: indefinite though not necessarily infinite. No surface sterilization possible.