Almost any agar can be used in vials or test tubes. If sealed tightly, active cultures will remain viable for several months but will require periodic transfer. Moorman's lab uses 5 ml glass vials kept at room temperature.
Recovery of Stored Isolates
Plate agar blocks or a colonized seed on a rich medium such as PDA. Growth from agar blocks may take 5-7 days to appear.
If no growth is observed, plate more agar blocks and more seed.
If still no growth is observed, shake the water tube well and pour small amounts out onto a rich medium.
If still no growth appears, having a grad student chant and dance around the plates for 1 hr per day for 3 days doesn't help!
If hyphal growth AND bacteria are observed, transfer some hyphae to water agar or a selective medium or water agar + rose bengal.
If only hyphal growth develops, transfer some to new plates. Transfer some to water agar and verify that it is actually Pythium. If it is Pythium, set up some new storage tubes.
Do not throw away a plate that has any hyphae until you are certain you successfully transferred the organism and it is definitely growing on the new plate.
Zoospore Induction
Boil leaf blade segments of rye, bent grass or other grass in distilled water for 10 minutes.
Suspend a few segments in a small Petri plate containing sterile 10% soil extract (see Part 2: Media for Pythium Culture for how soil extract is prepared) or sterile distilled water.
Incubate at room temperature under fluorescent lights or near a window with indirect light.
Some species (P. aphanidermatum) release zoospores within 12 hours (overnight) while others take longer. Usually zoospore release continues for several hours.
With some species, it is necessary to pour off the soil extract (or distilled water, add cold sterile extract (or cold distilled water) and place the plate in a 15-18°C dark incubator in order to obtain zoospore release in 4-5 hrs
