Preparing fresh Fusarium macrocondia (asexual
Inoculate example: 2 x 1 L Erlenmeyer bottles
with a baffled base,
each with 250 ml RA medium and 10 agar plugs (ø = 5 mm) from a fresh
at 20 oC, 150 rpm for 3 days
the cultures for spores by microscopy (the spores appear as banana
shaped structures) there should be many spores and few hyphae.
the culture through a single layer of sterile Miracloth placed in
a funnel and into sterile centrifuge tubes (400 ml) .
Stir with a sterile
spatula to avoid the filter from clotting.
the culture bottles with 250 ml sterile MilliQ water, and pour it
through the Miracloth filter.
the centrifuge tubes
Centrifuge at 13.000 g 4oC for 40 min
- Pour of
the supernatant (gently)
- Add 10
ml of sterile MilliQ and gently resuspend the pellet
the resuspended spores into two 50 ml
centrifuge tubes (Falcon tube or similar)
Centrifuge at 5000 g 4oC for 30 minutes
- Pour of
Resuspend the pellets in the two tubes with a total of 5 ml of sterile
MilliQ water and pool the volume in a single tube
- Take out
spore solution and dilute it with 900
MilliQ water (10 fold dilution)
Centrifuge the suspension from bullet 11 at 5000 g at 8 oC
for 30 minutes (the pellet will freeze at 4 oC)
Meanwhile count the spores
in the dilution from bullet no. 12.
Immediately preceding the centrifugation step remove the supernatant
Resuspend the pellet in 1-4 ml sterile 10 % glycerol to get a spore
concentration of 1 x 108 /ml
the spore solutions
at - 80oC (for long time storage) or keep
them at 4 oC where they will be good for a week or two.
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Dette sted blev sidst opdateret
14. July 2010