Preparing fresh Fusarium macrocondia (asexual spores)

Day 1 

1. 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 solid culture.
2. Incubate at 20 ^oC, 150 rpm for 3 days

Day 4 

1. Check the cultures for spores by microscopy (the spores appear as banana shaped structures) there should be many spores and few hyphae.
2. Filter 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.
3. Rinse the culture bottles with 250 ml sterile MilliQ water, and pour it through the Miracloth filter.
4. Balance the centrifuge tubes
5. Centrifuge at 13.000 g 4^oC for 40 min
6. Pour of the supernatant (gently)
7. Add 10 ml of sterile MilliQ and gently resuspend the pellet
8. Transfer the resuspended spores into two 50 ml centrifuge tubes (Falcon tube or similar)
9. Centrifuge at 5000 g 4^oC for 30 minutes
10. Pour of the supernatant
11. 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
12. Take out 100 ml spore solution and dilute it with 900 ml MilliQ water (10 fold dilution)
13. Centrifuge the suspension from bullet 11 at 5000 g at 8 ^oC for 30 minutes (the pellet will freeze at 4 ^oC)
14. Meanwhile count the spores in the dilution from bullet no. 12.
15. Immediately preceding the centrifugation step remove the supernatant
16. Resuspend the pellet in 1-4 ml sterile 10 % glycerol to get a spore concentration of 1 x 10⁸ /ml
17. Freeze the spore solutions at - 80^oC (for long time storage) or keep them at 4 ^oC where they will be good for a week or two.