Fungal Isolates (Method 1)
Autoclave Whatman No. 1 (7.09 cm) filter paper at 121°C (250°F) for 15 minutes.
Place sterile filter paper on top of PDA media in a petri dish. The moisture from the PDA thus saturates the filter paper. Place a small plug of anthracnose isolate on top of filter paper.
Incubate at room temperature for 6-8 days with alternating 12 hrs light/12 hours dark.
With sterile instrument, peel filter paper from PDA in petri dish and place inside an empty sterile petri dish – do not parafilm.
Put petri dish containing filter paper inside desiccator for 24-48 hours. The filter paper at this point has probably rolled up.
With sterile scissors, cut the filter paper into small squares (approximately 4mm x 4mm) and put into a sterile air tight screw-top vial. Refrigerate at 3-4°C (38-40°F).
When ready to use, put a square of filter paper onto a PDA plate and incubate at room temperature for 6-8 days with alternating 12 hrs light/12 hours dark.
All cultures are single spored upon initial collection.
Filter paper disks (grade 413, 55 mm diameter) are autoclaved in a glass petri dish for 30 minutes at 121C.
A single disk is placed on quarter strength Potato Dextrose Agar (these plates are 100 x 15 mm) with sterile forceps. The culture is then transferred to the outer edge of agar not covered with filter paper, one piece on each side of the plate. In this way, you can tell if the culture you transferred is contaminated. It will also grow if placed directly on the filter paper, but if your filter paper is contaminated, you will not be able to retransfer it.
After 1-3 weeks, when the culture has covered the filter paper from both sides and is sporulating, the disk can be peeled off using sterile forceps. Place it in a sterile (we use disposable plastic) petri dish and using the forceps and scalpel, tear/cut the disk into 20-30 small pieces. Leave this petri dish in the laminar flow hood with the lid on 3-4 days to dry completely. More than 10 days is not recommended because of the possibility of contamination. Place the pieces in a sterile 20 dram glass vial w/rubber sealed lid. (Of course, any kind of vial can be used, but the rubber sealed lids seem to keep out contamination better). We autoclave these 30 minutes also, with the lid loose, leave them in the hood to cool, then seal the caps tightly if we aren’t going to use them the same day. Make sure all the moisture is out of the vial before sealing it.
Cultures preserved this way can be stored in the refrigerator. Before storage, I always test one piece on quarter strength PDA for viability and to make sure it is not contaminated. I have some that I have stored in the freezer (a constant temperature freezer, not the kind that goes through defrost cycles!) for a year that were also all viable and uncontaminated, and sporulated quickly when restarted. To restart, just place one piece on quarter strength PDA, and place under 16 hr photoperiod lights. As I mentioned on the phone, I have used this method for Fusarium spp. also and have had good luck, except that they did not make it in the freezer.