TITLE: Inoculating and harvesting fungal isolates on cellophane overlay 
AUTHORS: Jana U'Ren, Lilly Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Some fungal isolates will embed into the solid media during growth, which makes it difficult to harvest agar-free mycelium for downstream applications such as DNA and RNA extraction. Here, we describe a protocol to (1) inoculate fungal isolates onto solid media with sterile cellophane overlays and (2) harvest the tissue for later DNA or RNA extraction. </div></div>

[STEPS]
?. [Preparing Cellophane]
Wipe down bench top and scissors with RNase away solution.
?. [Preparing Cellophane]
Cut cellophane into approximately 1 inch squares, taking care to measure squares to ensure they will fit in a 60mm Petri dish.
?. [Preparing Cellophane]
Place ~75 pieces into a 250mL glass beaker.
?. [Preparing Cellophane]
Fill beaker with 100 mL nanopure water to fully cover the cut cellophane pieces.
?. [Preparing Cellophane]
Cover beaker with aluminum foil and autoclave indicator tape. Autoclave for 30 minutes on liquid cycle at 121°C and allow to cool to room temperature.
?. [Preparing Plates with Cellophane]
Sterilize the biosafety cabinet and preheat the bactoincinerator.
?. [Preparing Plates with Cellophane]
Pre-sterilize fine-point forceps by submerging in 95% EtOH and placing for 5 seconds in the hot bactoincinerator.
?. [Preparing Plates with Cellophane]
While forceps cool, retrieve media plates from refrigerator and remove from sleeves, taking care to keep sleeves and media sterile since they will be re-bagged for future use.
?. [Preparing Plates with Cellophane]
In the biosafety cabinet, remove aluminum cover from cellophane beaker and place in the back of the biosafety cabinet to ensure that it does not become contaminated.
?. [Preparing Plates with Cellophane]
Utilize sterilized, cooled forceps to remove cellophane from beaker.
?. [Preparing Plates with Cellophane]
Once all plates are inoculated with cellophane, carefully re-bag media and place pink tape with initials and date to indicate that cellophane has already been added to plates.
?. [Preparing Plates with Cellophane]
Place the square of cellophane in the center of the plate. Be careful to lay the cellophane flat.
?. [Preparing Plates with Cellophane]
If cellophane remains in the beaker and the beaker has been kept sterile in the process of inoculating plates, place aluminum cover back on beaker and place on shelf for future use.
?. [Inoculating Cellophane Plates]
Pre-sterilize fine-point forceps by submerging in 95% EtOH and placing for 5 seconds in the hot bactoincinerator.
?. [Inoculating Cellophane Plates]
Sterilize the biosafety cabinet and preheat the bactoincinerator.
?. [Inoculating Cellophane Plates]
In your lab notebook, record the names of isolates to be inoculated onto cellophane plates.
?. [Inoculating Cellophane Plates]
While forceps are cooling, retrieve media plates from refrigerator and begin labeling plates with isolates to be inoculated.
?. [Inoculating Cellophane Plates]
Remove the Petri dish containing the fungal isolate to be inoculated from the stack. Remove the parafilm from the plate and discard. Place on the side of the hood, out of the way of where culturing is to occur.
?. [Inoculating Cellophane Plates]
Remove top lid from the new agar plate containing the cellophane overlay.  Place the lid such that when you work your hands/arms are not crossing over any of the plates.
?. [Inoculating Cellophane Plates]
Using the sterile scalpel, cut small piece of mycelium/agar from culture plate and carefully transfer it to the center of the piece of cellophane. Replace lid onto new cellophane plate and move to the side of the hood.
?. [Inoculating Cellophane Plates]
Replace lid on the source culture plate and stack away from samples that still need to be subcultured.
?. [Inoculating Cellophane Plates]
Repeat steps 18-21 until all isolates have been inoculated.
?. [Inoculating Cellophane Plates]
Once all samples are done, parafilm Petri dishes with two layers of parafilm, ensuring that the mycelium pieces on the new plate do not move.  Place the parafilmed Petri dishes on the proper culture tray.
?. [Inoculating Cellophane Plates]
Record any relevant notes on samples in lab notebook (i.e. whether the mycelium moved and had to be re-placed)
?. [Harvesting Cellophane Plates]
Sterilize the biosafety cabinet and preheat the bactoincinerator.
?. [Harvesting Cellophane Plates]
Pre-sterilize fine-point forceps by submerging in 95% EtOH and placing for 5 seconds in the hot bactoincinerator.
?. [Harvesting Cellophane Plates]
While the equipment is cooling, label the sides of 2 ml screwtop bead tubes with the following information:Name of isolateTube number  If it is for DNA or RNA Age of plateType of media it was grown on DateYour initials
?. [Harvesting Cellophane Plates]
Pour liquid nitrogen from large dewar into portable benchtop dewar.
?. [Harvesting Cellophane Plates]
Loosen cap(s) of tubes for one isolate (e.g. if one isolate has four voucher, loosen all at the same time.)
?. [Harvesting Cellophane Plates]
Using the sterile scalpel, carefully remove the plug that was used as the source inoculum. Place this piece on the side of the plate.  This will ensure that you are only collecting fresh mycelium without any agar.
?. [Harvesting Cellophane Plates]
Use the side of the forceps to lightly scrape the new mycelial tissue into a “bundle” (make sure not to tear into the cellophane).
?. [Harvesting Cellophane Plates]
Transfer the bundled tissue into labeled tube (this can be achieved by the use of the scalpel or a pair of forceps) and cap the tube.
?. [Harvesting Cellophane Plates]
Tap the tube on the benchtop to ensure the tissue is at the bottom of the tube on the beads.
?. [Harvesting Cellophane Plates]
Flash freeze tube by placing it in the liquid N in the portable dewar.
?. [Harvesting Cellophane Plates]
Repeat steps 30-34 until all samples have been harvested and frozen.
?. [Harvesting Cellophane Plates]
When all samples are done, remove the tubes from the dewer using long forceps and place them in their respective boxes in -80°C freezer.
?. [Harvesting Cellophane Plates]
Update the spreadsheet with harvesting information.Name of isolateTube number  If it is for DNA or RNA Age of plateType of media it was grown on DateYour name