Materials *not all of these materials are needed read the procedure first.
- Ø obtain a spore syringe or spore print of your choosing I will be using P. Cubensis strain
- Ø Agar Agar 20g
- Ø A 1-liter jar or vessel for sterilizing agar solution
- Ø Dextrose
- Ø Light malt extract
- Ø Food coloring (optional)
- Ø Scale
- Ø Pressure cooker
- Ø SAB (still air box)
- Ø Laminar Flow Hood
- Ø 10% Bleach Solution or 70% iso
- Ø Latex gloves (powder free)
- Ø Inoculation loop, exacto blade or scalpel
- Ø Micropore tape
- Ø Pressure cooker
- Ø 8oz pp5 deli containers
- Ø Pre-sterilized petri dishes or autoclavable petri dishes
- Step # 1: weigh out 20g agar agar powder, 10g light malt extract and 10g dextrose, there are other alternatives available just do some research and reading online. Add all your dry ingredients to a pot or sauce pan. Now add 1 liter of water distilled water works best but tap water is fine. Set the temperature to medium and stir mixture till dry ingredients are dissolved, can add food coloring to the mix it helps distinguish the mycelia on the agar plates/containers better but not necessary.
- Step # 2: Ounce all the dry ingredients are dissolved and you have a nice consistency you can either pour your mixture into 8oz pp5 deli containers with a small nail sized hole in the lid covered in 2 layers of micropore tape. You can also pour your mixture into a 1-liter vessel with a gas exchange hole covered in 2 layers of micropore tape. Then add a layer of tinfoil over top. You can use this to pour agar plates in front of a laminar flow hood ounce sterilized.
- Step # 3: pour your mixture about 1 cm thick in your pp5 deli containers place the lid on with your gas exchange hole covered in 2 layers of micropore tape, this will also be used for inoculation, cover with a layer of tinfoil and place them in your pressure cooker. Stack them so they are over lapping each other inside the pressure cooker or autoclave. If you are using a 1-liter vessel of solution and plan on pouring agar plates just place your vessel in the pressure cooker and sterilize.
- Step # 4: sterilize your plates or solution for 15-20 minutes at 15psi no longer. Allow pressure cooker to cool to room temperature. If you plan to pour plates remove your one-liter vessel before cooled to room temp. Ounce your PC is cool remove your pp5 containers from the cooker and place within your sab or Infront of your laminar flow hood for inoculation.
- Step # 5: Inoculating your pp5 containers or plates with a spore print or syringe. Inoculating pp5 containers with a spore syringe. Take your sterile spore syringe place the needle on your spore syringe and inoculate thru the micropore tape covered hole about 1 drop or 1cc depending on how you want to culture your spores. Place a new layer of tape over the hole. This can be done in a still air box or in front of a flow hood. Store your plates in a clean room temperature space. Similar procedure for agar plates just lift the lid slightly and inject your spore solution onto the plate (in front of a flow hood preferably).
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Step # 6: Inoculating using a sterile spore print. These are usually prints on tinfoil so unfold your print in your SAB or still air box. Flame sterilize your scalpel or inoculation loop and scrape small amounts of spores off into your desired culturing vessel, petri dishes or pp5 containers. This is to be done in a SAB or in front of a Flowood as well.
