Germinating Magic Mushroom Spores

Part 2 : Unit 1 : Germinating Mushroom Spores in Agar

Your mushroom cultivation journey starts with mushroom spores in one form or another. Spores are usually obtained in one of three ways – Spore Prints, Spore Syringes and Spore Swabs

Regardless of how these spores are obtained, they need to be germinated and allowed to grow into mycelium. The  germination medium used in this course is nutrient agar, stored in sterile petri dishes. You will prepare the agar and pour it into the petri dishes in our Still Air Box, in order to reduce the exposure to contaminants in the air. Once the agar has set, you will put the spores on top of the nutrient agar inside the sterile petri dish, and allow them to grow.

Germinating these spores in agar first (as opposed to germinating them directly in Liquid Culture) allows us to identify whether our spores were not contaminated. If they were, we should have clean, white mycelium growing in the petri dish…sections of which we will use in the next step, to inoculate Liquid Culture.

Magic mushroom mycelium in a petri dish
Magic Mushroom Mycelium in a Petri Dish

Sterilizing Agar and Pouring into Petri Dishes

Items needed
Notes & Tips

Video Tutorial : Making Nutrient Agar & Pouring it into Petri Dishes inside a SAB

The video markers above 👆🏼correspond to the the various Steps in this Unit. Click on them to skip to a Step.

Step 1 : Make the Nutrient Agar Liquid and Sterilize it

Weigh out the agar and LME powders and mix with 1 liter of non chlorinated water. Pour the liquid nutrient agar mix into the heat resistant flask. If you plan to use a magnetic stirrer, this is the time to place it in the flask. Loosely tighten the cap of the flask, and cover the cap and neck of the bottle with aluminium foil. Fill the PC with 1.5 inches of water, insert the rack and then place the agar bottle on the rack so that it doesn’t touch the sides of the pot. Sterilize the liquid by Pressure cooking it for 30 minutes. Allow the PC to cool off to no less than 42 °C or 108 °F , otherwise the agar will solidify. If it does solidify, simply warm up the bottle in a hot water bath (inside a pot) until the agar liquifies again.

Step 2 : Remove Sterile Agar bottle from PC, place in SAB and prepare for pouring Agar

While waiting for the PC to cool off, you can start cleaning up your workspace and SAB in anticipation for the Agar work ahead. Once the PC has cooled off to no less than 42 °C or 108 °F and is safe to open, remove the bottle of Agar, give it a gentle mix in the bottle (don’t shake). Sanitize and place the bottle in the SAB and wait for it to cool off some more. You want the Agar to be warm enough so that it stays liquid and doesn’t set in the bottle, but not too hot to hold for an extended period of time.

Next, sanitize the petri dish sleeve and place it inside the SAB so that the petri dishes are standing the right way up. Proceed to sanitize and place everything else you will need (scalpel, paper towels, etc) in the SAB and spray the air in the SAB with alcohol a few times to bring down particulates.

Step 3 : Pour Agar into Stacked Petri Dishes

Slice open the petri dish sleeve and stack the petri dishes in stacks of 5 or 10. With small movements, open each petri dish and pour in just enough liquid to cover the bottom of the dish. Try to keep the petri dish cover close to the base as you pour in the agar, so that the base is less exposed to falling contaminants in the air. Cover the dish quickly and proceed to open and pour the next petri dish. Complete all dishes in batches of 5 or 10, and allow them to cool off in the SAB for about 30 minutes. You can loosely cover the holes of the SAB with some paper towels, and keep the airflow low in the room as you wait for the agar in the petri dishes to solidify.

Step 4 : Wrap Petri Dishes with Parafilm and Store Aside

While you wait for the poured agar to harden, you can cut parafilm strips which you will use to wrap and seal your petri dishes.  I usually cut 6-8 inches of single strip per dish. Once you are ready to start wrapping your petri dishes, grab a parafilm strip and separate it from the paper. If you’re having trouble separating them, pinch off the corner of the parafilm strip to help remove its paper cover. Wrap each dish so that the whole circumference of the dish is covered in wrap. You can stretch the wrap as you pull it around the dish. Practice this a few times to get a feel for the elasticity of the parafilm. Once you have completed wrapping all the dishes, mark the date on each dish and store in a place where they won’t be disturbed for a few days. If any contamination entered the dishes during the pouring process, it will hopefully reveal itself in the next few days.

Troubleshooting : The Agar Making and Pouring Process

While you wait for the poured agar to harden, you can cut parafilm strips which you will use to wrap and seal your petri dishes.  I usually cut 6-8 inches of single strip per dish. Once you are ready to start wrapping your petri dishes, grab a parafilm strip and separate it from the paper. If you’re having trouble separating them, pinch off the corner of the parafilm strip to help remove its paper cover. Wrap each dish so that the whole circumference of the dish is covered in wrap. You can stretch the wrap as you pull it around the dish. Practice this a few times to get a feel for the elasticity of the parafilm. Once you have completed wrapping all the dishes, mark the date on each dish and store in a place where they won’t be disturbed for a few days. If any contamination entered the dishes during the pouring process, it will hopefully reveal itself in the next few days.

Spore Print or Spore Syringe to Agar

Items needed
Notes & Tips

Video Tutorial : Germinating Mushroom Spores in Agar Dishes

Step 1 : Clean and Prepare Work Environment, SAB and all required items

Clean and disinfect your workspace and SAB if needed. Disinfect all required items including spore prints or syringes, agar plates, sterile knife or scraper and paper towels, and put them into the SAB. Spray down the air in the SAB a few times to bring down particulates. Unwrap the parafilm from the nutrient agar petri dishes and give them a light sanitization by wiping them down with an alcohol soaked paper towel. Obviously, you don’t want any alcohol getting into the dishes, because alcohol kills the mycelium. 

Step 2 (from Spore Print) : Open Print Foil and Scrape Spores into Petri Dish

If you’re starting off with a spore print, this is your Step 2. Inside the SAB, spray down the air, then take the sterile blade out of its wrapper. Gently open the spore print foil and expose the print. Open the agar plate, and using the blade, scrape just a few spores into the middle of the agar petri dish. You don’t need many spores, just a speck will do. Once you have transferred a few spores, cover back the agar plate, fold back the foil print and place it in a clean ziploc bag. This ensures that the print stays as clean as possible for future use.

Step 2 (from Spore Syringe) : Shake Spore Syringe. Inject a drop onto each Agar Dish

If you’re starting off with a spore syringe, this is your Step 2. Inside the SAB, spray down the air a few times to bring down particulates. Sanitize your hands and and take the sterile blade out of its wrapper . Gently open the spore print foil and expose the print. With your other hand, open the agar plate and use the sterile blade to scrape just a few spores onto the middle of the agar in the petri dish. You don’t need many spores at all, just a few specks. Cover back the petri dish.

Step 3 : Close Spore Print Foil and Petri Dish

Wrap the petri dish in parafilm and place it in incubation at around 22-27 °C or 70-80 °F. Check them every couple of days for growth of mycelium or contamination. If any dishes show signs of contamination, discard them, or carefully attempt to cut out the healthy parts inside a SAB, and place the healthy piece in a new agar dish.

Pictures : Magic Mushroom Spores slowly Germinating inside Petri Dishes with Nutrient Agar

Petri dish with cubensis spores
Petri Dish With Cubensis Spores
Mycelium germinating in agar dish
Mycelium Germinating In Agar Dish
Mycelium growing in agar dish
Mycelium Growing In Agar Dish

Next Unit: Going from colonized agar to liquid culture. Learn how to choose clean, healthy mycelium from your colonizing petri dishes in order to inoculate Liquid Culture, create endless amounts of liquid mycelium.

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