Manure agar recipes for Panaeolus mushrooms

Charcoal Manure Agar recipes for Panaeolus and other dung loving mushroom species

Panaeolus on dung agar — Panaeolus mycelium and small pins on black charcoal manure agar media

If you are ready for the Panaeolus cultivation challenge, then the following two agar recipes can be a game changer. They are based on manure 💩 as one of the ingredients loved by Panaeolus mushrooms.

Recipe 1 - Charcoal Manure Agar Recipe

Ingredients

Dry manure (horse, cow, elephant, turtle, camel, zebra) - 50 g
Water - 500 ml
Agar - 10-12 g (Amazon)
Activated charcoal - 2g (Amazon)

Equipment

High-Precision digital scale - Amazon
Measuring cup
Saucepan and tablespoon
Canning jar with lid or Lab Bottle (Erlenmeyer Flask)
Funnel
Pressure Cooker or Autoclave, or Instant Pot, or Pot with Lid - Amazon
Infrared thermometer gun (optional) - Amazon
Petri dishes - Amazon
Parafilm or PE food film or Clear Packing Tape
Still Air Box, or Fan Filter Unit, or Laminar Flow Hood for sterile work

Preparation

Prepare dry manure. You don't need composted manure, you can dry fresh poo in direct sunlight.
It’s important to dry it, because wet manure has too much ammonia nitrogen which can be harmful for mycelium
Turn dry dung into powder. You can grind it, or use a pounder, or wear gloves and crush with your fingers.
Dry poo doesn't have a bad smell and looks like peat soil for plants
Boil dry manure in water for 10 minutes.
Let it sit for 16-20 hours.
Filter out solids (you can use cheese-cloth or coffee filters).
Bring back to 500 ml.
Heat it again, add agar 10-12g and bring to boil.
Add activated charcoal - 2g (or food color)
Activated charcoal is optional, however black color is perfect for working with Panaeolus mycelium, which has grayish tint and is much thinner than Cubensis mycelium ropes. Color contrast between black agar and thin Panaeolus mycelium is extremely helpful for your further mycology work (transfers, culture isolation, contamination id etc.)

Recipe 2 - Dung Agar media recipe with additional ingredients

Main ingredients

Dry manure (horse, cow, elephant, turtle, camel poo) - 50 g
Water - 1 Liter

Additional ingredients

Malt Extract - 5 g/L (Amazon)
Yeast Extract - 2 g/L (Amazon)
Calcium Sulfate (aka gypsum) - 0.7 g/L (Amazon)
Peptone - 0.10 g/L (Amazon)
Agar - 20 g/L (Amazon)
Activated charcoal powder - 4g or black/blue food color (Amazon)

Preparation steps

Turn dry manure into powder - 50 g.
Boil manure in water (1L) for 10 mins.
Let it sit for 16-20 hours.
Filter out solids (you can use cheesecloth or paper coffee filters).
Bring the mix to 1 Liter.
Heat it again and add additional dry ingredients.
Bring the agar media to a boil, hence all ingredients and agar will be dissolved.

Sterilization and preparation for agar inoculation

Sterilize prepared agar media in a pressure cooker for 30 minutes at 15 psi (250ºF or 121ºC). In instant pot or saucepan with lid for 45-60 minutes.
Let the PC to depressurize completely, open the lid and remove bottle with hot agar.
Let the agar cool down to 130-140°F or 45-50ºC before pouring. At the lower temperatures agar clumps appear. Agar solidifies completely at 86°F or 30°C. Use an infrared thermometer gun to monitor the temperature.
Pour agar in a sterile Petri dishes or ketchup cups.
Let it harden and cool down to room temperature.
Inoculate agar with Panaeolus genetic material (spore swab, spore syringe, liquid culture, agar to agar transfer).
Cover agar plates with food film, or use parafilm or tape.
Keep agar plates in a dark place within temperature range 73-86°F or 23°..30°C.
Sit back and wait for mycelium growth.