Grow mushroom cultures from spore

mycelium running

If you purchased a spore print or a culture syringe to start your mushroom cultivation, your next step would be to prepare AGAR media and pour that into Petri dishes.. Agar is a seaweed extract, a gelatinous substance that will solidify at room temperature. It can be sterilized by pressure and is the ideal food source for your mushroom mycelium to thrive on. You will need a sterile space, a glove box or a laminar flow hood to do agar work.

Prepare 500 ml of Agar solution:

You will need:

  • a pressure vessel
  • a stove top
  • an erlenmeyer flask (500 ml)
  • quality grade PDA (potatoe dextrose agar)
  • some tinned foil
  • roll of tissue paper
  • clean non chlorinated water
  • a digital scale

The process:

  • place your Erlenmeyer flask on the scale and turn it on.
  • measure 20 grams of PDA powder into the flask
  • then pour 200 ml water into the flask
  • swirl the solution for a while until the powder has properly dissolved
  • add the remaining water to the flask – up to 500 ml or measure on scale to 500 grams.
  • again swirl the solution.
  • cut a piece of tinned foil – rectangular shape – 30 X 15 cm – fold this double
  • coil this around the mouth of your flask and place  some rolled up tissue paper in the mouth of the flask. Now fold the tinned foil over the tissue paper and create a tight filtered lid. Take another 5 x 5 cm tinned foil and place that over the lid you just created.
  • place your flask in the pressure vessel
  • I have a rounded base in my pressure vessel to lift the flask off the direct heat
  • add plenty off water, enough to cover the bottom 1/4 of the Erlenmeyer flask, closed the lid
  • bring to boil and allow to steam for 5 – 10 minutes before closing the pressure valve.
  • bring to 15 psi and take note of the time
  • pressure sterilize your medium now for 45 minutes
  • once done move the pressure vessel to a clean environment and allow to cool for at least an hour before use.

Pouring the dishes:

Use latex gloves and sterilize your hands regularly using Isopropyl alcohol (have a ready mixed bottle at hand – I mix 10% water to the alcohol to allow better spread of alcohol).

  • in a sterile environment remove the liquid agar from your pressure vessel. Ready the receiving petri-dishes.
  • in a rhythmic manner pour some liquid into the your dishes, starting from the bottom dishes upwards
  • you should have enough liquid to pour 30 dishes
  • allow the dishes to cool in a sterile environment (either in sterile glove box or in front of a running flow hood).
  • Once cooled I store my dishes in Sterile zip lock bags until use.

Culturing from spore:

rhizomorphic mycelium
a Good example of rhizomorphic mycelium
  • in a sterile environment, remove some agar plates from the zip lock bags.
  • have your spore print ready
  • with the tip of an inoculation loop, gently rub the surface of the agar with your loop, this will collect a little moisture.
  • now rub the tip of the loop over a section of the spore print to collect the spores
  • now rub the spores in a zig zag over the surface of the agar dish and close the dish
  • name your dish with the mushroom culture and place the date on it.
  • repeat this procedure and make 2 or three more from the same print.
  • if you are using disposable inoculation loops, you can use a new loop for each dish to limit cross contamination
  • I seal the sides of my dishes with para film but you can also use Micropore tape.
  • incubate the dishes at 20°C for 7 – 10 days

Easy incubator:

  • purchase and use 2 small black tubs with a lid (approximately 20 liters in volume)
  • place a water proof fish tank heater in the base of plastic tub one, pour enough water in the base of this tub for the heater to be submerged (keep submerged and top up with water regularly).
  • place tub 2 into tub 1 – place all your dishes for incubation into this tub and close with lid.
  • have a thermometer ready in your tub to measure the incubation temperature and adjust the heater accordingly.

Sub-culturing:

You are in fact creating a new strain or a couple of new strains of your chosen mushroom when culturing from spore. When looking at my incubating dish, I look for sectors of growth that are fast and rhizomorphic. Rhizomorphic mycelium may resemble a river delta, each artery of the mycelium in search of food.

  • in a sterile environment, place the culture dish you prefer
  • ready some fresh agar dishes to receive sections of your multi-spore culture dish
  • sterilize the tip of your scalpel with an alcohol or small gas flame until red hot.
  • choose a sector of your growing mycelium that is fast and rhizomorphic, cut a square piece of culture from this sector and transfer this to the waiting agar dish
  • repeat until you have 2 or 3 samples
  • close the dishes with tape and incubate for 7 – 10 days
  • keep an eye on the mycelium and mark the best performing mycelium.
  • you will see more sectors forming as the mycelium expands, these sectors can again be sub-cultured using the same method as above. In no time at all you can have many dishes incubating, my advice would be, work sparingly.
  • once you have a chosen mushroom culture on agar you are ready to expand the mycelium to seed.

Making your mushroom spawn:

Read sterilizing seed for mushroom cultivation

  • prepare your seed matrix and sterilize as per the above article.
  • in a sterile environment, ready your sterilized seed and chosen mushroom culture.
  • sterilize the tip of your scalpel with an alcohol or small gas flame until red hot.
  • allow to cool, then cut and transfer a wedge or two of mycelium on agar to the waiting seed matrix.
  • close the seed jar or bag and incubate for 7 – 14 days.
  • after 3 – 4 days inspect the seed and growth rate of the mycelium. At this stage I like to break up the mycelium – seed growth and disperse this through out the bag.
  • in another 3 or 4 days the mycelium should have run through all the seed. I now break the seed up into individuals again and incubate a couple more days before I use the mushroom spawn.

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