Workshop: Culturing Mycelium

Mycelium cultures and how to look after them by Ashley Granter from Natura Design Studios.

This protocol is designed by Ashley Granter from Natura Design Studios for the Royal College of Art biomaterial platform & Open Cell.

Agar cultures are intended for the storing, cloning and classifying of fungi. It provides a sterile surface, free from competitors, where the mycelium can grow in order to expand the amount of material. The process follows the mixing and sterilising of an agar recipe, which can vary greatly between species of fungi or even the mycologist doing the work. After the medium has been sterilised in an autoclave, it is left to cool to just about 50 degrees Celsius, where it can then be poured into pre-sterilised petri dishes in a controlled environment. The plates are then left to cool where the medium solidifies; these can now be inoculated with culture or stored for later use.

Once inoculated, the plates are stored in monitored conditions that match the species preferred environment, these are then left to grow for up to 2 weeks (once the mycelium has reached the edge of the plate. This finished plate can then either be stored as a sample, used for the creation of spawn or cloned further to create more plates.

Link to github and full protocol

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MYCELIUM CULTURES AND HOW TO LOOK AFTER THEM

by Ashley Granter from Natura Design Studios

Agar plate procedures

90mm plastic petri dishes/ plates
Agar agar
Malt barley extract
Nutritional Yeast
Parafilm tape
Dye (optional)
Isopropyl alcohol (70%)

Equipment needed:

Autoclave/ Pressure cooker
Weighing scales (0.1g accuracy)
Pyrex beakers (needs to fit in the pressure cooker)
Sterile area (Still air box/ flow hood/ Bunsen burner)
Scalpel

MEA Agar recipe

10g Agar agar
10g Malt barley extract
1g Nutritional yeast
500ml boiling water (preferably distilled)
Dye (optional – Varies depending on strength)

Note: Around 750ml fills exactly 25 x 90mm plates. Change the recipe accordingly.

Procedure

Weigh out ingredients and add boiling water, mix thoroughly.
Pour into a container that can withstand the pressure cooker. A Pyrex beaker is recommended or some kind of glass flask that is made from heat resistant materials, additionally make sure that it pours without going everywhere. Wrap the opening of the container with tin foil to reduce the moisture loss through boiling.
Add items to the pressure cooker, it may be helpful to add jars around the sides to stop the beaker from falling over. Make sure that the beaker is not in contact with the metal walls.
Pressure cook the agar for 45 minutes at 15psi.
Let the pressure cooker return to ambient pressure and let the agar cool down to a point where it can be handled.
Sterilize all the equipment needed and pre-cut the parafilm into strips for the plates (usually about 2 inches by 3/4 inches). Wiping down the plastic encapsulating the petri dishes, cut the bag open and slide the stack out upright.
Pour agar into plates in a stacked motion, starting with the bottom plate first. This reduced the amount of condensation on the plate lids. (It is recommended to pour the agar when it reaches around 50 degrees Celsius, for beginners, this may be hard to achieve and take some trial and error).
Let plates cool and set in the sterile area.
If using the plates immediately, inoculate straight away, if not, wrap the individual plates using the parafilm strips and then store the plates in a cool, dark area until needed, upside down to limit condensation. Note: Don't forget to use sterile technique from step 4 onwards; even just the slightest speck of dust will compromise the plates with the consequences possibly arising much further down the line.

Plate Culture Transfer

This stage is intended for the multiplication and expansion of living cultures whilst also allowing long term storage and maintenance of a database of species. Plate cultures can be transferred up to 50 times in sequence before it is recommended to end the specimen; this is due to the fact that over time as the culture ages and grows further and further, minor mutations begin to form in the DNA, most of which can be ignored, but some will cause the growth to fail, reduction in speed or other changes that can be detrimental to your process. Once you have the plates cooled and ready to be transferred to, you can begin this stage.

Procedure

Similar to all processes in mycology, start by making sure everything is set up, wiped down and planned.
Once the area is sterilised (with the Bunsen burner on if not using a flow hood/ still air box), take your fully colonised culture plate that is of interest and unwrap the parafilm. Ashley Granter – Co-founder & Materials Specialist @ Natura Design Studios | 2019
Flame sterilise your scalpel and plunge the hot tip into one of the sterile clean plates to cool it down.
Take a small square cutting from the plate of interest (POI) and transfer it to the fresh plates, do so in groups of 3-4 and flame sterilise the blade in-between to ensure that you don’t spread contamination from plate to plate. If you suspect that the blade is compromised, flame sterilise after and then continue.
Once transferred, in batches, wrap the plates individually, label and store for growth. Tissue Culture Cloning Tissue culturing is one of the most prominent skills needed as a mycologist, as it allows for clones to be taken from wild/ commercial mushrooms which may express desirable traits such as speed of growth, resilience to adverse weather or its ability to digest substrates that are a priority. One note to make about this process is that the whole aim is to get a clone, so avoid spores, as propagating spores will result in a random group of traits that will most likely be completely different to its parent mushroom. Samples of tissue from mushroom fruit bodies will always produce clones if extracted in the right manner. To add to this process, you can adapt it further following from step 8. Once you have plates poured and cooled, set these aside to prepare your fungal specimen.

Procedure

With the mushroom fruitbody, cut/brush away any soil or debris that may be attached to the base of the mushroom and spray down the whole fruitbody with isopropyl alcohol.
Wipe down the surfaces and using an empty petri or dish with a lid, separate the main stem from the cap and place the stem in the dish and cover with a clean object.
Prepare the workstation for transferring the tissue on to sterile, cooled agar plates, turning the Bunsen burner on and placing all relevant items in close proximity to ensure they stay sterile.
Spray hands down with alcohol and heat the blade of the scalpel till becomes red hot.
Cut down the length of the stem but ensure that this isn’t exposed yet; flame sterilise the tip of the scalpel again.
Cut away a piece of tissue from the centre of the stem, being careful not to touch the scalpel blade with the outside of the stem or any other surface/object that could compromise the sterility.
Once you’ve managed to cut a small sample away, place it on the centre of the petri dish carefully and repeat this step will you’ve finished with the current sample.
Wrap and seal all the petri dishes with parafilm, label and store. Note: Make sure to keep a close eye on the development of your plates within the next 3-4 days. You are hoping to see some white mycelium growth stemming from the tissue sample, as soon as you see enough that you can take a culture transfer from, do so immediately. Tissue cultures are one of the hardest processes to master as it requires you to carefully transfer the fresh, clean mycelium multiple times to ensure that any spore or contaminants that have likely been transferred on the plates too can be isolated away; this is called ‘running’ the culture, in order to finish with a pure, clean mycelium culture.