Kick start your Myco-Culture

Kick start you Myco-Culture, mycelium cultivation grow club

Path	Target	Duration	Skills
Discovery,Curiosity	18-25, ++ 8-12 people	Variable	Bio-Fabrication

Introduction and context

One of the most emerging developments that is currently growing rapidly in the field of industrial design, fashion and art is the creation of biobased materials, bio-fabricated in laboratories from microorganisms, bacteria and biopolymers. The industry is already incubating solutions to create new products that propose alternatives to materials derived from petroleum or animals, with examples from small and large companies such as Deserto, Piñatex, Bolt Threads, Gelatex.

The ¨Mycelium Growth¨ club is a demonstration and hands-on activity to create awareness on the processes involved in creating new materials with fungi. In this case we can explore the possibilities of fungi producing mycelium as a substrate bonder, through a focused hands-on activity on some of the stages of the entire process.

INTRO This learning path is about Mycelium growth. It raises awareness and basic knowledge of the processes involved in creating new materials with mycelium as a grown bonder for a substrate.

The activity is designed for a moderate number of participants, for being a hands-on activity each participant will get the opportunity to exercise in different stages of the preparation and inoculation processes under direct supervision. The ideal number is 8 to 12 people.

Learning Outcomes: - How to work in a DIY sterile environment. - How to prepare the nutrient broth to create the growing medium. - How to inoculate the medium. - How to prepare the substrate. - How to inoculate the substrate.

Target audience and context of use
Creatives and professionals, such as producers, designers, architects, biologists, material scientists, SMEs, and companies with interest in sustainability and alternative innovative biobased materials.

You can run the activity in different places like a university, a lab, a cultural center, a museum, etc. Set up a collaboration between your lab, material centres, the department of biology of universities, cultural centers or a museum.

Recommended number of attendees (8 to 12) and facilitators(2 to 3). Consider and agree in advance who will be the photographer for the event.

Preparation

Dive into the references provided in this page, share and comment safety instructions with facilitators, who at all times must remember the participants to respect them. Organize some testing sessions and brief meetings for coordinating with facilitators.

Organize the Step by Step of the activity. Write a general description of the activity to invite attendees, and promote the event on social media.

Be careful. Check you have all the tools and materials needed and that you will have time to wrap up your tools, and set up the logistics for the event room.
Prepare in advance:
- Materials and Equipment
- Incubation of the mycelium if both Steps are done in one session
- Check you have enough supplies
- Setup recording and Remote - Learning Equipment if necessary

General Equipment - 4 tables, chairs (one table for central display, three for students)
- Video recording and photo shoot set up for distance live or remote observation if necessary.
- Safety Equipment : 95% Ethanol,Kitchen paper, Disposable gloves, Lab Coats

Part 1: MEDIUM (NUTRIENT BROTH) PREPARATION

Preparation and materials

Tools and Materials

Tools

• Pressure Cooker
• Autoclave bags
• Glass or plastic Petri dishes
• Scalpel
• Bunsen Burner (or camping gas stove) + Lighter Gloves
• 250 ml Reagent Bottles with blue lid
• Permanent marker
• Parafilm
• Alcohol

Materials

• Spores, Spawn from a company, or fresh mushroom of choice (Reishi and Trametes Multicolor are recommended).
• For 4 petris of 100 mm diameter :
• 200 ml distilled water
• 4g Nutrient Agar
• 4g of Malt extract Powder
• 0.5g of Yeast
• 0.2g of Peptone if available Note: scale the amounts according to the total amount of petris to prepare for the total of attendants to the activity.

Step by Step Instructions

STEP BY STEP overview

step 1: Prepare the medium

• Mix all the ingredients in a blue lid Reagent bottle.
• Firmly close the Reagent bottle and shake until dissolved.
• Calculate 250ml for 4 petri dishes, and prepare according to the number of participants.

step 2: Sterilize the Medium

• Pour 1 litre of water in the pressure cooker.
• Place the autoclave bag.
• Place the bottles vertically with the lid half unscrewed inside the autoclave bag.
• Close and pressure cook for 15 minutes.
• Release the steam carefully away from the face before opening.
• Use silicone kitchen gloves to close the lid firmly and take out the bottle to cool down. Note : If you use glass petri dishes, sterilize them closed by pressure cooking them.

step 3: Sterilize your working area

• Clean the table with alcohol and paper.
• Pour alcohol in a circle.
• Place the Bunsen burner in the center and turn it on.
• Let it burn for a few minutes before starting the next step. Note : if you are working in a laboratory, you can also work under a sterile hood, or a glove box, instead of using the burner.

step 4: Pour the medium in the petris

• Place the closed sterilized petri dishes around the burner within the alcohol circle (DIY flame sterilization technique)
• Unscrew the bottle’s lid close to the burner and sterilise its edge placing it directly over the flame.
• Quickly open the petri dish and pour 4-5mm height of the medium
• Close and let it cool down.

step 5: Inoculate the petri dish

• Use the same setup as step 3.
• Sterilize your hands (wearing gloves) with alcohol.
• Place the petri containing the medium within the alcohol circle.
• Sterilize the scalpel with the flame until the blade is red hot.
• Cut a 1-3 cm2 piece from purchased spawn, or a small triangle from fresh mushroom gills.
• Open the petri towards the flame with your free hand and with the scalpel place the piece on the medium.
• Close the petri with Parafilm.
• Mark each petri dish with date, mushroom strain, and type of medium. Note : You can also use spores or liquid culture.

step 6: Incubate for two weeks

• Place the petris in a dark closed controlled environment.
• The temperature should stay from 24-27°C, and relative humidity (RH) between 80-90%.
• Increased CO2 concentration in the air has a positive effect on mycelium growth.
• Open for fresh air exchange every 1-2 days.
• Incubate for two weeks.

Part 2: PREPARE AND INOCULATE SPAWN

Preparation and materials

Equipment, Tools and Materials

Tools

• Glass jars

Materials

• Substrate for spawn : half a cup of dried rye grain or barley is recommended

Step by Step Instructions

Mycelium spawn is a nutritious substrate that has been fully colonised by mycelium, it usually comes in the form of grains or bird seeds, as the smaller particles work better when inoculating larger substrates.

STEP BY STEP overview

step 1: Prepare the Spawn’s substrate

• Thoroughly rinse half a cup of dry rye grain or barley until the water runs clear.
• Soak the grain in 1 cup of distilled water for 12- 24 hours, adding a quarter teaspoon of gypsum for calcium carbonate.
• Put the grain in a pot, bring to a boil and simmer for 10 minutes.
• Strain the grain and shake to help it dry.
• Fill up from 3 to 4 glass jars to three quarters with grain (depending on how much the grain swelled).

step 2: Sterilize in the pressure cooker

• Place the lids loosely on the jars. Lids should have air exchange holes using either micropore tape or filter wool to cover.
• Place foil over lids to ensure no water droplets fall on lids.
• Add approximately 1,5 litres of water in the pressure cooker (or enough for 1,5hrs).
• Place the autoclave bag inside the pressure cooker and place the jars vertically with the loose lid inside the bag.
• Close and pressure cook for 1,5 hours at 15 psi
• Remove the pressure cooker lid gently. Release the steam carefully away from the face before opening.
• Tighten jar lids when they are cool enough to touch.
• Remove jars and let cool.

step 3: Sterilize your working area

• Clean the table with alcohol and paper.
• Pour alcohol in a circle.
• Place the Bunsen burner in the center and turn it on.
• Let it burn for a few minutes before starting the next step. Note : if you are working in a laboratory, you can also work under a sterile hood, or a glove box, instead of using the burner.

step 4: Inoculate the Spawn

• Sterilize your hands (wearing gloves) with alcohol.
• Place the jar containing sterilized grain within the alcohol circle.
• Sterilize the scalpel with the flame until the blade is red hot.
• Cut a 1-3 cm2 piece from the mycelium petri dish, from purchased spawn, or a small triangle from fresh mushroom gills.
• Open the jar towards the flame with your free hand and with the scalpel place the piece on the grain.
• Close the jar with Parafilm.
• Mark each jar with date, mushroom strain, and type of spawn substrate.

step 5: Incubate for three weeks

• Find a ventilated, humid and dark space to place the inoculated grain to grow.
• The room or space’s temperature should stay from 21°C to 27°C, with near to 70% humidity. Or use an incubator.
• Open for fresh air exchange every 1-2 days.
• Leave for three weeks to grow.

Part 3: PREPARE AND INOCULATE THE BULK SUBSTRATE

Preparation and materials

Equipment, Tools and Materials

Tools

• Large metallic bowls
• Sprayer bottle with water
• Large Pot
• Stove

Materials

• For substrate : Spent grain from the beer industry is recommended to start with. You can also use hemp, coffee grounds , sawdust, wood chippings, straw, cow manure, etc. (Most organic matter that can be sourced as by product of other processes, agricultural waste or surplus.) For hemp, sawdust and wood chipping make sure to add flour to the mix.
• Prepared spawn or mycelium culture.

Step by Step Instructions

STEP BY STEP overview

step 1: Prepare the substrate

• Use different materials that contain cellulose, such as : spent grain from the beer industry, sawdust, woodchips, straw, hemp, coffee grounds, wheat husks, manure, etc.
• Search for components with different granulometries so that there is air for the mycelium to grow into.
• Mix all ingredients in a clean bowl and add flour.
• Add distilled water with a sprayer bottle, until the mix is humid but not dripping. Note : It is recommended to start by using spent grain from the beer industry. If you get it fresh, it will already be pasteurized from the beer making process. Also, if you use hemp from animal shops, it’s normally already been pasteurized.

step 2: Pasteurize the substrate

• Place the substrate in a mesh bag, nylon canvas should work.
• Find a pot big enough to fit the substrate volume.
• Fill halfway with tap water and bring to a boil.
• Once boiling, bring the temperature down, place the substrate in the pot and let simmer for an hour.
• Ideally maintain a temperature of 75°C - 80°C.
• Turn heat off and remove the bag when cool enough.
• After the substrate has been pasteurized, let it cool completely.
• Rinse excess of water.
• Place the bag on a drying tray to let the excess water evaporate.

.

=== "step 3: Disinfect the container for growth"
    * Select a container for mycelium to grow strong before 3D forming, generally a grow bag or a glass jar
    * Disinfect the container using alcohol.



=== "step 4: Inoculate the Substrate"
    * Place all your materials in a sterile environment. It can be a separate room cleaned with bleach and alcohol.
    * Use protective gear inside the sterile environment.
    * Sterilize your tools and working surface with alcohol.
    * Place the pasteurized substrate inside the disinfected container.
    * Place your bisturi in the flame to sterilize.
    * Cut small triangles from the mycelium petri dish, or previously prepared spawn, and distribute in the bulk substrate.
    * Mix and close the bag or glass jar. Make sure that the bag has a vent window or the jar has holes covered with cotton for allowing the air exchange

=== "step 5: Incubate for two weeks"
    * To incubate the substrate, keep the inoculated substrate bags from 21°C to 27°C and 70% level of humidity.
    * Fresh Air exchange 
    * Leave in a dark room under these conditions for 2 weeks to grow.
    Note : If after a few days, if you see mold growing in the jar (red, green, black spots) you can try to remove it to let the mycelium grow but it might well be that you need to throw away your substrate and start again the process of inoculation because it has been contaminated. If the substrate generates a strong smell, that’s also a sign that the mycelium is not growing well and it’s rotting.  
    === "step 6 : Transfer to the mold"
    * Once the mycelium has grown strong and healthy, transfer to the molds.
    * Place a growing pasteurized substrate inside the mold.
    * Inoculate (=put several pieces of mycelium in the mold) the substrate
    * Press well and fill the mold completely with the substrate.
    * Don’t hesitate to break the pieces of mycelium, it’s actually good to develop new mycelium quicker.
    * Wrap or cover the mold with plastic film but let some air circulate (for example by making holes in the plastic film)
    * Incubate for 2 weeks in a dark and humid place.
    * Once the sample is fully colonized, take off the mold and let the sample dry for 1-3 days.
    * Put the sample inside the oven at 70°C for 5 min to 1 hour depending on the size of your mold to stop the growth.

Safety rules & Tips

• Wash your hands, then disinfect them with alcohol-based hand sanitizer before and after the process.
• Sterilise your working area before working, cleaning with alcohol, throughout and after the process.

Flyer for participants

Tips to facilitate the activity in context (to-do / not do)

• Set up an eventbrite for registration and communicate via your channels.
• Due to Covid regulations, participants were not allowed to come to stations and watch the demonstrations from a close distance. As a solution, a TV with a tripod and a camera was installed over the stations and participants could see what was happening from the top through the screen
• For the better understanding of the materials, it is recommended to have samples to demonstrate the outcome of the recipes

Estimated cost
We recommend purchasing tools and materials locally, the cost is approximate and the list serves as reference. You might plan some costs for getting the materials and tools but also some fees linked to your collaboration with the place you run the workshop.

Item	Quantity	Cost (€)
Spawn
Pack of Fresh mushroom	1u	2
6 glass Petri Dishes 100 mm diameter	1u	70
20 Plastic Petri Dishes 100 mm diameter	1u	60
Distilled water	1l	1
Nutrient Agar	100g	28
Malt extract Powder	100g	33
Yeast	7g	1
Peptone	100g	22
Pressure Cooker	1u	50
Autoclave bags	100	47
Scalpel	10u	5
Bunsen Burner (or camping gas stove)	1u	30
Pack of Disposable Gloves	1u	4
250 ml Reagent Bottles with blue lid	2u	10
Permanent marker	1u	2
Parafilm	38m	32
95% Ethanol	1u	5
Dried rye grain or barley	1kg	3
Glass jars	1u	2
Spent grain, hemp, coffee grounds , sawdust, wood chippings, straw, etc.	3u	6
Flour	1-2kg	2
Large metallic bowls	1u	3
Sprayer bottle	1u	1
Large Pot	1u	25
Stove	1u	30
Kitchen paper	1u	2

References

Links to photos and recorded materials

A selection of readings around sustainable biomaterials and material design by Anastasia Pistofidou
1. Kickstart you Myco-culture!
2.How to grow Oyster Mushrooms
3.[cultivation of good and healthy mushrooms] (https://www.boletsdesoca.com/)

Licence and credits

Attribution — ShareAlike CC BY-SA This activity has been designed by Anastasia Pistofidou for shemakes.eu. It is based on Fabricademy´s learning content of Biofabricating Materials, FabTextiles´ open publications and Iaac Fab Lab Barcelona´s learning experiences methodology.

Related and supporting activities/modules
It can be combined with the other topics within the Sustainability Package of Fabricademy, such as Biochromes and Textile Scaffold. See clubs, Bioshades, Kombucha

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Last update: September 15, 2022