Once you have been growing your own oyster mushrooms successfully and enjoying the fruits of your labour, you may like to complete the cycle and become independent by producing your own oyster mushroom spawn.
This instructable describes how to propagate oyster mushroom spawn via grain spawn transfer, agar tissue culture transfer and liquid inoculation methods. These methods are all low tech (requiring only basic equipment), covering the pleurotus ostreatus (winter) and pleurotus pulmonarius (summer) varieties.
See related instructable - How to Grow Oyster Mushrooms (Low Tech)
Step 1: Materials
You will need...
Wide Mouthed Jars (1 litre)
Pressure Cooker with pressure gauge installed (22 litre – to fit approximately 8 Jars)
Seed or Grain (birdseed or millet seemed to work well)
Cotton Wool (to filter out contaminants)
Bowls (to soak grain)
Sieve and Ladle (to rinse and drain grain)
Spoon, Knife and Fork (for working with spawn)
Aluminium Foil (for wrapping jar lids etc in pressure cooker)
Drill (to provide air hole in jar lids)
For Grain Spawn Transfer:Original Spawn Master (Pleurotus ostreatus (for winter) or
Pleurotus pulmonarius (for summer) buy online and have it delivered
For Agar Tissue Culture: Scalpel, Alcohol Burner, Petri Dishes, Nutrient Agar and Young Mushrooms
For spore-mass/liquid inoculation:Syringe (5ml), Mushroom Spores, Jar of Sterilised Water
Clean Room:Wood, Plastic, Silicon Sealer, Nails, Staples, Bleach
Step 2: Prepare Clean Room
To perform the grain inoculations, you require a sterile environment. Air is full of impurities and so it is important to reduce the level of containments where possible. This can be achieved by constructing a simple clean room. Using thin lengths of wood, nail these together to make four wall panels. Cover these panels with plastic sheeting (use staples to attach). Screw the four panels together using sealant along the joins. Run sealant between the base of the walls and the floor. Finally, tape a plastic sheet over the top of the room to form its ceiling. For an opening, you can cut a suitably sized slit, attaching duct tape tabs with stick on Velcro acting as fasteners. The room should be able to be easily wiped down with a bleach solution. The room also requires a work bench and storage shelves for incubating your jars of spawn.
Most mushroom clean rooms have a HEPA filter installed to provide clean oxygenated air. This instructable uses a low tech approach and will give you alternative methods for minimising air contaminants.
Step 3: Prepare Jars
Preserving jars are ideal for preparing mushroom spawn. You should be able to find 1-2 litre jars at your local preserves outlet (I found using 1 litre jars better for fitting into the pressure cooker/autoclave). If you are unable to buy suitable jars, then recycling old jars is even better (we used 1 litre olive jars). Make sure you clean your jars thoroughly, inside and out.
Taking a drill, make an 8mm hole in each jar lid. Later, this will allow the mushroom spawn to breath.
Step 4: Prepare Grain
Measure out approximately 2/3 jar of grain per required jar (the grain will expand slightly as it is soaked). Soak the grain for 24 hours in a bowl of water (use enough water to cover the grain completely. If you are using larger grains, like wheat or corn, I found simmering the grain for 30 minutes instead of soaking helped to prevent growth of unwanted moulds. Rinse and drain the grain thoroughly. Litre jars should be 3/4 filled with grain and lids (with breathing hole) fitted and capped with aluminium foil.
If required (see Step 8 Liquid Inoculation Methods), prepare a jar of clean water to be sterilised (with a few coins added - these help to cut up grain spawn later when shaking the jar), fit lid and cap with aluminium foil.
Step 5: Sterilisation
Place the prepared grain jars into the modified pressure cooker, with an aluminium parcel of utensils (spoon, knife & fork) to use during the inoculation steps. Add approximately 3 litres of water, pouring it carefully around the jars. Pressure cook the jars at 15 psi for 60 minutes. If the pressure cooker doesn't retain a vacuum, cover the valve with an alcohol or bleach soaked cloth as it cools. Wash your hands with antibacterial hand-wash and in the clean room remove jars (while still warm), giving them a shake to allow the grain to flow freely. Allow the jars of sterilised grain to cool completely. Place the parcel of sterilsed utensils on your work bench for later.
Step 6: Inoculation I (Grain Spawn Transfer)
There are a number of methods to inoculate the sterilised grain. One of the most straight forward and successful methods, is grain spawn transfer. You can purchase your initial grain spawn online and have it delivered.
Spray down the clean room walls with a 1:20 ratio (5%) of bleach to water (It is suggested that a HEPA filter be employed to clean the air, however, this instructable is low tech). Make sure you have showered and are wearing clean clothes. Clean your hands with antibacterial soap or wear sterile gloves. A face mask and hair cap will also help reduce contamination (we are very dirty creatures). Open your spawn bag (or jar) and taking your sterile utensil of preference, break up the grains ready to transfer. Remove the aluminium foil and lid of your jar. Transfer 1-2 desert spoons of the spawn into your 1 litre jar of sterile grain. Quickly, push a small amount of cotton wool through the lid's breathing hole and attach to the jar. Finally, shake the jar vigorously to disperse the grain spawn throughout the jar. Place on a shaded shelf within the clean room to incubate. For pleurotus ostreatus incubate at 24°C (75°F) and for pleurotus pulmonarius (summer) 24°C to 30°C (75°F to 85°F).
Step 7: Inoculation II (Agar Tissue Culture Transfer)
Another method to inoculate your grain, is by first propagating the mushroom tissue on Agar (or cloning). Measure out 5.75 grams of nutrient agar powder to 1 cup of clean water (ample for 5 or more Petri dishes). Begin to heat and stir until the agar is completely dissolved. As it begins to boil, continue to stir for a minute and then remove from the heat. Pour a thin layer into your Petri dishes and cover with lids. Wrap in aluminium foil and pressure cook with your grain (or for at least 30 minutes at 15 psi). Move your Petri dishes, mushroom tissue and other equipment to the clean room. Allow the Petri dishes to cool completely. Spray the clean room walls, benches and floors with 5% bleach solution (as before wear clean clothes, wash your hands etc). A laminar flow bench and hepa filter would reduce contamination during this stage, but it is possible (with a higher contamination rate) to succeed without one.
Taking the mushroom by its base, carefully spit it in two. Place the mushroom (outside down) on to a clean surface, making sure you keep the inside tissue from touching anything. Sterilise the scalpel blade by holding it within the alcohol burner's flame. Lift the lid of the Petri dish and cool the scalpel blade by placing it centrally into your agar. With the scalpel, carefully cut a small square from the newly exposed mushroom tissue. Place the square of tissue centrally into the agar and cover with the Petri dish lid. You may wish to tape the lid (with a clean breathable tape) to reduce the chance of contamination. Repeat the process, making sure to sterilise the scalpel before each transfer. Leave the Petri dishes to incubate.
Incubate for pleurotus ostreatus at 24°C (75°F) and for pleurotus pulmonarius (summer) 24°C to 30°C (75°F to 85°F). Colonisation should take approximately 8 to 10 days.
During this time, remove any Petri dishes that appears to be contaminated with other moulds. Once fully colonised (mycelium nearing the edges), it is time to transfer the agar to the sterilised grain. Choose only the most healthy cultures for the inoculations. Sterilise the scalpel blade, remove the Petri dish lid and cut 2 wedges from the centre of the dish. Remove the aluminium foil and lid of the grain jar. Using the scalpel, transfer the wedges to the sterile grain. Quickly, push a small amount of cotton wool through the lid's breathing hole and attach to the jar. Finally, shake the jar vigorously to disperse the mycelium from the agar throughout the grain. Place on a shaded shelf within the clean room to incubate (temperatures as above). Repeat for each jar of sterilised grain.
Note: Cloning repeatedly (without introducing new strains) may lead to replicate fading, with subsequent cultures eventually losing vitality and therefore producing less mushrooms. In contrast to cloning, spores when germinated (see Step 8b), create many different strains that compete with each other. The resulting mushroom characteristics may therefore vary from culture to culture.
Step 8: Inoculation III (Liquid Inoculation Methods)
One simple way of making your purchased mushroom spawn go further, is to add it to sterilised water, making a liquid inoculant. Taking the sterilised jar of water (see step 5), remove the aluminium foil, open the lid and add a number of desert spoonfuls of grain spawn (the more spawn you use, the faster your liquid inoculations will colonise and the less chance there is for contamination). Attach the jar's lid and reattach the aluminium foil and shake vigorously. Adding some coins to your water jar, before its sterilisation, helps to cut up the grain spawn as you shake. Take the syringe (5ml) and piercing the jar's aluminium (through the lid's hole), draw 5ml of the liquid inoculant. Take the sterilised grain jar, with cotton wool plugging its breathing hole and inject the inoculant past the cotton and into your grain. Shake the grain, mixing the inoculant throughout the jar. Place on a shaded shelf within the clean room to incubate. For pleurotus ostreatus at 24°C (75°F) and for pleurotus pulmonarius (summer) 24°C to 30°C (75°F to 85°F). You can store the liquid inoculant in the fridge until needed.
Another method of making liquid inoculant, is mixing sterilised water with mushroom spores. The trick with this method is to capture the spores. Pick a young mushroom, you may notice evidence of it having released some spores (fine white residue or powder found on the substrate below the mushroom). Wrap the mushroom in aluminium foil and leave it over night. Open the foil and remove the mushroom to reveal its spore print. Take the syringe and draw out 5ml of sterilised water. Mix some of this solution with the spore print and draw it back up into the syringe. You can then replace the lid and store it in the fridge until needed or simply inoculate as above.
Both methods above are to be performed within the clean room as in steps 6 and 7.
Step 9: Inspect Jars
With grain spawn transfer, you should notice popcorn sized colonisation evenly spread throughout the grain after 1-2 days. If needed, give the jars a shake to evenly distribute the mycelium to aid colonisation. Once the mycelium is relatively uniform throughout the jar, leave it to incubate. The grain should be fully colonised with mycelium in seven to ten days.
With agar tissue culture transfer and liquid inoculation, you should notice an initial fine mycelial network, jars may need to be shaken after 4-5 days. Colonisation should be complete by day ten.
Remove any jars showing signs of contamination (such as green mould) from the clean room. Once the jar is fully colonised, you can use it to inoculate more jars, or store it at low temperatures until needed and of course you can use it to inoculate straw substrate to grow mushrooms (see instructable).
So there you go... not exactly rocket science.
Note: It is considered good practise not to propagate spawn beyond the third generation to prevent higher contamination rates. One litre of master culture should be able to produce 10 litres of generation one, 100 litres of generation two and finally 1000 litres of generation three. Contamination rates of 10% or less are considered acceptable.
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