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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.
<p>Hi, Can one use a smaller pressure cooker? Dose the cotton wool have to be sterilised, if so how? and when growing mushrooms, can one sterilise the straw in a microwave oven ? Thanks</p>
<p>A larger pressure cooker simply fits more jars - sure a smaller pressure cooker is fine. Depending on availability, you can buy medical grade cotton which is sterile. Microwaving the substrate (very high temperatures killing everything) will sterilise it, which is better than nothing, but will be more susceptible to contamination than pasteurisation.</p>
<p>Hi, the prepared jars i'm using are 1 year old. The growth of the mycelium inside the 3 jars is very different.</p><p>Maybe is because during the last year water inside the jars has gone?<br>Can I add more water now? Thanks</p>
<p>learn what you teach, and you are producing mushrooms, thank </p>
I'm a beginner and need stuffs to start growing different kind of oyster mushrooms in my room this winter, as a practice. Who do I contact or where shall I shop wheat straw, mycelium, spawn, etc.?
<p>Sorry I can't help you much here, it really depends on the country where you live. Have a look on the web for others growing in your area, perhaps a forum and ask there.</p>
<p>BUY some mushrooms in a grocery store, chop them up real fine and toss them is some boiled straw in a canning bottle with a coffee filter over the top when finished held on by the Canning ring.<br><br>I have about 10 of them working well - I scoop out about 2/3 and refill them - either with straw or coffee grounds, and they are off to the races again . . .<br><br>I have moved up to filing boxes (11 x 12x 17) with sterilized straw - and a quart of mycelium in each - kept in unties garbage bags. SO far its working</p>
I believe it or not bought my oysters @ Home Depot. These are my first mushrooms and they are doing awesome. I wanted to get my feet wet so to speak, but definitely hooked. :)
<p>Okay, I'm thinking out loud here and need help clarifying my thinking....</p><p>Spores are seeds and agar is a sterile growing medium. Why can't you just put spores into agar?</p>
<p>Hey Jen, yes you are right, however the key to a healthy uncontaminated culture is to reduce the exposure time during spore transfer. If you are simply transferring from a spore print, the time the agar is at risk of contaminants can be minimized. Or at least that's the idea, you can always experiment to see what works for you.</p>
<p>Hello! Great instructions. I have a question regarding sterilization of straw. I have been told that instead of soaking and then boiled in jar it could work heating it directly with water for an hour at 140 F. What is your view about it?</p><p>Also does the straw have to be fully dry for inoculation? And if yes, what is a right method?(letting it dry on a cloth in the clean room, for example) The same question comes using your method, does it have to be fully dry for sterilization?</p>
<p>Hi there, </p><p>I would say for the growing medium (straw), that boiling would be ok, but with soaking first and draining, then heating/steaming, it is all ready to go for inoculation with less chance of contamination. The medium should not be dry (gravity draining is fine). Equally, with seed and spawn propagation, I would suggest it should be drained then steamed to create less chances for contamination.</p><p>But really, you can try different things and let us know how you go, it's all a learning experience. :) All the best!</p>
<p>Hey Rocketsurgery,</p><p>Good article good information so thanks to those who contributed. I have several questions but I will start with one. Getting ready to start my venture. Let's say I have innoculated jars that have colonized with mycelium. These were made from spore syringes. I am now ready to mix with substrate (straw in this case). I am going to use grow bags in dark grow room. In the dark grow room what temp and humidity do I need for the bags to colonize? Again I have many questions but instead of bombarding you all at once I will ask one at a time.</p><p>Thanks for your help in advance,</p><p>swimmingupstream</p>
<p>Hi there, Have a look at my other instructable &quot;<a href="https://www.instructables.com/id/1-How-to-Grow-Oyster-Mushrooms-Low-Tech/">How to grow Oyster Mushrooms</a>&quot; it lists the different temperatures and humidity for the various stages of both Winter and Summer varieties of Oyster.</p>
<p>Thanks a lot for yourt instructable. Me and a friend are in the process of following this. But we have some questions:</p><p>1-Would this low tech approach also work for the less agressive shiitake? Or would we need a HEPA filter for that?</p><p>2-Doesn't the humidity in the clean room need to be controlled in any way?</p>
<p>Shiitake should be fine (depending on your source and level of senescence). I would recommend trying the cloning method with Agar as I have seen this work well with store bought Shiitake. The humidity of the clean room is not too much of a concern as you are propagating in enclosed containers, so a comfortable room humidity 60-70% would be fine. Let us know how it goes.</p>
Hey Rocketsurgery, great instructable! <br><br>I did a course in growing exotic mushrooms some years ago, but ive forgotten most it. This was very helpful in bringing to memory again what i had learnt.<br><br>Out of those three methods, which is in your oppinion the most reliable? <br><br>Since space in my house is quite limited, i was thinking of building a clean cabinet (instead of a room), like they use for sand blasting or in medical labs. I could maybe put a light bulb and thermostat, and it could double as an incubator. Do you think that will work? Have you seen something similar being used before?
<p>Hey there, I found the grain transfer to be the most robust and reliable, however, Agar Tissue Culture (cloning) seemed to also work well, but definitely requires a super clean environment. A clean cabinet is a very good idea to avoid contamination and you could add an air filter (hepa - pass-through) to help avoid contamination if you find that to be a problem. I'm not sure why but working with spores seemed a less vigorous and reliable method for me (or perhaps I was too impatient), it worked but often resulted with a high contamination level. This method is best to promote diversity (and actually should result with strong vigorous mycelium networks) but relies on good spawn capture methods. All the best.</p>
<p>I want to grow on agar.. Can I just get a mushroom from the market and take a piece from that to inoculate the agar? Or does it have to be fresher? </p>
<p>Hey, You can certainly clone market bought mushrooms. You could also try to harvest a spore print. The issue that you might find is initial slow growth due to the age and stage of the mushroom (growing old or senescence) - but certainly give it a try.</p>
<p>Thanks for going to all the effort putting up this info. It has been very useful to me.</p>
<p>Thanks for taking the time to say thanks :) All the best</p>
How to maintain humidity in 44 degrees .
<p>Maintain humidity by growing in a contained environment (growing room) - but remember to flush the room with fresh air regularly or your CO2 levels will start to effect growth.</p>
<p>nice and ready to share with my youth group</p>
Please sent the process in my mail [omchoudhary135@gmait.com]
Hi! First of all thanks for the info, you sure know what you're talking about! And very well explained which is a pleasure :) <br>I am just starting with the mushrooms and I've been looking around to see which would be the best way in my case. I am based in Ireland, and was thinking of starting small probably with grey oyster and maybe king oyster, which sound like the easiest. I have access to large amounts od woodchips and also sawdust, so i was planning on using that as a substrate. Would the sawdust work for doing the spawn u describe here?<br>And i also wanted to ask, maybe a silly question, but... can you not just have a small batch of mushrooms that you 'let to go to seed' and collect the spores of to keep inoculating new sustrates? Whats is the downhill on that? <br>Thanks! And thanks again for the instructable!<br>Marta
Please send this process in mail [omchoudhary135@gmail.com]
<p>Hi Marta,</p><p>Someone else asked a similar question regarding substrate on my <a href="https://www.instructables.com/id/1-How-to-Grow-Oyster-Mushrooms-Low-Tech/">How to Grow Oyster Mushrooms</a> <a href="https://www.instructables.com/id/1-How-to-Grow-Oyster-Mushrooms-Low-Tech/">instructable</a>, they were looking at 79% Sawdust, 15% Rice Bran, 2% Lime, 2% Gypsum and 2% Milled Corn substrate mix. <a href="http://www.bapress.ca/eah/v1n2/Tajudeen%20O.%20Oseni.pdf">Here</a> is a good guide with regard to additional additives producing larger yields in sawdust. Anyway, I would assume a similar mix would be best for spawn production, although I have never tried it - that's where your experimenting will come to the fore. Have a little fun, try some things and see. Try pure sawdust (no additives) and compare. There are no silly questions, in Step 8: Inoculation III (Liquid Inoculation Methods), I mention how you can use mushroom spores to inoculate your substrate, so you could try that, although for me this was the least reliable - but perhaps you would be more successful. Using mushroom spores, as opposed to cloning directly from the mushroom, can lead to a stronger more diverse collection of spawn, due to the survival of the strongest/fittest.</p><p>I do wish you all the best in your mushroom growing future :) Thanks for your encouragement too! If you think of it, please let me know how you go.</p>
<p>This is actually a very well written and illustrated piece of work. I admire the author for taking the time to put this together. I wish I had found it before I tried to make my first batch. I am now more experienced, but still looking at all types of mushrooms and their propagation.</p>
<p>Thanks for your encouragement!</p>
<p>I really need to learn about how to Grow Oyster Mushroom Spawn. So ,please give the way or mathods. thank a lot.</p>
<p>This instructable describes how to propagate oyster mushroom spawn via grain spawn transfer, agar tissue culture transfer and liquid inoculation methods. If you move through the steps by clicking the &quot;Next&quot; button, you will hopefully learn a few different ways to grow oyster mushroom spawn. All the best.</p>
<p>How can I study about grow Oyster Mushroom Spawn.I want learn about Grow Oyster Mushroom Spawn.</p>
<p>nice and ready to share with my youth group</p>
<p>Thanks - hope your youth group enjoys!</p>
<p>nice and ready to share with my youth group</p>
We can grow morel mushrooms like this too?
<p>actually, morels are a bit more complicated and harder to cultivate. they require more care and different resources.</p>
<p>Yes with a few differences. Have a look at this well written step by step guide for morels. http://www.thefarm.org/mushroom/morel.html</p>
So if you can't &quot;propagate spawn beyond the third generation&quot; how can you truly close the loop? Would germinate spores with sterilized water from mushrooms you produced be viable? Is it possible to not have to depend on a lab for fresh cultures?<br>Thanks for the instructable!
Hey fpe&iuml;&iquest;&frac12;aloza, <br> <br>I'm so sorry for not replying sooner - I only just spotted this comment. <br> <br>The key thing when it comes to mushroom propagation is diversity. You may well have a great batch of mushrooms, but unless you keep the gene pool diverse you have a high probability of mutation and disease. There is no reason not to try emulating a wild mushroom environment (and their propagation techniques), but your yield might not be as great. <br> <br>Thanks for your encouragement!
<p>i have a similar question. <br>Someone, somewhere, is making the spawn you start with. Surely they have some way of ensuring the generation that you get is #1 and not #15000. how do they do it? Do they have some magic mother patch, which the get spores from each year, do they go hunting for edibles, or do they have some other method?</p>
<p>There are two methods for creating new organisms: cloning, and reproduction. The mycelium that you grow mushrooms on is not the roots, it is the entire organism. mushrooms are the reproductive organs of the fungus. the gills on the mushroom have billions of spores inside and those can be collected and spread on the agar medium and germinated into mycelium. The other method is cloning. in this method, you can take bits of the mushroom's stem and put it into agar or coffee grounds, or any other medium for growing mushrooms in, and that will grow in mycelium</p>
<p>My understanding is that if you have a good culture going you can preserve it by making a master slant. That is a test tube full of agar with a bit of wood in it that can keep viable in the fridge for years. Then when you start a new batch you cut out a tiny bit of the master slant with a sterilized scalpel and put it on a petri dish and use that to inoculate your grain jars.</p>
<p>It is about spawn diversity and incredibly sterile working environments. You can now see that some of the major spawn producers/sellers are teaming up to increase diversity and spawn quality. You know, humans have the same issues with propagated defective genes, you simply don't notice the issues until the population size is small and perhaps where marrying first cousins is the norm. We all need to preserve and cellebrate diversity, wherever we find it.</p>
<p>Fantastic Instructable! Your thoroughness in imagery and descriptions were very helpful and I appreciate all the time you put into creating this!</p>
<p>Thanks for taking the time to say so!</p>
<p>Nice one!, all the basics, i recomend to read statmets books for extra information, let me share some of my work <a href="http://www.setasdesiecha.com" rel="nofollow">www.setasdesiecha.com</a> is in spanish.</p>
<p>Thanks, yes I have read lots of Paul Stamets... he is the great mushroom guru and certainly the source of most of my mushroom knowledge... thanks for your encouragement :) Thanks for your link, I'll check it out.</p>

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