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Non-sterile experiment


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#1 newmoon

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Posted 26 February 2019 - 01:18 AM

Hello all,

 

After a long time of reading this site but having nothing to share, here's something new. This experiment is still in its early stages, but perhaps it will be of interest. Any replication or refinement is much appreciated.

 

The goal is to find a completely non-sterile method of cultivating mushrooms, based on a couple of observations:

1. Established mycelial growth is resistant to contamination

2. Some substrates are resistant to contamination, but won't directly allow germination.

 

The idea, then, is to germinate the fungi on a minimal amount of material, and then transfer to a resistant substrate and build up mass gradually from there.

 

Materials used:

1. Oatmeal - "old fashioned rolled oats"

2. Coir

3. Worm castings

4. Small capped test tubes (15ml or less) or tiny jars.

5. P. cubensis "B+" spore syringe from a commercial vendor

 

Nothing is sterilized or pasteurized.

 

Place one oat at the bottom of each tube, dry. Add one drop of liquid from the syringe (this is enough to hydrate the oat). Seal the tube and let sit at room temperature. Once the oat is mostly colonized, add a small amount of 50/50 coir/castings mix on top. Once that's colonized, go from there. Alternately, transfer the colonized oat to damp cardboard and expand that way.

 

After a week, 14/20 tubes have shown growth, 4/20 have grown mold, and 2/20 have grown bacteria. A picture is attached of one tube starting to colonize the "bulk" substrate. More replicates will be performed and better pictures added as time allows.

 

This also appears to work with Psilocybe cyanescens, transferring to cardboard after the oat is colonized.

one_grain.JPG


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#2 Myc

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Posted 26 February 2019 - 09:51 AM

Welcome newmoon!



#3 newmoon

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Posted 26 February 2019 - 10:21 AM

Thanks, Myc! I've learned a ton from your posts over the years, especially about wood lovers.

 

Here's a picture of an oat that was transferred to cardboard two days ago. Toilet paper roll moistened with reverse osmosis water. This one is also B+ cubensis.

 

tp_oat.JPG


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#4 Microbe

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Posted 26 February 2019 - 07:21 PM

I love your thought process. The growth on the cardboard, looks damn good. I love experiments and will surely be checking in on this frequently. Im about to resume all my experiments i had to place on hold because of life and cold ass weather!

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#5 newmoon

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Posted 28 February 2019 - 12:33 PM

Thanks, Microbe!

 

The tube in the first picture (B+ cubensis) has been coming along nicely, so today I transferred it to a 1/2 pint mason jar and added a layer of coir/castings on top. Hoping that will colonize well.

cube_b.JPG

 

The B+ cubensis oat on cardboard has also expanded well. I imagine one could do non-sterile strain isolation and other agar techniques on cardboard like this (I know that other people have experimented with this before), but I'm not going to bother this time around. So, I trimmed the cardboard, gave it a day to keep growing, and then put it in a jar with coir/castings.

cube_card.JPG

 

Finally, here's an oat colonized by Psilocybe cyanescens deciding whether it wants to move to cardboard. This is also non-sterile, I just put it in a petri dish with a parafilm wrap because it was a handy container.

ps_cyan.JPG


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#6 newmoon

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Posted 03 March 2019 - 03:36 PM

Unsurprisingly, the "bulk" jar with the first B+ tube started growing mold. I did some surgery to see if I can recover anything, but I'm not optimistic. More where that came from, anyway.

 

This did make me realize that I'm still thinking too much in the sterile way, where one grows inside a jar with a few air holes covered in micropore tape. This doesn't work here, obviously. So, in the future I'm going to try making it closer to an open air situation after the initial piece of grain is colonized.

 

I've also started an experiment to get a better understanding of the germination rates with this approach.

 

tubes - Copy.JPG



#7 newmoon

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Posted 12 March 2019 - 11:15 AM

I feel pretty comfortable saying that starting on an oat and then transferring to cardboard is a viable non-sterile first step for both ps. cubensis and ps. cyanescens. Somewhere around 1/3 of the oats get to this point, after which I transfer to cardboard:

tubes.JPG

 

I think the tubes are still too large, and so moisture control is difficult. I'll be trying some more oats in much smaller containers, and I expect that the success rate will be higher. Even with 1/3 success, I think it's ok. The cost is pretty much free and it doesn't require any time to set up.

 

Ps. cyanescens, just moved to a bigger jar of damp cardboard:

cy.JPG . I plan to expand this out on cardboard/paper/wood chips.

 

Ps. cubensis after a few days on cardboard:

cube.JPG cube2.JPG

 

I'm still playing around with different ideas for the next step for cubensis. I'll also be starting some other wood lovers and oysters to see how those work.


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#8 Sumgi

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Posted 12 March 2019 - 12:26 PM

That's looking great, thanks for keeping us informed! 



#9 newmoon

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Posted 20 March 2019 - 12:49 PM

The coir/castings mix seems prone to mold in sealed containers, so next I'm going to try castings alone. I've kept castings in airtight containers for storage for many months with no sign of growth.

 

Took this oat on cardboard:

growth - Copy.png

 

Cut it approximately as the sectoring suggested:

cut - Copy.png

 

Put each piece in a jar of castings:

jars - Copy.png

Each lid has a 9/32" hole, with a single layer of micropore tape to keep humidity in and insects out. The castings have been stored in freezing conditions to hopefully kill off any invertebrates.


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#10 newmoon

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Posted 26 March 2019 - 02:25 PM

I've started a good number of jars like the one in the previous post. Here's the one with the most growth:

growth.JPG

The hyphae are moving through the castings at a reasonable speed, but the growth isn't dense - just tendrils. There are no obvious signs of other molds competing. Does anyone have an interpretation of this? I'm wondering if it could be a lag time switching from eating cardboard to eating castings, but I don't know.



#11 picapau

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Posted 26 March 2019 - 08:15 PM

This is a fascinating experiment.

 

I have no answers to your last post...but I do have a question.

 

Have you considered a thin top  layer of vermiculite  in the jar of castings?

 

Would that allow for the jar to remain open even?

 

 

Just curious...


Edited by picapau, 26 March 2019 - 08:16 PM.

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#12 newmoon

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Posted 26 March 2019 - 10:10 PM

Interesting idea! What you're saying, I think, is that a layer of vermiculite would keep insects out and prevent it drying out while still allowing greater air flow? In this context, of course, blocking other spores and contaminants isn't worth bothering about, since they'll probably be in the castings already.

 

It might work - I'm not sure if adding vermiculite would make the castings more likely to start growing mold. My intuition hasn't been very good about this, I've been learning. I don't have any vermiculite on hand at present, but might pick some up to try it. I also wonder if there's a difference between a vermiculite barrier and adding more air holes.

 

I am trying one completely open air approach: I transplanted a houseplant recently, so I buried three of the colonized test tubes (like the first picture of post #5 in this thread) in the soil mix. Not sure if it'll lead to anything, but thought it worth a try. I've had luck burying slices of conventional brf cakes in indoor flower pots previously.



#13 picapau

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Posted 27 March 2019 - 10:38 AM

My understanding of the vermiculite is that because it has no real nutritional value  bacteria and such won't grow in it, so it makes a good barrier and helps keep things moist while allowing air exchange.

 

The more experienced peeps here would know for sure.

 

But I'm intrigued by your experiment. Obviously, the mushrooms find ways to thrive in 'non-sterile', open air, natural environments.  Everything I've heard about the way mycelial networks grow, which isn't a whole lot, suggests that they have an intelligence that is unique to the environment they are growing in. So, I would guess that if you kept building one of your cultures up in strength that it would ultimately begin to thrive  in your specific environment. Like the way sourdough starter works to some degree.

 

I might be waxing simplistic in that regard but I will keep reading your updates and may have to make time to do my own experiment.    


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#14 Athanasius

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Posted 28 March 2019 - 01:40 PM

Posts like this are evidence that shrooms definitely aren't hurting intelligence.  Great experiement!  If this works I might give it a shot on some non-psilly shrooms.  I wonder how many more people would grow oysters at home if they weren't restricted by having a sterile environment. 


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#15 newmoon

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Posted 31 March 2019 - 06:32 PM

My apologies if this is boring - I know it's sort of a bunch the same thing without any conclusions.

 

I've been trying 1.5mL micro centrifuge tubes for starting the oats, and so far I think they're the best choice. $0.03 each. They're the perfect size for one oat and a drop or two of spore solution. Trich shows up before the psilocybe germinates, so far, and so it's easy to weed out the failed tubes. I have a batch of cubensis from a syringe and a batch of azurescens from a print in progress in these tubes, will post pictures once they're more photogenic. I'm still finding success rates of around 1/3, which I think is fine.

 

I tried some other small containers which turned out to slowly absorb water, leading to everything drying out. Whoops.

 

Everything is moving slowly due to the low temperatures in my space, but hopefully I'll have progress to share soon.

 

Here's a picture of a very rhizomorphic P. cubensis on cardboard.

rhizo.JPG

 



#16 Sumgi

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Posted 02 April 2019 - 02:51 PM

Good stuff!

Not only are you trying new and interesting things, you're actually following through with keeping updates rolling!

I already have a PC on-hand and a hepa filter on order, but years ago when I started growing mushrooms I was a real penny-pincher. You have a set-up that could get someone up and running for under $30, including a spore syringe! 

I'm interested in seeing how this continues on; maybe I'll have to get some centrifuge tubes myself and start playing around with this...

I've not worked much with mushrooms needing casings, but had thought the casing to be a non-nutritive addition that creates the environment to allow for fruiting not an actual food-source in and of itself. Will you be adding more nutrition to the cardboard/casing set-up? Again, I'm fairly inexperienced with these methods so this is coming strictly out of curiosity.

 


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#17 newmoon

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Posted 02 April 2019 - 07:53 PM

If you have a little extra solution left over after noccing up your next round of jars, please do give it a try! It'd be great seeing how this works in someone else's situation. I think those little flip top 1.5mL centrifuge tubes are good, but any tiny container that doesn't absorb moisture should work fine. Seems like one oat and one or two drops of solution is good in a container that small. Something bigger will need some more moisture. I've been having better luck with an eye dropper than a syringe, but you might be less clumsy and not have a problem. I've been finding that it takes a couple/three weeks for growth to start, but it's cold where I'm living. Mold seems to show up before that if it's going to, luckily.

 

One mL is about 20 drops, so it doesn't take much of the syringe to do a few dozen oats.

 

My thought is that cubensis seems to do pretty well eating worm castings. I've had luck in the past spawning grain to castings or castings + coir (with conventional grows). So, I've been thinking of the castings as a bulk substrate rather than a casing. I might try some other thoroughly composted manures if this doesn't work, since those can be pretty resistant to molds too if they're old enough. I like worm castings because they're free once you get a couple rubbermaid totes and some red wigglers :biggrin: .

 

I'm surprised by how well cubensis does on unsupplemented cardboard, so I might keep that one in the last post going and see what happens. It's low-quality cardboard, so I'm guessing it has a higher cellulose:lignin ratio than good stuff. Maybe that's why the cube's eating it, dunno. Interesting stuff to me, anyway.

 

As far as motivation, besides finding a cheaper way, I'm kind of turned off conceptually by sterile approaches to growing life. I'd rather be growing plants in dirt & worm shit than in a test tube or in the pesticide/agrochemical nightmare we're in now, and I feel the same about fungi, if it's possible. If there's a way to work with the microbes instead of nuking them I'll feel pretty happy when I can sell my PC. Sorry if that's too off topic!


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#18 Sumgi

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Posted 06 April 2019 - 09:16 PM

Haha, I've been seeing "castings" but been reading it just as "casings." It all makes sense to me now  :biggrin:!

I like the idea of getting some different pets besides various myceliums; I may have to give red wigglers another try. I finally birthed our sealed accumulated compost buckets into the pile yesterday since it's finally thawed out; maybe the worms could keep up with everything indoors over winter otherwise and I wouldn't need to replicate this springtime ritual... Plus they'd be working in pursuit of a higher purpose!

I get what you're saying with the sterile culture thing; everything evolved to live as part of an ecosystem so there should be a balance you can find that it works with. I manage a small organic orchard and I try to avoid any of the broad spectrum pesticides because that reset of killing everything just sets me up for more pest problems. I'm trying to incorporate an ever increasing number of native plants in an insectary to create as much biodiversity as possible to limit the open niches that allow my bigger problem pests to thrive and hopefully create a balance that is tolerable and doesn't require my intervention. 

I have some stuff on order but don't know how soon everything will show up. The challenge for me will be finding the balance between sterile culture complete lassaize faire. Like, wear gloves or not, wipe down everything with alcohol, only some things, or no things. Black and white is easy, gray is an unbounded expanse...

At the very least it seems like what you're doing now has already shown that it can, indeed, be done!  :smile:

 


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#19 newmoon

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Posted 07 April 2019 - 11:37 AM

Ha, that makes more sense. Everyone in the compost world calls them castings, so I've gotten stuck in that vocabulary. Pretty easy in this context to miss that "t", though :biggrin:

 

Vermicompost is great. Low maintenance, good product in the end, very friendly environmentally (except for the invasive potential of some of the worm species in some ecosystems, but that's manageable with a little care). The greenhouse gas emissions are lower than mesophilic composting and comparable to thermophilic, but it's much easier to optimize at a home scale than thermophilic composting.

 

Very cool about your orchard! I've seen research indicating that native plant biodiversity can increase yields by suppressing pests, as you mention. Agroforestry and agroecology seem like the best hope for sustainable food production in our future.

 

 

I think this is a way to get some good mycelium on cardboard without any equipment or concern for sterile procedure, but I'm still not sure about the best place to go from there. I'm planning to play around with this over the next year as I'm able. Maybe you'll figure something out! If my current attempts with jars of worm poop fail, I'm thinking I might try a variation of Hyphaenation's puck tek next. I'm also trying some open air flower pot grows.

 

Sterile procedure is crucial to the normal approaches, of course. Doing the PF tek or grain or agar without good lab technique will just end in mold. But, with this different type of approach I've not taken any precautions beyond washing my hands.

 

Here are some of those 1.5mL centrifuge tubes I mentioned. These will be ready to move to cardboard in a few days.

little_tubes.JPG little_tubes2.JPG


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#20 rockybia

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Posted 07 April 2019 - 03:32 PM

This is some very cool stuff, newmoon! Please keep it up. If I were a betting person, I'd say an effective, non-sterile means could be super helpful for people living off the grid, maybe in areas where outdoor cultivation isn't possible. While cleanliness IS the key to success, there are also impracticalities for some folks. Cant wait to see where this goes.


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