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Offlinedoctor_kandee
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A dead horse subject: Liquid Mycelium, a new approach?
    #4085512 - 04/22/05 05:37 PM (12 years, 1 month ago)

Ok, so I've read many people's attempts at culturing liquid mycelium as the only source of psychoactives as outlined in the Psilocybin Production Manual or something like it. This was, for those who are unfamiliar, a manual for producing 5000 doses of psilocybin in a small room with easily obtained materials. Ok, after having read many people's trials and errors, and reading a few things about liquid cultures in bioreactors, I have a question.
Is areation really the only issue that comes in to play here? I understand that liquid mycelium doesn't contain as much psilocybin blah blah blah. But the time it takes to supposedly grow liquid mycelium would more than make up for this fact. Ok, so all of these things have been said a million times. Here's my question.
If we somehow increased airflow throughout the entire vessel that contains the liquid mycelium, would we have a greater chance of culturing an entire jar of fluffy white mycelium that could be dried? I have the book Psilocybin Producers Guide or something by Stamets, I don't have it in front of me. Anyways, in the book, there are pictures of a quart jar completely full of fluffy white mycelium without rye grains or anything. Assume we were to introduce clean air for a 12 hour a day flow, does one think this could happen? I'm picturing using a filter flask like pictured here: http://www.coleparmer.com/catalog/large_image.asp?img=3455821.jpg and adding an aquarium air pump filtered, to add a constant supply of clean air. I'd stop up the top with cotton or another suitable substance to allow gas exchange. Does this seem like it may solve the problems with liquid culture? I'm speaking of liquid culture for harvesting of the mycelium, not for using as a starter for pf-cakes. What do you all think? I figured this may be a way to make a very basic bio-reactor environment with home materials.
Sorry this post was so long, but I wanted to cover everything anyone could ask, and stay on my goal: harvesting liquid mycelium for personal use.


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Offlineprototypical_man
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: doctor_kandee]
    #4086397 - 04/22/05 10:34 PM (12 years, 1 month ago)

the amount of myc yeilded this way would be insignificant


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Offlinepsilocyben
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: prototypical_man]
    #4086411 - 04/22/05 10:39 PM (12 years, 1 month ago)

half of your post count can be accounted for with this thread:what:


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InvisibleSoopaX
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: prototypical_man]
    #4086917 - 04/23/05 01:00 AM (12 years, 1 month ago)

Why the fuck would you say something like that ?Either answer his question or keep your opinions to yourself. Jesus fucking christ.


IF you set up a quart mason jar with Karo syrup and had oxygen bubbling though it via an aquarium pump with an in-line filter, you'll grow the damn thing near full of mycelia. It's not that worth it, but thats how you do it.


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InvisibleArsey
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: doctor_kandee]
    #4087996 - 04/23/05 11:45 AM (12 years, 1 month ago)

The design concept you mention is poor and the aertion would work better if it ran contineuosly versus only half the time.

A max conc. of 1% psilocybin occured after seven days in one study and then dropped off significantly. It was stated the larger the vessel the greater the loss of psilocybin possible due to poor aeration.

The design you are proposing appears to be only one step up from a stirred Fernbach. However, stirring with introduced air would allow for some increase of media volume in such a flask. Although, the flask you introduced as an example is not a fernbach. That flask is a filter flask, similar to an Erlenmeyer. With either flask, the air would be better if introduced directly into the media, just like a fish tank. So, really there's no need for a filter flask however, a multi hole stopper and some glas tubing would be in order.

If that's the size and caliber of bioreactor you're considering I suggest you review this website. The practicle fermentation guide may have some more details on the reactor. There's is a bit pricey @ 90pounds but you could follow their design as a basis for your model.

For really significant volumes of media I think you'd need a column type airlift/bubble reactor. Imagine a tube inside of a tube, the mycelium hitch a ride on the bubbles as they rise in one tube. when they reach the top the air exits via the filter and the mycelium drift down the inner tube or outer shell depending upon the configuration. I imagine you could build a five litre model out of PVC and use either peroxyacetic acid liquid flush or a formaldehyde gas to sterilize it. The media could be prepared ina small concentrate and the overall volume of water in a pressure cooker. The nute conc would go in via syringe port and the water could be vacuumed in directly from the PC with some minor modifications. Of course you'd need a five litre or better pc.

For intermediate volumes of media you may want to consider making an attemp at modifying a spawn bag. Imagine the largest bag laying sideways with a shallow bed of media. A .03um filtered air line is introduced through the injection port to keep the bag inflated and provide aeration. A series of these bags could be kept on a rocking table or vibrating table for greater agitation.

One final thought, the amount of starter culture becomes an issue with larger reaction vessels as well.


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Offlinedoctor_kandee
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: Arsey]
    #4088909 - 04/23/05 05:38 PM (12 years, 1 month ago)

Ok here goes another long post, divided into 2
Part 1
Quote:

Arsey said:
A max conc. of 1% psilocybin occured after seven days in one study and then dropped off significantly. It was stated the larger the vessel the greater the loss of psilocybin possible due to poor aeration.





Ok, so I'm really interested just from a standpoint of what Stamets was thinking to try this out.  Would one do better perhaps with pint sized jars with 2 holes punched, one for areation with a tube (as described earlier) going 24/7, and one stoppered with polyfil or something similar to allow gas exchange.  The smaller the size, the lesser loss of psilocybin, yes?  Also, according to Stamet's book, which again, I'm hearing is crap, he proposes 20-35MG of Psilocybin/Psilocin could be harvested from each quart jar.  This, according to other sources, equals about 2 conservative doses or one powerful dose.  So if I cut the size down to pint, could I expect 1 pint to yield one dose?

Quote:

Arsey said:
The design you are proposing appears to be only one step up from a stirred Fernbach. However, stirring with introduced air would allow for some increase of media volume in such a flask. Although, the flask you introduced as an example is not a fernbach. That flask is a filter flask, similar to an Erlenmeyer. With either flask, the air would be better if introduced directly into the media, just like a fish tank. So, really there's no need for a filter flask however, a multi hole stopper and some glass tubing would be in order.




You say glass tubing would be an order, would that be for the air to flow through?  Why would glass be a better alternative to simply cheap plastic tubing I can get at the hardware store?  Also, if I were to just shove some tubing down the stopper, I imagine it should touch the bottom, yes?  Just trying to get this together in my head.  I thank you very much for the valueable links.  I'm looking at the bioreactor site you suggested now, and I'm thinking I could do that cheaply...Maybe I'll try it and make a tek on it...

Quote:

Arsey said:
For intermediate volumes of media you may want to consider making an attemp at modifying a spawn bag. Imagine the largest bag laying sideways with a shallow bed of media. A .03um filtered air line is introduced through the injection port to keep the bag inflated and provide aeration. A series of these bags could be kept on a rocking table or vibrating table for greater agitation.




This sounds like something that could be done rather cheaply, which is what I'm attempting, something for the home cultivator looking to cultivate the mycelium without having to fruit the mushroom. 

Part 2
Quote:

SoopaX said:
IF you set up a quart mason jar with Karo syrup and had oxygen bubbling though it via an aquarium pump with an in-line filter, you'll grow the damn thing near full of mycelia.  It's not that worth it, but thats how you do it.




Would you say it's not worth it due to the lower concentration of psychoactives?  Would the larger amount of mycelium be also of poorer quality?  I'm just wondering why no one ever does extraction of mycelium, as it seems like it would yield a purer product, if in less mass.  Also, according to most things I've read, mycelium can be grown in a week as opposed to longer time period required for fruiting and etc. 
Thanks for answering.
:thumbup:
I thank everyone for their input on a subject I'm becoming interested in, and trying to find usable information on, which is unfortunately lacking on many sites.


Edited by doctor_kandee (04/23/05 05:42 PM)


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Invisiblemycofile
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: doctor_kandee]
    #4093064 - 04/24/05 10:38 PM (12 years, 1 month ago)

Stamets didn't write that book. Stamets wrote books about growing fungi, not some wacked out hippie ass pipe dreams. I'm not saying that liquid fermentation and bioreactors are in themselves wack, just that the 5000 dose book had no real research put into it more than hey lets smoke a joint and play what if.
Just wanted to clear that up. It wasn't Stamets.


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InvisibleMrMaddHatter
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: mycofile]
    #4093713 - 04/25/05 01:23 AM (12 years, 1 month ago)

Quote:

mycofile said:
not some wacked out hippie ass pipe dreams.  I'm not saying that liquid fermentation and bioreactors are in themselves wack, just that the 5000 dose book had no real research put into it more than hey lets smoke a joint and play what if.




:lol: :thumbup: :thumbup:


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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: MrMaddHatter]
    #4094844 - 04/25/05 01:12 PM (12 years, 1 month ago)

Actually, I just read that and it sounded kind of rude. No offense intended, just making a point of clarification. All apologies.


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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: mycofile]
    #4094883 - 04/25/05 01:28 PM (12 years, 1 month ago)

Quote:

mycofile said:
Actually, I just read that and it sounded kind of rude. No offense intended, just making a point of clarification. All apologies.




Nada wrong with telling it like IT IS, as you did.


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InvisibleIDontGrow
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: agar]
    #4095342 - 04/25/05 04:04 PM (12 years, 1 month ago)

your contam rate would be damn near 100% just fruit some 1/2 pints invitro...still the best easy and space saveing way i can think of...


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Offlinedoctor_kandee
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: mycofile]
    #4095952 - 04/25/05 06:19 PM (12 years, 1 month ago)

Quote:

mycofile said:
Stamets didn't write that book. Stamets wrote books about growing fungi, not some wacked out hippie ass pipe dreams. Just wanted to clear that up. It wasn't Stamets.




Sorry for the confusion of who did what. I have the mushroom cultivator by stamets and I confused the two. However, I do feel there is some practical something to be found in Gottlieb's proposition, after all, mycelium will grow in liquid in small doses, so...
Ok, so after thinking about this even further more than I should, I propose this set up, and please, if this is completely useless, someone tell me.

1. I go to Igloo or wal mart, and purchase a 2 gallon/5 gallon cooler for water like for construction/industrial use, with a spout. I also purchase some vaccuum tubing, a 10 gallon aquarium aereator, and a grow light. Also I buy some silicone sealer, and a plug to run tubing through. Total purchase: $20 if I buy everything brand new at retail cost.
2. I make up batches of liquid substrate, like malt extract liquid or potato dextrose yeast liquid. I pressure cook this in quart jars, sterilizing the medium. Meanwhile, I wash out cooler with 1% bleach solution, and also pc my vaccuum tubing, maybe flush with bleach solution.
3. Now I run tubing into bottom of cooler with silicone sealant, creating a water tight entryway into the cooler. I take my liquid substrate, stick it in cooler, inoculate under most sterile conditions found, maybe even incoluate individual jars for complete sterility, then dump in big container. Now I put aquarium areator, mount lightbulb on top, put lightbulb on 12 hour timer, and let sit. Mycelium is areated, light is present, we could get about 2 gallons of mycelium per pop. Assuming a 12 day cycle, we could get about 2 gallons worth of mycelium areated properly, and then harvested, dried, extracted.
Does this seem like it would work? I pose to you all any suggestions? Areation always seems to be the issue, if we had adequate areation, could not mycelium be made? According to Arsey this is the problem with large scale mycelium production. Let me know if I'm going about this incorrectly.


Edited by doctor_kandee (04/25/05 06:29 PM)


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InvisibleSoopaX
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: doctor_kandee]
    #4096550 - 04/25/05 08:38 PM (12 years, 1 month ago)

Use a large glass jar for your fermentation vessel. Mix 1 part Karo syrup with 9 parts B O I L I N G water. I'd recommend sanitizing the carboy first with rubbing alcohol or iodoform. Rinse it out, pour in the proper amount of karo, then add your BOILING water. Put a cap over it. When it's cool, inoculate with a whole lot of spores. I'm thinking that a 5 gallon carboy would work for this, and for that much, you'd need TONS of spores. Maybe 5-6 LARGE prints worth. Now, for aeration. You'll need a L O N G hose with an air stone at the end. Sanitize this contraption and place it in your inoculated carboy. Now, instead of having the air pump blow air directly into your sugar water, you'll want the air to be sanitized. From your aquarium hose, put an inline filter. These are sold with the aquarium shit at your local aquarium shit store. It's just a plastic tube with some polyfil. This is an OK prefilter, but you are going to want the air to be much cleaner. The hose from your bubbler, after the in-line, will need to be run into a jar that is about half filled with h2o2 (straight from the bottle). Now, the hose in the h2o2 will be connected to an air stone so that smaller bubbles are formed. This h2o2 jar will be kept in the dark so that the h2o2 doesn't decompose as much. The lid should be very tight and the in-hose should be caulked around so that no air escapes. Now you have air being bubbled through h2o2. Now, the lid on the h2o2 jar is going to have to be air tight so that no air leaks out, and it's going to have to have an "out" hose for the sanitized air to go through. Just make it air tight, the hose sticking about 1/2" inside the jar. The sanitized air will be pumped out through this hose and into your carboy.

Will this work? I can't say.

Should it work? Most likely.

If it DOESNT work, set up another jar with 70% rubbing alcohol, bubble through that, then through the h2o2.

This should work. You'll need a near air-tight seal on the top of your carboy as well to let the air out. Figure something out.


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InvisibleSoopaX
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: SoopaX]
    #4096566 - 04/25/05 08:41 PM (12 years, 1 month ago)

I'd also recommend using the most potent strain of mycelia that you can find, probably, from baby_hitlers writings, azures.


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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: SoopaX]
    #4096831 - 04/25/05 09:42 PM (12 years, 1 month ago)

bubling air through peroxide will not sterilize or sanitize it. It *might* kill any contaminate spores, but it won't kill any living contaminants. Even alcohol A doubt would do the trick unless you really atomized the air bubles. Contams can get a free ride right through the alcy inside an air bubble.

They do make HEPA inline air filters. Fungi Perfecti has 'em, I think they are in the $80 range and I have no idea if your standard aquarium pump would be able to pass air through it. Anyway, that's what I'd recomend for sterilizing the air going through the bubbler.

But going back to kandee:
Quote:

Does this seem like it would work?



sorry, no it doesn't. It sounds like it will contam. I guess it depends on what you mean when you say the most sterile conditions possible, but I would think that the fact that you are using a cooler would mean you probably don't have a clean room, just guessing though.

Quote:

Areation always seems to be the issue, if we had adequate areation, could not mycelium be made?



I don't think areation is the only issue, I think you will run into trouble with sterile technique. Assuming you didn't, and aeration is the only issue, then it's possible. Use the above mentioned inline hepa filter. But then in the end, how good are your extraction skills? How expensive are solvents? Do you already have a fume hood, a sep funnel, a vacuum filter setup?

I don't want to sound like I'm knocking you down. I'm just mentioning things that you will need to address sooner or later. If you already have, good for you and good luck.


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I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: mycofile]
    #4096857 - 04/25/05 09:46 PM (12 years, 30 days ago)

Oh yeah, it sounds like you may not have read all the posts on this dead horse subject. Do some thorough searching and you will find a wealth of ideas and what if's, and a few people with experience under their belt.

Also, you do know that you can do a couple liters of liquid inoculate as long as you use a wide squat flask, filter the top, and provide constant stirring on a stir plate. You knew that right? Cause if you're only shooting for a couple gallons, you could do that with a couple 6 liter flasks and 2 stir plates.....


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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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InvisibleSoopaX
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: mycofile]
    #4097156 - 04/25/05 10:55 PM (12 years, 30 days ago)

Quote:

mycofile said:
bubling air through peroxide will not sterilize or sanitize it. It *might* kill any contaminate spores, but it won't kill any living contaminants. Even alcohol A doubt would do the trick unless you really atomized the air bubles. Contams can get a free ride right through the alcy inside an air bubble.




I'm 95% sure that this WOULD work, actually. I suppose I'll have to try it at some point, but I don't have my digicam now, nor do I really have any room to do it. I will at some point, and I bet it will work. I have a pretty low spore count in my room anyway.
Quote:


They do make HEPA inline air filters. Fungi Perfecti has 'em, I think they are in the $80 range and I have no idea if your standard aquarium pump would be able to pass air through it. Anyway, that's what I'd recomend for sterilizing the air going through the bubbler.




Yea, that might do it too. I think that a prefilter of some TIGHTLY packed polyfil, then bubbling it through some cold IPA and cold h2o2 would do it. I'm really sure that it would.
Quote:


I don't think areation is the only issue, I think you will run into trouble with sterile technique. Assuming you didn't, and aeration is the only issue, then it's possible. Use the above mentioned inline hepa filter. But then in the end, how good are your extraction skills? How expensive are solvents? Do you already have a fume hood, a sep funnel, a vacuum filter setup?




Yea, thats another thing. I'm not sure how well the extraction methods work. Why would you need a separatory funnel? You aren't separating polar layers here.


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Offlineblackout
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: SoopaX]
    #4097909 - 04/26/05 04:08 AM (12 years, 30 days ago)

I once grew some out in a gallon jar with peptone and malt extract. My idea was to keep adding sugars as they got used up in the broth. Since the mycelium got thicker it would be able to use up the sugars quicker. There has to be a limit to this since it should eventually poison itself with its own by-products. I ended up with a huge mass of mycelium with a thick layer on top.
I ended up tossing it since it smelt a bit odd, didn't strain or weigh it. I microwaved it in the gallon jar


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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: blackout]
    #4174045 - 05/14/05 11:40 AM (12 years, 12 days ago)

You may want to check Beer, Beer, and More Beer website (www.morebeer.com) for their aeration system used to culture yeast Part No FE380. This, combined with a suitable container, such as a 3 gallon carboy and a stopper with two holes (1 for the diffusion stone and 1 for an exit tube) may be all you need to construct your own liquid fermentation vessel (the design of which you can find under Ward's Scientific Item No. 14 V 0302. Alternatively, you can construct a stir plate/erlenmeyer flask set up as described in www.primetab.com/yeaststarter.html. or use the procedures described in Stamets, The Mushroom Cultivator.

As to the science surrounding the production of active compounds like psilocybin, there are quite a few studies that have been done that have looked at the actual production ratio's in liquid culture. The first lab work done was by Heim and Cailleux, 1957, followed by Albert Hofman . There is a simple explanation of their experiments in "Magic Mushrooms around the World" by Jochen Gartz, 1996. The take home message from this book is to use either Cubensis or Mexicana, as they produce the most mycelium of any strain. "In Basel, it was also possible to cultivate the mycelia from different species on liquid solutions of malt extract. The mycelial tissue was found to produce psilocybin without having to go through the fruiting process... Even through the mycelial tissue grown from the Mexican species contained only half as much psilocybin (0.1-0.2% of dry weight) as the mushrooms did, high yields (20g/liter) along with the easy and rapid cultivation of mycelial tissue more than compensated for the lower psylocybin content. ...It is interesting to note that mycelia from the North American species of Psilocybe cyanescens will turn blue and accumulate psylocybin when grown on a medium of solid malt agar, while both of these traits disappear completely when the same strain is cultivated on liquid medium."

There was also an interesting doctoral thesis done more recently by Niels Jensen "Attempted Molecular Cloning of Enzymes from the Psilocybin Biosynthesis Pathway in Psilocybe tampanensis", which contains some methods for liquid and solid agar media preparation. For some reason, he was unable to get psylocibin to produce in his liquid cultures. His work is interesting because it shows bee pollen has a significant impact on growth and fruiting. (see "Growth-Promoting Effect of a Brassinosteroid in Mycelial Cultures of the Fungus Psilocybe cubensis" J. Gartz, 1990 as well)

Here is an excerpt from Niels paper -

Effect of media on growth and alkaloid content of Psilocybe strains

Ps. cubensis, Ps. tampanensis, Ps. azurescens, and Ps. cyanescens were grown in different media in 6 cm diameter petri dishes filled with 25 ml medium containing 2% agar if not otherwise stated. After 14 days the mycelium was harvested by scraping off from the agar. The 500 ml submerged cultures were inoculated with a finely cut 1 cm2 agar block and shaken at 28?C at 100 rpm. The mycelia were lyophilized, weighted, extracted, standardized, and HPLC analyzed for psilocin and psilocybin content (Table 6).

The tested Psilocybe strains showed variable preferences for the tested media. In general, the defined Leatham?s media, sugar-cane medium, and beer-based media were poor substrates compared to malt extract based media. The submerged culture produced the highest amounts of biomass, followed by the surface cultures on soft agar (0.2%). But under both conditions the mycelia did not produce measurable amounts of alkaloids. The same was true for Ps. tampanensis submerged cultures in 6% malt extract with no supplement, 0.3% yeast extract, 2% pollen, or both added (data not shown).

Psilocybe cubensis was grown in Leatham?s medium without glucuronic acid and salicylic acid. After 14 days the mycelium growth was inspected and analyzed for its psilocin and psilocybin content.
Ps. tampanensis produced sclerotia on a medium containing 6% malt syrup and 0.3% yeast extract. These sclerotia were growing up to a diameter of 12 mm and often continued growing after block transfer onto fresh medium. They built preferably directly beneath the inoculation block or at a concentric border between a fluffy mycelium around the block and a surrounding strandy mycelium. The sclerotia had a dry weight of 27% determined by lyophilization of fresh sclerotia for 2 d and contained comparable amounts of psilocybin per dry weight to the reference material, Ps. cyanescens fruiting bodies. From all tested conditions on solid agar the sclerotia had the had the highest weight of biomass and contained the highest amounts of psilocybin.

After longer incubation of Ps. tampanensis for about eight weeks small and incompletely developed fruiting bodies formed. They originated from a fluffy brownish, white-spotted mycelium and showed a a strong bluing reaction on pressure.


Ps cubensis, Ps. tampanensis, Ps. azurescens, and Ps. cyanescens were grown in different media. After 14 days the mycelium was harvested, weighed, extracted, standardized, and analyzed for psilocin and psilocybin content.
Ps. cubensis produced psilocin, but no psilocybin under the conditions analyzed. In contrast Ps. tampanensis and Ps. azurescens produced both alkaloids. Ps. azurescens and Ps. cyanescens were growing very slowly on all media tested.

(all quotes taken from sources without permission of authors)


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InvisibleSoopaX
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Re: A dead horse subject: Liquid Mycelium, a new approach? [Re: strangefruit]
    #4176886 - 05/15/05 02:48 AM (12 years, 11 days ago)

AWESOME! My priest told me about that the NIGHT that I read this post, but I was too drunk to remember the source.


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