07-04-03 13:48
No 444469
Hi!
Swim is in the lucky position that he will receive a vial of Claviceps Paspali - Stevens & Hall F-140 in a few weeks. He will follow the patent: US3038840 for the growth and extraction of the goodies. If all goes well it could yeild up to 1600 mg's of (iso)lysergic acid / L of broth.
But, swim is not totally hot in field of fungii growth, so a little advice from more skilled microbiologist's is really appreciated :)
The final growth is going to happen in a protected(such that it does not release Zn++ and Fe++ ions into the soln., which is supposed to inhibit the formation of lysergic acid derivates) 30 L steel vessel. It'll have built in clean oxygen supply etc. The setup for that one is quite obvious, but untill that point, there are some things which swim need a little advice on:
1)
innoculation
Once the critters arrive, they are to be innoculated onto some agar plates. How sterile does the environment have to be to protect them enough? Compared to innoculating e.coli. on agar, how sensitive are paspali(or fungii generally speaking). Swim was thinking of constructing a glovebox, which filtered the air though a NaOH soln. and NaOCl to sterilize the environment for working with the agar plates.
2)
Incubation liquid + Prefermentation liquid:
In the patent the utillize a rotary shaker for these two steps. It should be able to do the steps without... They just need a little longer i would think. But the growth is supposed to be aerobic throughout the process, and yet the do not mention this in the patent for these steps? Does the shaking from the rotary shaker provide enough oxygen for the little beauties? Maybe a homemade clean-air supply should be utillized to compensate?
I'll probably(most definately) post more questions, as the project evolves! Wish swim luck, with the shortage of everyones favorite material and lack of precursor material we need some serious work in this field. If it really is as easy as the patent seems, there are fun times ahead!
Regards
Bandil
Size doesn't matter, it's the sound you make when hitting the ground after smoking it, that matters!
(Moderator)
07-04-03 15:15
No 444487
Never skip the stepwise increase in volume. You do need a separate 'incubation liquid' culture. Don't ask me why
, but it doesn't work well without.
(Hive Bee)
07-04-03 20:49
No 444531
Swim was thinking of constructing a glovebox, which filtered the air though a NaOH soln. and NaOCl to sterilize the environment for working with the agar plates.
Maybe you should consider the use of a negative ion generator.
Yes, the Ion is much better for sterile work than a flow
hood. When trying to clone a mushroom under a flow hood,
all the blowing air blows contaminants off of the mushroom
everywhere. The Ion does not do this. The Ion also removes
down to .001 micron......no flow hood made can do that.
http://www.mushroomsfmrc.com/cgi-bin/web
http://www.poedecoder.com/Qrisse/works/d
(Hive Bee)
07-04-03 21:03
No 444533
Hmmm... they do seem to like it in there :) But i can't see through their sales talk. Is it really better to use than all sorts of other filters? If it is, it sure would be an elegant solution.
Edit: They only accept cash/checks, and it'll probably take about forever to deliver. I think i have seen some electrical smoke removers somewhere around here. Isn't that built on the same principles?
Size doesn't matter, it's the sound you make when hitting the ground after smoking it, that matters!
(Hive Bee)
07-04-03 22:55
No 444552
Hmmm... they do seem to like it in there :) But i can't see through their sales talk. Is it really better to use than all sorts of other filters? If it is, it sure would be an elegant solution.
Yeah, I know, they are full of sales talk, but the guy that runs the place (Stephen L. Peele) is considered very knowledgeable on the subject of mushroom growing. He's been growing mushrooms for more than 30 years and even discovered a new psilocybin-containing mushroom, named after him.
This is indeed the same as the electronical smoke removers.
Basically an air ionizer generates negative ions that collect the contaminants on the nearest positive charge, at it's base. If used in a room then all particles will fall on the floor.
I would still use a glove box and the air flow setup you described, but you could use the ionizer inside the glovebox as an extra protection.
Also, adding an antibiotic like gentamycin to your agar could also reduce your chances of contamination. Don't remember the % but you can find this info at sites like the Shroomery.
http://www.poedecoder.com/Qrisse/works/d
(Hive Adickt)
07-05-03 02:48
No 444587
In my shroom time I made a 'glovebox from playwood and plexiglass. When all my autoclavated good's were in the box I sprayed it with 30% hydrogenperoxide, kill's everyething inside. Then opened my autoclavated bags and start working. Claviceps was much more wigour than psilocybe (10-100 times) so I don't think that's a big problem. When you go to the liquid work, just filter the air throug a HEPA filter ( the P3 from gasmask's) it remove eaven virus (much smaler than fungus). Have fun.
(Hive Bee)
07-06-03 17:43
No 444891
Having worked with C. Paspali and C.purpurea, here are some suggestions:
1) You really need to get some practical experience working with fungi and sterile culture. A good "50 cent education" would be to get a book like "The Mushroom Cultivator" by Paul Stamets. It covers in depth alot of the techniques, equipment, etc. that you will need.
2) A glove box is OK, but even better is to construct a HEPA Filter chamber. It's basically a fan pushing air thru a micron filter (filter out mold spores, etc.) This way you have a sterile air flow in which to work with the fungi.
You can work without either a glovebox or HEPA chamber, however you'll get some cultures that are contaminated. Make up for that by making many more agar plates, tubes. etc.
3) Working with C. paspali in liquid culture is NOT as easy as you think. You will definitely need a rotary shaker or at least some way to get sterile air into the liquid media while stirring. A rotary shaker is used because one can get air into the media AND control the temperature at same time. Claviceps in liquid culture needs to be grown at certain temps (24-27 C.) or it will NOT produce any appreciable amounts of lysergic compounds.
Also, keep in mind that as the Claviceps grows, it produces compounds into the media call Glucans. Glucans are thickening agents; i.e. it will make the media very viscous and thick, making it hard to aireate and stir! I use a bioreactor/fermenter to overcome this problem.
4) C. paspali needs to be gradually "scaled up". That is you have to start the culture on agar, then move to small amounts in liquid on Rotary Shaker, then larger amount on Shaker, then larger amount in fermenter.
5) Check out more than one Patent. Do a search on Claviceps. There are many patents that go into great detail about the conditions and media, etc. required for Claviceps growth.
(Hive Adickt)
07-06-03 19:25
No 444913
Don't claviceps produce aflatoxines under some conditions ?
Bubbleplate, are you saying that bubbling (sterile)air throug a 25L container isn't posible ?
(Hive Bee)
07-06-03 20:02
No 444924
Swim will build in an overhead stirrer, and of course an aerator in the bottom. Does it really get so think, that it's practically unstirrable, and thus unaerable?
Size doesn't matter, it's the sound you make when hitting the ground after smoking it, that matters!
(Hive Bee)
07-06-03 20:16
No 444925
Stirring overhead is fine, but keep in mind that no matter what size fermenter vessel you have it will need to be:
1) Airtight - air in/out only through the filtered airlines.
2) Sterilized - EVERYTHING that comes into contact with the fungus/media will need to be sterile and kept that way while growing fungus.
I filter air pumped into fermenter by using 50mm PTFE type round filters on the air intake and vent lines.
You will need to pump steam somehow into your large vessel for 60 minutes or more to sterilize it.
One cannot imagine just how thick and viscous a fermentation can get until you actually do it! Foaming/bubbles is also a problem. Pumping air thru media will suffice to provide enough O2 and stirring for days 1 through 5, but after that good mechanical stirring is required!
(Hive Bee)
07-06-03 20:26
No 444928
ergot fungi produce aflatoxin's. I think you're thinking of Aspergillus species, many of which produce deadly aflatoxins.
(Stranger)
07-10-03 18:15
No 446215
Hey while your waiting for the shipment, you should look at all the alkaloid production enhancers that have been mentioned around here in the last year or 2.
And to think that just this morning in the shower I was thinking that all current labs use diverted ET pills, and that indeed that ergot cultivation was a waste of time...maybe you can revive (at least my) interest with a successful experiment!
Post 246864 (bujinkan: "Re: ergot and agar", Tryptamine Chemistry) Post 430459 (Bubbleplate: "five-fold increase in total alkaloid production", Tryptamine Chemistry)
Biojammer, have you hands on experience with ergot? is it hardy enough to use more ghetto procedures in your opinion?
(Hive Addict)
07-10-03 23:56
No 446271
CL spent some time programming pilot plant genetic engineering fermenter control systems...
The growth of fungi/yeast always requires agitation. In the fermenters this is usually done w/ stirring.
Monitoring of the gases (dissolved O2), ph and feed rates was always done, since feed rates have to be increased as culture exponentially grows..
Sterile air was brought in through filters.. a syringe filter of appropriate micron size should work for this.. but you'll need to have the culture shaken..
Ph will tell you when your waste products are being generated. Phosphoric acid was usually used to increase the pH.
So spend some time and look up the parameters and feed rates for your organism and make sure you have that data before you go ahead..
Infinite Radiant Light - THKRA
(Hive Bee)
07-29-03 15:51
No 450555
The F240 is coming along quite nicely now. The agarplates has been innoculated 7 days ago, and are full of nice fluffy white colonies all over. Two got infected, but these where saved by cutting away the green mold with a sterile knife. Seems to be doing ok now! They are quite easy to work with compared to cubensis strain.
The plates will be copied onto several other plates shortly, in order to experiment further with this delightful fungii. The best colonies will be placed in the submerged medium at the end of the week!
Regards
Bandil
Size doesn't matter, it's the sound you make when hitting the ground after smoking it, that matters!