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LSA bioreactor superthread (C. Paspali)


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#21 director of sound

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Posted 29 August 2010 - 12:15 AM

ok heres the bioreactor head. should be easy enough to make, the gas/thermo ports would be thin walled tubing so a piece of glass tubing can fit through it for a bubbler. and the track for the tubing (area H) would have silicone in it too. the base would be allmost the same but solid. the bearing should be able to be epoxied in place. steralization on this peice could be done by steam flushing through a gas port while leaving one of the cap screws out. the liquid media could be added while still hot and allowed to cool after the steam steralization before adding the culture 'IV' style through an injection port. i figure a brewing style air port/trap could be used for the gasses to escape through.

http://mycotopia.net...=1&d=1283058913

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Edited by director of sound, 29 August 2010 - 12:22 AM.


#22 Erkee

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Posted 29 August 2010 - 08:41 AM

Wouldn't a carboy work? Or a jug?

I'm grooving over your design work though!
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#23 cosmoline

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Posted 29 August 2010 - 08:58 AM

Yeah, please remember that I never claimed to come up with this info...


DOS - Good work man! I'm a technophile so looking at your design made me drool. Very well designed. And really interested in sterilizing by bubbling steam through, seems very viable for a large reactor!!

A few things for your design: would you really want a ~5 foot GLASS tube full of gangrenous fungus (if using ergot or non mutated paspali). Even if it is pyrex, one mistake and the fungus surfs straight at your bloodstream on a board of razor sharp glass. If you are looking at those volumes why not a carboy, modified pressure cooker, aluminum keg, a half keg, soda making cannisters, etc.

This design would be a lot more appropriate for seed cultures, the top can't be made of HDPE if its getting thrown into a PC tho. Keep up the good thinking guys :headbang: :rasta:

#24 director of sound

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Posted 29 August 2010 - 11:39 AM

saftey is one of my greatest concernes when working with ergot so the utmost care will be taken to make sure the reactor/column can not be tipped over or bumped. it would also be well hidden form prying eyes in a dark dank basement in a climate controlled closet somewhere in the middle of bum fuck egypt. pyrex is quite durable but still able to be broken, a thick coat of liquid laytex could be applied to the outside to keep the shards togather in the event the reactor was to break. many reagents i work with are quite dangerous (concentrated acids and bases, highly toxic metal salts, oxidizers) and the large 1gal reagant bottles they come in are coated with laytex. tyvek suits are a god send for repealing splashes of dangerous chemicals and they make oversize nitrile rubber gloves and absorbant powders for spill cleanup. i suppose a carboy would work in a pinch but might be a pain too, a rubber bung to close it dosent have as much space for a stirrer and bubbler to go through and that would be the minimum needed to get it to work. the bung may come loose from the stirrer spinning if not constantly oiled or greased and introduce contamination. i mean if you are to go as far as making a culture might as well do it right the first time...

Edited by director of sound, 29 August 2010 - 12:19 PM.

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#25 director of sound

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Posted 02 September 2010 - 11:07 PM

ok so heres a paper i came across that shows some HUGE promise... i mean like getting close to 2mg from 1ml of the culture with a non mutated strain!! that would corespond to 1.5-2g per 1L of culture and my 18L bioreactor design would turn out a whopping 32g (+/- 5) every 2 weeks. the study dealt with determining why phosphorus depleated cultures produced poorly. by adding extra phosphates in the form of sodium phosphate at crucial times in the fermentation the culture that would normally produce 300-500ug/1ml would produce 800-1000ug/ml. then they went farther with substituting the phosphate with very small ammounts of an arsenate (specificaly potassium arsenate) and the P depleated culture jumped into the 1200's. even further than that and cultures supplemented with both phosphates and arsenates (non mutated i might add) would produce in the 1400-1500ug/ml range. now im talking 1.4-1.5mg /ml with a non mutated strain of claviceps purpurea. imagine if one were to mutate or obtain a mutated strain and use it in this manner. could one see 2.5-3mg/ml? more? what a feat it would be to get 3g or more per a liter of culture in 13 days..........:headbang:

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#26 rhiolyte

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Posted 03 September 2010 - 09:17 AM

super great thread!

but 3 things are bugging me.

1. where to get a c. paspali culture?
2. where to get 8 Ki's hbwr?
3. 13 days sure is a long time to be shitting bricks..

slam bam thank you ma'am via total synthesis, then destroy the evidence in this game, right?
to bypass those shiny bracelets they want you in...

#27 Guitardude

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Posted 03 September 2010 - 10:38 AM

Me dad always used to say.... "more than one way to skin a cat"
LSD LC's for the masses.... am I off track here or is this like an lsd "sourdough starter"..... you guys rock!
Peace
GD
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#28 bear

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Posted 03 September 2010 - 11:28 AM

Obligatory St. Anthony's Gangrene warning.

Thanks for the schematic, it's actually perfect for a number of uses.

#29 cosmoline

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Posted 03 September 2010 - 03:49 PM

ok so heres a paper i came across that shows some HUGE promise... i mean like getting close to 2mg from 1ml of the culture with a non mutated strain!! that would corespond to 1.5-2g per 1L of culture and my 18L bioreactor design would turn out a whopping 32g (+/- 5) every 2 weeks. the study dealt with determining why phosphorus depleated cultures produced poorly. by adding extra phosphates in the form of sodium phosphate at crucial times in the fermentation the culture that would normally produce 300-500ug/1ml would produce 800-1000ug/ml. then they went farther with substituting the phosphate with very small ammounts of an arsenate (specificaly potassium arsenate) and the P depleated culture jumped into the 1200's. even further than that and cultures supplemented with both phosphates and arsenates (non mutated i might add) would produce in the 1400-1500ug/ml range. now im talking 1.4-1.5mg /ml with a non mutated strain of claviceps purpurea. imagine if one were to mutate or obtain a mutated strain and use it in this manner. could one see 2.5-3mg/ml? more? what a feat it would be to get 3g or more per a liter of culture in 13 days..........



Yo man, that is what is got me very excited about this method, was posted in the original thread:

Addition of sodium arsenate at levels between 1/50 - 1/20th the molar concentration of phosphate, 100% increase in yield - Ref R3. This is a real easy mod, overdoing the phosphate too much wont hurt yields, so set a level of phosphate and add KH2PO4:Na2HAaO4 at 50:1-20:1 ratios keeping your reasonable level of phosphate in mind. This is more about ratios then amounts.



The amounts in the original post are completely understated, with proper modifications you can boost the yield to unbelievable amounts.

Obligatory St. Anthony's Gangrene warning.


Bear, I was under the assumption that if you had a true "alkaloid-blocked" mutant of C. Paspali it would only produce LSA,iso-LSA, LSH and iso-LSH as far as ergoline alkaloids go. Feel free to correct me but nothing in a correctly maintained, mutated paspali culture should cause gangrene.

#30 Erkee

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Posted 25 September 2010 - 12:27 PM

Just found this:

DSM 833 - Claviceps paspali Stevens & Hall Name: Claviceps paspali Stevens & Hall DSM No.: 833 Other collection no. ATCC 13893 Information: <- K. Kieslich <- ATCC <- A. Tonolo, F 237. Paspalum distichum. Produces lysergic acid derivatives (U.S. Pat. 3,038,840). (Medium 129, 26°C) Isolated from: Paspalum distichum Medium: 129, 26°C Literature: 3610 Supplied as: actively growing culture (on agar or in liquid medium, depending on the strain) Risk group: 1 (classification according to German TRBA) Price: EURO 65 (non-profit making institutions),
EURO 100 (other institutions): Normal price,
active culture

medium 129:

129. POTATO DEXTROSE AGAR
Infusion from potatoes (see below) 1000.0 ml
Glucose 20.0 g
Agar 15.0 g
Potato infusion:
Boil 200 g scrubbed and sliced potatoes in 1000 ml water for 1 hour. Pass through fine sieve. Avoid new potatoes.

from [here]

#31 director of sound

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Posted 25 September 2010 - 10:56 PM

so i found my ergot, unknown strain but deffiently an ergot. growing on a grass of sorts. i collected a few speicmines but the whole field is infected so ill be able to get more if the agar cultures ill be trying fail. check it out!:headbang:

http://mycotopia.net...=1&d=1285473318

http://mycotopia.net...=1&d=1285473318

http://mycotopia.net...=1&d=1285473318

http://mycotopia.net...=1&d=1285473318

http://mycotopia.net...=1&d=1285473318

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#32 Erkee

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Posted 26 September 2010 - 12:12 AM

:headbang:

#33 tregar

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Posted 26 September 2010 - 12:32 AM

Great find! nice pics too.
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#34 Phlux-

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Posted 26 September 2010 - 02:15 AM

just been keepin an eye on this thread - useless post but check this

Posted Image

with this playin

[Direct Link]


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#35 fungiman

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Posted 26 September 2010 - 10:52 AM

I've already posted about Ergot, here's my repeat.
http://mycotopia.net...-claviceps.html

#36 cosmoline

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Posted 30 September 2010 - 12:12 AM

erkee: that right there is my wetdream, wish I was in germany so I could have it shipped to me.

DOS: nice find, I guess the first step would be to identify the type of plant that fungus has infected. I believe thats C. purpurea, def not paspali.

#37 cosmoline

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Posted 03 April 2011 - 06:04 PM

There is Claviceps paspali for sale in the shroomery marketplace right now surprised more people arent jumping all over it.
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#38 Erkee

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Posted 03 April 2011 - 06:21 PM

There is Claviceps paspali for sale in the shroomery marketplace right now surprised more people arent jumping all over it.

Bonus!
I put a bunch of grass stalks gone to seed in a covered plastic bucket with an inch of water, last fall, cold storage, see what would come up.
Misted every week for a month, then forgot.
Something took it on.
And took it over.
Small kidneys 0.5~1mm long, grey black, on thin stalks, by thousands.
Thinking not the clavicep though.

#39 director of sound

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Posted 04 April 2011 - 04:35 AM

pics? fruiting ergot sclerotum will look like this.... if you come across horns of ergot in a field first quickly clean them in a dilute bleach/peroxide solution, 1cap bleach and about 20ml 3% peroxide to 1L of clean or distilled water. pat dry with a clean paper towel and wrap in that and toss in the freezer for about 2 months. ergot needs a period of cold to trigger fruiting. after the freeze put some clean sand in a small fish tank and sprinkle in the sclerota and cover over with a thin layer of sand. with luck if kept at room temp and misted daily after about 2 weeks to a month it will fruit (cover over with plastic wrap to keep up the humidity). you can then carefully remove the fruited sclerotum and rinse in the same bleach/peroxide solution before carefully lancinf the fruit heads with a glass microscope slide cover slip held in front of it. the "honeydew" will squirt onto the cover slip which you can then drop into your sterile culture medium....


http://mycotopia.net...=1&d=1301909905

Edited by director of sound, 04 April 2011 - 04:46 AM.


#40 Frequency

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Posted 04 April 2011 - 06:52 PM

pics? fruiting ergot sclerotum will look like this.... if you come across horns of ergot in a field first quickly clean them in a dilute bleach/peroxide solution, 1cap bleach and about 20ml 3% peroxide to 1L of clean or distilled water. pat dry with a clean paper towel and wrap in that and toss in the freezer for about 2 months. ergot needs a period of cold to trigger fruiting. after the freeze put some clean sand in a small fish tank and sprinkle in the sclerota and cover over with a thin layer of sand. with luck if kept at room temp and misted daily after about 2 weeks to a month it will fruit (cover over with plastic wrap to keep up the humidity). you can then carefully remove the fruited sclerotum and rinse in the same bleach/peroxide solution before carefully lancinf the fruit heads with a glass microscope slide cover slip held in front of it. the "honeydew" will squirt onto the cover slip which you can then drop into your sterile culture medium....

DOS your posts are always awesome. :bow:
Thanks for sharing the pic and the culturing tek! A+




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