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Invisiblemicro
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Registered: 05/09/03
Posts: 7,532
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What worked best for us (why poo cont'd)
    #22395939 - 10/17/15 11:44 PM (8 years, 3 months ago)

Well, this is what I've found works the best:

Materials:

All American Pressure Cooker
Autoclavable bags with self-healing inoculation port and .2um filter patches
Heat sealer
Autoclavable bottles with septum lids
Glass syringes (these are reusable and autoclavable)
Parboiled rice (cheap as hell in any grocery store)
Spore syringes (you can go by prints as well but it is up to you to ensure sterility)
Plastic bins and either saran wrap or plastic sheeting, depending on the size of the bins
Peat and lime (to make casing, a la Stamets et al.)
Heater
Paper clips
Rubbing alcohol (preferably ethanol) either 70% or 91%
Spore syringes
Regular commercial bleach
Vermiculite

Optional materials:

Bale(s) of straw
Materials to make liquid media (I stole LB media from the lab I worked at but potato dextrose broth is better anyway)
Lots of plastic sheeting, staple gun, duct tape, mini hepa filter for room
Autoclave tape (useful when sterilizing liquids)
Bunsen burner
Glass petri dishes (or sterile disposable plastic ones)
Erlenmeyer flask(s)
Aluminum foil
Hepa filter

Protocol:

Optionally sector growth using bunsen burner for 10cm radius of sterile work area and pyrex petri dishes
If you do this you'd use PDA, Sterilize for 30 minutes along with the empty petris from an Erlenmeyer flask covered in foil
Cook some parboiled rice using 3/4 of the water listed in the directions. The parboiled rice is precooked a bit and it comes out less sticky and more airy than brown rice
Put a liberal amount in the bags but not so much it is filled more than a third of the way up to the filter patch
Gently fold the top of the bag 1/2" down 4 or 5 times after letting the air out and secure them that way with a paper clip on each end
Sterilize for 90 minutes at 15 PSI (make sure it is there before counting)
Always let the PC cool before opening it up
Open it up and heat seal the bags close.  You will need to adjust the heat sealer so it won't burn the bag apart so practice with a test bag first a few times until you get the setting right
Once the bags all have a GOOD seal (if they split open they are unusable) then let them cool to room temperature on the inside as well as the outside
Clean a room with 2% commercial bleach (probably should do this beforehand; we always brought the pressure cooker into a cleaned room before opening it and heat sealed it in there)
Optionally the clean room can be lined with the plastic sheeting (see the positive pressure flow room link in my sig and the optional materials applicable)
In the clean room, with either clean gloves or scrubbed hands, wipe down the inoculation site with your ethanol and then knock it up with the spore syringe
With the bunsen burner (optional) or a flame, sterilize the syringe and replace the cap and let it cool before knocking up the next bag.  think we only used 1 or 2 cc's per bag; you don't need a lot
Colonize the bags at 80F or so. I was really ghetto about this and used a space heater in a closet and opened the door a certain amount to control the heat
Let the bags colonize to about 15% and then break it apart and mix around the mycelium so it will colonize exponentially faster
Once the bags are completely colonized, in clean plastic bins, line the bottom with a thin layer of vermiculite and crumble up the colonized mycelium from a bag in an even layer, but not *too* even (if it is too even, you will get smaller mushrooms and a greater flush but this is not ideal
). I usually only had an inch or so of colonized material in each bin and you won't get as many flushes this way but I see that as a good thing since you get everything at once this way.
Mix the peat and 25% lime by volume and add enough water that some drips out when you squeeze it.

Or - optionally spawn to bulk using the straw; chop it up (thanks to azur for pointing this out) pasteurize at 160F for 45 minutes

Squeeze out the water and crumble up the peat on top of the mycelium so it is airy, into maybe a quarter to a half inch of a layer on top. You don't want overlay -- the mycelium should poke up through the casing layer but not form a mat on top (this is also associated with high CO2 levels and low humidity).
Save a bag, sterilize a bottle of water in a bottle with a septum cap and autoclave tape for a half hour and the tape should indicate it is sterilized
Shoot the ddH2O into the bag through the injection port after rubbing with alcohol, mix it up using the outside of the bag and suck it back up, using a sterilized glass syringe
Save it or optionally knock up a jar of sterilized PDB and let it colonize using a setum-top bottle (loosen the lid a bit to sterilize it, adding autoclave tape if you have it)
We just misted and fanned; substitute methods as you feel fit

This isn't a tek anyway; I don't believe in them

Everyone has different needs based on geographical location and other shit anyway

Have fun!


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


Edited by micro (10/18/15 11:11 AM)


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Offlineinvitro

Registered: 05/03/13
Posts: 2,529
Last seen: 1 month, 21 days
Re: What worked best for us (why poo cont'd) [Re: micro]
    #22395943 - 10/17/15 11:47 PM (8 years, 3 months ago)

:popcorn:


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Invisiblemicro
bunbun has a gungun
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Registered: 05/09/03
Posts: 7,532
Loc: Brick City Flag
Re: What worked best for us (why poo cont'd) [Re: invitro]
    #22396972 - 10/18/15 10:24 AM (8 years, 3 months ago)

bump, it's done

have fun, kids!

i'm not growing cubes again anyway


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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