|Liquid Culture Notes|
Liquid Inoculant from Live Mushrooms
Using a blender and mason jar to prepare liquid inoculant from a ripe mushroom and sterile water. Suitable for making mycelium syringes or for direct inoculation of jars or bulk substrates.
Standard-mouth mason jars fit onto the bases of Oster, Proctor-Silex, and Hamilton Beach blenders (I've tried those three) and probably onto other manufacturers' blender bases. Wide-mouth mason jars do not fit blender bases.
|Spare Blender Blades and Gaskets are available at Ace Hardware and other outlets for about $10. Blades and Gaskets can be attached to mason jars with the usual jar lid rings. It's not neccessary to use the blender base. It's even possible to hold the jars firmly in place and blend them with the jar ring on instead of the blender base, or the jar ring can be removed and the blender base attached for blending.|
When these mushrooms [Psilocybe cyanescens and Psilocybe pelliculosa]
are grown under liquid media, they do not produce any psilocybin or psilocin.
This was also found to be true by P. Catalfomo and V.E. Tyler, Jr..
They published the same findings about
Psilocybe cyanescens and Psilocybe pelliculosa......they do not produce
psilocybin or any other analogs (Catalfomo, P. and V.E. Tyler, Jr. _The
production of psilocybin in submerged culture of Psilocybe cubensis_
LLoydia 27:53-63, 1964). However, once the mycelium of one of these two
is transferred to an agar or grain media, it does produce psilocybin and
psilocin. This finding sets the stage for a landmark statement by myself.
Using Psilocybe cyanescens and Ps. pelliculosa for standards, I suggest
that "in some types of mushrooms the mycelium may find it's way to a new
feeding substrate and this new substrate that is not normally used by the
particular mushroom or will support complete growth of said mushroom,
allows the mushroom to produce compounds not normally produced because of
the addition of needed nutrients. In these cases, the mushroom was always
capable of producing such compounds but the needed nutrients were never
there on the normal substrate."
"Liquid Culture Mycelium" (alt.drugs.mushrooms)
Osmotek Bioreactor (industrial liquid culture systems)
From: firstname.lastname@example.org (email@example.com) Subject: liquid culture using malt extract... Newsgroups: alt.drugs.mushrooms Date: 1999/02/17 tried liquid culture years ago as per "The Psilocybin Producer's Guide"(1976 by Kistone Press) by Adam Gottlieb which purported to "produce 5000 doses of organc psilocybin every week in a small room". It did not work well. clumps of mycellia grew,resembling jellyfish.none got larger than approx. 1/2 inch diameter and total yields were abyssmal. problems include airation of the medium and since it's liquid, contams spread very fast, esp. when you shake the jars to aerate. Gottlieb never tried his own tek i'm told & it doesn't work well in home lab type conditions. peace *** Posted from RemarQ - http://www.remarq.com - Discussions Start Here (tm) ***
From: Ville Tuomi (firstname.lastname@example.org) Subject: Re: growing mycelium in a liquid culture from tissue Newsgroups: bionet.mycology Date: 1998/07/17 As an addition to culturing pure culture from agar slant in liquid you could try adding some sawdust to media. This way you can obtain smaller particles of mycelium as there is some solid material present. This works well for wood lowers if the media is continuously mixed. Furthermore I've succeeded by blowing air to the media through a 0,2 um filter. The air is blown to the bottom of the bottle via a steel pipe and it gets out through a filter in a cap. This way the air mixes the media and sterilization is easy as the air moves through the filter. Also, fungi and most other micro-organisms grow much faster in aerobic conditions. Hope this helps. VT Tad wrote: > Has anyone had any expereince growing mycelium in a liquid culture? > specifically using mycelium tissue taken from inside of a mushroom? > > any insights on feasibility and best way to attempt this... would be > appreciated. > > thank you
From: email@example.com (firstname.lastname@example.org) Subject: Re: Repost: Easy to make mycelium syringes Newsgroups: alt.drugs.mushrooms View: Complete Thread (3 articles) | Original Format Date: 2000/02/17 In article <email@example.com> Karl Brooks
writes: >From: Karl Brooks >Subject: Repost: Easy to make mycelium syringes >Date: Thu, 17 Feb 2000 17:32:27 GMT >Here's the original post from Hippie3, in case he's still lost in >Usenet Hell. >take a spore syringe from yer favorite vendor. >split it up into 10 syringes of 1 cc each. >prepare a jar (w/filter on cap) of honey water (1 tsp. honey: 1 pint >distilled water) by placing the jar in a pan of boiling water for 30 >minutes. >remove to a clean area & let cool to room temp. >after cool, draw 9 cc of sterile honey water into each syringe >containing 1 cc spore water. >keep in clean conditions at 85*F until mycellia grows, then use to >innoculate as usual. I thought that the mycelia should show up first in the liquid culture? If it doesn't - what is the use with this? I would use malt syrup dissolved in the water for a better liquid culture medium. That is what all the liquid culture things use. MALT SYRUP. and I would wait until mycelia is all through the liquid media - then inoculate jars or whatever. HEY
From: Chris Wright
Newsgroups: bionet.mycology Subject: Re: growing mycelium in a liquid culture from tissue Date: 4 Jul 1998 13:26:27 -0700 Organization: Center for Microbial Ecology Growing mycelium in a liquid medium is not difficult (providing that it is a fungus which is culturable). I would suggest isolating the culture on an agar medium first to insure you are working with a (putative) pure culture. Then use the same formula you used in the agar medium except hold the agar. Grow the mycelium in sterile erlenmyer flasks which have a foam plug or some type gas exchange apparatus. Do not fill the flasks too full. There is a tradeoff between surface area and amount of substrate. Mycelium can be grown either static or shaken. Good luck, Chris
From: The Walrus (firstname.lastname@example.org) Subject: liquid mycelium Newsgroups: alt.nature.mushrooms View: Complete Thread (2 articles) | Original Format Date: 1997/06/05 CaMagic1 wrote: >Does anyone have experience with starting a liquid culture from spores. >I'm currently trying it with a recipe of 20g malt extract 20g dextrose 1L >distilled water. I want to know if I need to adjust for Ph. How well >will mycelium grow and for how long in this medium -- if at all. If >anybody could give me some insights on the procedures and rules for >growing PC mycelium on liquid sugar that would be great. The media you describe should work. If you use distilled water, you don't need to adjust pH. The medium I use is 4% (by weight) malt extract powder in distilled water. It is sterilized at 15psi for about 20 minutes, allowed to cool overnight, and then innoculated with 1%-2% (by weight) with spore syringe solution. The culture is then incubated at 30C for about 4 days with frequent agitation. During this time, you should notice the solution becoming milky and towards the end of the incubation, there will be fluffy lint-like pieces of mycelium floating around. I assume you are familiar with sterile technique, in which case, you need to watch for a couple things: 1L of bulk liquid is a lot to sterilize all at once in a pressure cooker because it will heat up unevenly and you will end up with some of it being undercooked unless you sterilize it for a longer time (maybe 40min.). You must also agitate the solution enough to aerate it and break up the mycelium. This method will not work well if the media is not agitated. I use the completed solution to innoculate other substrates. I use 5-10ml per PF jar. This is a very large amount of innoculant, but the jars colonize very fast and it doesn't matter because there isn't a shortage of innoculant. When I run low, I prepare more malt media and add 10ml of the current mycelium solution as a starter culture and proceed as before. So, from 1-2ml of spore water, I end up with several hundred ml of heavily colonized mycelium solution. I don't try to harvest the mycelium from the solutions. Also, I haven't tried growing the solution for more than 7 days. Hope this is useful. The Walrus ------------ coocoocachoo ------------
Culture source and cloning
2) If one cuts the peeled chunk of stem almost all the way thru', then one can lift the blender parts just enough to stick the stem thru and finish severing the sterile chunk from the rest of the stem with the edge of the jar and the blender blade assy.
4) The knife and tweezers are sterilized in Al foil sheaths in the pressure cooker.
5) Eventually the cloned strain will lose stamina and stop producing and growing vigorously. It is a good idea to take a spore print and start a new culture every year or so (at least.) See the spore print procedure.
Mycelium can be cultured in a liquid (as opposed to a solid like vermiculite/brown rice flour) which you can then use to easily inoculate substrate and start mycelium growth much faster than if you were using spores.
You should first go to your local homebrew supply store and buy the lightest colored powdered malt extract they have. Mix 4% (by weight) malt extract powder with distilled water (boil it, and then mix in the powder. No need to let it cool to room temperature first). Pour this medium into any large jar with a metal lid, and make one small hole in the lid with a nail. Cover the top tightly with aluminum foil, and either boil the jar in water for an hour (be sure to put the jar on a washcloth and not directly on the bottom of the pot) or use a pressure cooker set at 15psi for 20 minutes. Let the jar cool overnight.
The next day take your spore syringe, stick the needle through the hole in the lid, and squeeze in the solution. Cover the top of the jar with aluminum foil again, only not too tightly since you'll want to allow gas exchange. Keep the jar somewhere warm and dark, and agitate it every day. You should notice the solution becoming milky, and towards the end of the incubation you'll see fluffy pieces of mycelium floating around in it. At this point you can keep it in the fridge until you're ready to use it. When you want to start a mushroom culture somewhere, flame-sterilize the needle of a 10cc syringe, let it cool, stick it through the hole in the top of the lid, and pull in as much solution as you need. You can then inject this into your substrate and it will start growing immediately. Any time you run low on mycelium solution, go through the same process with another jar only instead of injecting spores inject the last of your mycelium solution. Be on the lookout for any contamination!
Have all materials at hand so you can work quickly. Put about 1 1/8 cups water in the measuring cup. Add about 1 tsp honey to the water and stir until dissolved. Place the cup and the half-pint jar in the microwave. Fry on high for five minutes. Open the microwave do not remove the cup and jar. Working quickly within the microwave and using the potholder to protect your hand, pour water into the jar to about the bottom of the threads. Stretch saran wrap over the top of the jar and secure with a rubber band. Let cool to room temperature. Clean a working surface such as a table with a disinfectant such as Lysol or bleach water. Mist the surrounding air with the disinfectant. Working quickly inoculate the jar through the saran wrap with a sterile spore syringe using about 2 cc and cover hole with masking tape. Incubate at 80 degrees Fahrenheit. Once the mycelium has grown, shake to distribute the mycelium, and poke thru the saran wrap with a sterile syringe and suck up the white mycelium, cover the hole with tape and save for further use.
A fast way to colonize your jars
The achievement of this tek is to shorten the time needed to colonize a jar as you'll see signs of growth in 24/36 hours, instead waiting 3/5 days for spore germination.
What you need:
How to do it:
Some optional improvements:
250ml Eberbach Container
500ml Eberbach Container
1000ml Eberbach Container
Waring High-Speed Blender Base