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.

Cloning Mushrooms via Liquid Inoculant

Blender Usage

Sterile Tissue Cloning Procedure (coffee grinder, hydrogen peroxide & syringe)

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.

http://diseyes.lycaeum.org/teo/peele.txt
[item by Stephen Peele of FMRC/Florida Mycology Research Center]

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."


Catalfomo, P. and V.E. Tyler, Jr.
The production of psilocybin in submerged culture of Psilocybe cubensis
LLoydia 27:53-63, 1964

Research by Gartz (1989) showed alkaloid synthesis in Psilocybe cubensis (Earle) Singer is suppressed when the mycelium is grown using agar media supplemented with more than 10 % malt sugar. P. azurescens reacts similarly. Research has also shown that alkaloid content is generally low in the mycelia compared to the fruitbodies. With Psilocybe cubensis, the main alkaloid synthesis occurs during the differentiation of the mycelia to the fruitbodies. (Gartz & Muller 1989). Further, younger fruitbodies frequently have higher alkaloid levels than more mature ones. (Gartz 1992/1993). As Table Ill shows, specimens grown outdoors in Germany

"Liquid Culture Mycelium" (alt.drugs.mushrooms)
Osmotek Bioreactor (industrial liquid culture systems)



From: hippie3@geocities.com (hippie3@geocities.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 (ville.tuomi@kolumbus.fi)
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: anon@anon.not (anon@anon.not)
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 <88hbbc$d7r$1@nnrp1.deja.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 (walrus@nym.alias.net)
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
------------

Sterile Tissue Cloning Procedure

Using a Coffee Grinder, Peroxide and Mushroom Tissue

  1. Create a paste of brown rice flour and water (or your choice of substrate), spread evenly in the bottom of a canning jar, seal, pressure cook at 15 psi for 30 minutes.
  2. Pour about 1/3 cup of 3% h2o2 into a jar.
  3. Obtain a clean coffee grinder; fill with water to the brim of the metal cup, insert a fresh and cleaned mushroom stem (no cap) and proceed to grind for 10-15 seconds.
  4. Use a clean spoon to transfer some of the resulting mixture into the h2o2 jar.
  5. Let it fizzle for a couple minutes, then draw into a syringe.
  6. Inoculate jar created in step #1, or simply inoculate pf-style jars.

Tips if Culture Contaminates

  1. Extract a piece of healthy mycelium.
  2. Cut mycelium into tiny pieces with razor blade.
  3. Transfer pieces to a small pool of h2o2 and then suck into a syringe.
  4. Inoculate another culture jar with it.

Blender Usage

This is what happens when one tries to sterilize a regular ol' acrylic blender jar.   This can lead to spousal disenchantment with your creativity.  It was at this point that it was "discovered" that the size and thread of a regular mouth mason jar match the blender parts.  ( This didn't impress her either since she's known that since high school Home Ec class.)
Remove the base, seal, and blade assembly from the blender container.   Put the seal and blade assy. on the 8 oz mason jar ( 2/3 full of H2O) and cover with Al foil, then sterilize.   The tab sticking out to the side is just to make it easier to remove the foil in the glove box.
The base doesn't come into use until after performing the shroom water procedure.  The base part doesn't need to be sterile.  Just clean with lysol occasionally for general cleanliness.  Be sure everything is tight and seated properly, then put the assembly pictured below on the blender and blend on the highest speed for 3 or 4 seconds, allow to stop and repeat for another 3 or 4 seconds.   Next remove the base and leave the blade assembly and seal in place.  Place it back in the chamber to have the blade removed and to be covered with the baggie for drawing syringes.

Culture source and cloning

Culture source:
 Either acquire a spore syringe or get started from a fresh fruit body.  Spores can be injected into the whole grain recipe or the MMGG recipe.  It /seems/ that spores work better in the MMGG recipe, but more tests need to be made. After getting a strain started, you can farm it for a long time with the procedure below.
 
Cloning procedure:
   Take a healthy mushroom for cloning around the time that the veil breaks.  You want one that is still growing vigorously, not an abhort.  Place it in the transfer chamber.  Avoid fruit with brown streaks on the stem.  Use a sterilized Xacto knife and tweezers to extract a clean chunk of stem to add to sterile water in a mason jar.
 This is done by holding the freshly picked shroom near the cap, and using the sterile knife to cut a ring around the stem about 2" from the bottom.  Only cut a little way into the stem.
Use sterile tweezers to grab and peel the surface layer back toward the base of the stem until it resembles a peeled banana.
 Then cut the stem just above the peels.  Once you've removed the Al foil from the sterilized blade assembly and mason jar, You have a sterile table to work on for cutting off the stub.
 Then slice off the clean chunk and let it fall into a mason jar (8 oz regular mouth) 2/3 full of sterilized water.   The jar is prepared by covering with the metal blade assembly and seal from the blender and that is covered with Al foil and sterilized.  The transfer is done in the glove box.  For more details see the "Blender Usage" page.
It turns out that a regular mouth mason jar has the same size and thread as the plastic blender container part from an Osterizer blender (probably other brands as well), so after dropping in the piece of stem, remove the jar from the chamber, then screw on the base of the blender and proceed to blend for about 3 or 4  seconds at the fastest speed.  Allow to stop, then repeat.   Be sure to hold the jar while blending.  When done, remove the blender base,  put the jar (w/seal and blades) in the chamber,  remove the blades and seal, then immediately cover with a new baggie (in the transfer chamber).  Then  draw syringes of the inoculant water by piercing the plastic bag.   This procedure has proved to be very effective.
For pictures of the details of the blender parts and mason jar assembly go to the Blender usage page.
Additional info/tips:
1) It is best to use a rubber band around the shroom water jar/baggie to keep out contaminants.
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.
3) The shroom water has been stored for 1 week with no harm and it's gone bad in 2 days.  I have no idea how long it can remain viable.   It is MUCH better to make up the shroom water and use it immediately.  The growth rates are incredible when the shroom water is fresh.  One pint cakes (rice only) have been birthed 10 days after inoculation.  14 to 21 days is more common.
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.

from `The Mushroom Cultivator' by Stamets & Chilton - Tissue Cloning


Liquid Growth Medium

Huge thanks to The Walrus!

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!


AMD’S Easy No PC Honey Water Tek

Materials


Procedure

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.


CLuB99'S Honeywater Mycelium Tek

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:

Optional:

How to do it:

  1. Drill a small hole in the lid, large enough to pass through with a needle.
  2. Mix a small quantity of honey (less than a teaspoon) in the water, the water must be very light yellow coloured.
  3. Sterilize for 30 minutes and let it cool (don't over sterilize or the sugars in the honey caramelize).
  4. Inject a small quantity of spore solution, 1-2 cc it's enough.
  5. Put in a dark warm place, better to stir a bit once a day, and wait until you see small white hairs floating in the solution,
  6. wait a day or 2 then add some H2O2 , few cc's, (I would put 2 cc in a 1/2 pint jar) then put the jar in the fridge. This helps to keep out contaminants. Specially bacteria.
  7. Now the honeywater it's ready. To use it simply suck some water with a sterile syringe and inoculate your MMGG/PF TEK style jars as usual.
  8. The solution will last at least 2 weeks, but anyway I wouldn't use a solution much older as the risks of contamination increases.

Some optional improvements:

honeylid.jpg (14311 bytes)


LIQUID INOCULATION TOOLS

Eberbach Containers
Fungi Perfecti stocks Eberbach containers for devotees of the liquid inoculation technique. These containers are autoclavable. Partially fill with water, autoclave in your pressure cooker. Transfer an entire dish of mushroom mycelium into it. Place the container on a stirrer, activate, and in five seconds or less there are millions of tiny fragments of hyphae which can be transferred into sterilized grain. This technique minimizes contamination, accelerates colonization, maximizes the potential of your mycelia, and tremendously speeds up the inoculation/incubation process. The cultures and time you save will more than make the cost worthwhile. One culture grown on a 100 x 15mm petri dish can effectively inoculate 100-300 cups of grain using these stirrers! In fact, some mushroom species grow too slowly via the traditional methods of propagation, and defeat would-be spawn makers. With these stirrers, mycelium of difficult-to-grow species can be expanded quickly and efficiently. In our opinion, these stirrers are integral to any professional spawn laboratory.

250ml Eberbach Containers

250ml Eberbach Container
This is an all-stainless-steel Eberbach container, fitted with an airtight lid.
EB250 $239.00

500ml Eberbach Containers

500ml Eberbach Container
An all-glass container, featuring a screw-on metal cover and stainless steel blades. (Note: the manufacturer suggests that this unit be autoclaved unassembled. All-metal containers can be autoclaved assembled.)
EB500 $179.00

1000ml Eberbach Container
An all-stainless-steel container, equipped with a 1/2 inch diameter airtight sampling plug that is perfect for the insertion of a syringe or pipette with minimal risk of contamination. Comes with a threaded cover. We highly recommend this unit.
EB1000 $529.00

Blenders

Waring High-Speed Blender Base
This is a durable, single-speed, professional quality blender base designed for use with the 250, 500 and 1000ml Eberbach containers. 20,000rpm, consumes 360 watts.
WB700 $179.00
WB700E for 230V/50Hz applications $279.00