-Fahtty Isolation/Mycelium syringe tek- *REVISED*

[fahtster]

ok... so i'm bored and thought i would repost this tek so it can go into the new vaults... also revamped it a bit. i think it should be tied to this thread somehow... http://forums.mycotopia.net/showthread.php?t=5795 (same ol' same ol': dried tissue mycellial syringes)
ok.... on with the show.
-Fahtty Isolation/Mycelium syringe tek-
1. Overview - This is a tek that I use to isolate substrains and make mycelium syringes
at the same time without the use of a glovebox or flowhood. Although it is required that
you are experienced with grain substrates. This can be done in a few minutes while making
you spawn grain jars/bags what have you.
First take a clean 1/2 pint jar and fill the bottom with a 1/2 inch or so of hydrated grain substrate.
(Note- I used dry popcorn kernels just for demonstration purposes only... you are going to use
hydrated grain). Here are a couple examples..

Now cover with two layers of tyvek and lid band (this is what i use, you can use polyfil or any number of ways
to seal the jar) and PC normal, 15 psi for 45 min...

When this is done and cool remove from PC and clean the center of the tyvek with rubbing alcohol....

Now poke thru the center of the tyvek with a spore syringe (sterile of course) and ONLY PUT ONE DROP IN THE MIDDLE...
I have found that this is VERY hard to do.. lol but it is ESSENTIAL for this tek to work....

Now DO NOW SHAKE, DISTURB, OR EVEN MOVE the jar... if you can, innoculate the jar in the exact place
you are going to let it colonize. The reason that you don't want the jar to be disturbed is that you
want the spores to germinate as close together as you can to allow ONE dominate substrain to 'eat' the
rest. Don't even look at the jar for five days. This how you get a substrain.
Now you are going to let this substrain colonize the little bit of grain on the bottom of the 1/2 pint.
Remember to tape up the tyvek 95% or your grain will dry out.
When finished it should look something like this..

Now it's time to make the syringes you are going to use to innoc. your actual substrate. I usually make three to four syringes
per 'faht jar'. I'll demonstrate how to make three. Take a clean, empty, 1/2 pint jar and put 25 ml. of water in it.
This is the water that is going to fill up your three syringes that are going to innoc. your substrate. cover with two
layers of tyvek, a band, and foil over the whole deal and set in pc.
Now fill one syringe with distilled water and put in a 1/2 pint jar, needle first filled 3/4 of the way with distilled water.
(basically you'll have a syringe sitting in a jar filled with water.) Now set this into the pc as well. what this does is keeps the
syringe full of water.. I don't know about you, but opening a pc to find an empty syringe when you wanted to find a full
one sucks. this happens cuz the pressure in the cooker forces the water out of the syringe... then the pc cools
down and the pressure equals out, but if the needle is submerged in water, it will suck up the water instead of air.
Now take your other two syringes and wrap them up in foil. place those in the pc also. pc at 15 psi for 30 min.
Once the pc has cooled, remove the jar of water and three syringes (one full of water and the other two wrapped in foil)
Now take your colonized 1/2 pint iso. and break it up in the jar by hitting the jar against your hand,
It shouldn't take much...

Now clean off an injection spot on the tyvek with Rubbing alcohol...

Now take your one syringe full of water and flame the needle. then inject the water into the jar...

Now remove the needle and quickly place a piece of tape over the needle hole in the tyvek...

Now kind of gently shake the jar to get the mycelium to come off the kernels and into the water
from your plain water syringe. This acts kind of like a organic eberbach container. :-) ...

Once completed, clean off another injection spot with R.A. and take your now empty plain water
syringe and poke thru the tyvek (you are going to want to do this as close to the edge of the jar lid as possible)..

push the needle up against the side of the jar. tilt the jar so you get as much
water by the syringe as you can and suck up the mycelium ladened water... when done your syringe should
look something like this....

As you can see the syringe is now only half full. This is because some of the water 'stuck' to the corn kernels..
this is ok.. as you can also see, there is so much mycelium in the syringe that it has a blue tint to it
and you can't see thru it. :-)
Now this is where that jar of water and the other two syringes come in. take the syringe with the myc. laiden water in it
and inject that into the jar with the water for the other two syringes. just squirt that first syringe in there and suck up
a full syringe of the newly mixed water.. this is why you put in 25 ml.. 20 ml is for two syringes and the 5 ml. is for the other
half of the myc. laiden syringe. clean off two more injection spots for the other two syringes and you have three fully
stocked myc. syringes. be sure to cover any holes in the water jar until all syringes have been filled as not to contaminate
your myc. water.
this tek works great for multiple reasons... you can use it to get a substrain.. or to get mycelium syringes.
you can also use this tek for cloning.. just drop in a piece of mushy from the inside of a stem that was dipped in h2o2 for a couple seconds.
and there you go! (works best if done in a glovebox cuz the jar must be opened)
you can also use this tek to store substrains.. mycelium can be dried and reanimated with water. just take off the tape on the tyvek
(minus the one that is covering your innoc. point) that you use to keep the grain hydrated and let dry out.. thats it. I've had very little
problems with this tek.
fahtster

[motograter]

I remember reading this a while ago... I was WAY too new and pretty intimidated by th idea of working with grains, or trying to isolate. Think I'll give it a shot now, it seems pretty easy.

Nice work!

I love this tek, and used it often before I got started with agar. It works so amazingly well! Thanks for the re-post Fahtster!!!!

[SharkieJones]

Yeah cool tek.

[fahtster]

thanks yall.
motogator: yeah, the tek is super easy to do. grain prep. is pretty straight forward too. First, put your corn in the pc without your jar rack (the plate the pc comes with to keep the jars off the bottom of the pc.) and cover with at least three inches of water.
you can probably go a little less once you get the hang of how much the dry kernels will absorb. on average the kernel will expand 3 times it's size.
Now pc at 15 psi for 1 hour. after an hour, turn off the pc and left the pressure adjust back to zero... once the safety nob drops, you can unscrew the lid. be very careful.. the pc will still be very hot. don't let the steam get ya in the face.
Now pour the corn into a strainer. I rinse off the corn with hot water first. the kernels will have lots of starch on them.. if you rinse with cold water first the starch will coegulate making it a bitch to get off. rinsing is a very important part of the process... you want to get the kernels as free from starch as possible. once you've rinsed with hot water... rinse with cold. this will cool the kernels down making them easier to handle and also 'tighten' the kernel up making it not so mooshy.
once rinsed, i let the corn sit for a few minutes to drain. Next i lay down a piece of paper towel and lay the corn on top of that in a single kernel layer. then i place another piece of paper towel on top of the corn layer and roll my hands lightly over the corn. this will wipe off excess water and starch from the corn before i place it in the 'faht jars'.
next the new faht jars go into the pc again for another 45 mins. allow the pc to cool this time before opening. make sure you shake the jars once they are out of the pc to redistribute the starchy water that will indefinately end up poolin on the bottom of the jar and thats it.
fahtster

[Elf Salvation]

Hooray, thank ya again faht


Elf

[reverend trips]

[Hippie3]

it's great to see you're active again, faht.

[fahtster]

fahtster

[Soliver1]

Sweet post faht -

It is indeed great to see you back around.
I still can't think of a cake grow without seeing
those pics of your 30+ cake gardens with shrooms
sprouting out of every inch . . .



soliver

[golly]

Glad u reposted this tec fhat...i have had very good results with your method.
Works with all kinds of grains...
Great way to store a culture.
Cloning works well using a small amount of grains in multiple jars..
Best of all ,it allows u to test questionable prints without going to agar or gambling on whole qts...
Also is easy to detect contaminants b4 making a Myc syringe ,which is not the case with normal LC....Thanx agin Mon....