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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1395
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 02:52 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

-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..
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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...
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When this is done and cool remove from PC and clean the center of the tyvek with rubbing alcohol....
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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....
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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..
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Now you are going to want to prepare plain water syringes... I do four per 1/2 pint isolate... BUT you are
going to want to prepare five and ill tell you why in bit. Also, leave 1 ml out of four of those syringes

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...
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Now clean off an injection spot on the tyvek with Rubbing alcohol...
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Now take the one plain water syringe that you didn't leave any water out of. flame the needle and inject
the water into the jar...
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Now remove the needle and quickly place a piece of tape over the needle hole in the tyvek...
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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. :-) ...
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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)..
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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....
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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. :-)

Ok, now comes in the rest of your syringes... This one myc. syringe you just made has enough myc. in it to
fill the rest of your syringes... When you PC your syringes, also PC one empty 1/2 pint jar with
two layers of tyvek and band. Use this jar to combine all four plain water syringes (the ones missing the 1 ml
of water) and the one myc. syringe. Then suck up the water and you will have four myc. laden syringes. The only
downfall of this that you have to use the syringes right away or the myc. will die in the syringes. But this is
a great way to get isolates without the use of a glovebox or flowhood. my yeilds have increase at least 30 percent
since i switched to isolates over multispore. Here is a couple of pix of qts i did with this tek.. works like a
charm. first one is Matius Romero and the second is a Texas isolate... :-)

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Enjoy! :-)

fahtster

(Message edited by fahtphish on June 21, 2004)
Yo. :-)
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JJ Downstream (Jj_downstream)
Member
Username: Jj_downstream

Post Number: 50
Registered: 06-2004
Posted on Sunday, June 20, 2004 - 02:56 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Beautiful!
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MushieDoobyDoo (Desideratum)
Junior Member
Username: Desideratum

Post Number: 12
Registered: 05-2004
Posted on Sunday, June 20, 2004 - 03:24 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

GREAT TEK!!!!
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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1397
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 03:30 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

oh yeah... don't forget to make new 'Jar Masters' from those four syringes to keep in foil and in the fridge in case you get a strain that you want to extend. :-)

fahtster
Yo. :-)
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grasshopper wilkins (Triked)
Senior Member
Username: Triked

Post Number: 453
Registered: 01-2004
Posted on Sunday, June 20, 2004 - 04:44 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

nice.Upload
"Little boy lost takes himself so seriously. Brags of his misery, he likes to live life dangerously." -Bob Dylan
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HF (Highflyer)
Moderator
Username: Highflyer

Post Number: 2014
Registered: 12-2002
Posted on Sunday, June 20, 2004 - 05:06 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

It would be best to make around 5-10 of these jars from spores, and grow out each substrain, so you could get the best isolate. Doing 1 jar will give 1 substrain, and it may not be the best fruiting one available.

I do something similar to this with agar. Ill just knock up quarts with spores, and transfer a kernel of corn to an agar plate once its colonized. Doing this gives you a substrain to save, and allows you to test that substrain immediately from the colonized jar.
"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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grasshopper wilkins (Triked)
Senior Member
Username: Triked

Post Number: 463
Registered: 01-2004
Posted on Sunday, June 20, 2004 - 05:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)


i went to jim morrisons grave while i was in france. made it by like 10 min. too they were locking gates as i was walking out.
your pic reminded me.
"Little boy lost takes himself so seriously. Brags of his misery, he likes to live life dangerously." -Bob Dylan
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rodger rabbit (Skyypilot)
Moderator
Username: Skyypilot

Post Number: 2723
Registered: 02-2003
Posted on Sunday, June 20, 2004 - 05:34 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I still fail to see where you are isolating strains however. It looks like several different jars of multispore injection? If you put a drop in each jar, the most dominant substrain will gobble up the rest. This is what happens everytime you inoculate jars with a syringe. A different substrain might become dominant in each jar, but it's still multispore inoculation. Did I miss something?

The only way I know to truly isolate strains is to use the two dimensional space of an agar dish. You can actually see the different strains, and transfer each substrain to a new dish to grow out. This way, the most dominant one doesn't take over the rest. Each substrain can then be fruited to see which one is best.

"Whatever it is, that girl put a spell on me". . .jimi hendrix
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HF (Highflyer)
Moderator
Username: Highflyer

Post Number: 2018
Registered: 12-2002
Posted on Sunday, June 20, 2004 - 05:51 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)


quote:

A different substrain might become dominant in each jar, but it's still multispore inoculation. Did I miss something?



Thats the theory....the most dominant substrain will be the only one left in the jar. Giving you one isolated substrain. Do this with multiple jars, and you are sure to find a very good fruiting, very aggressive colonizing substrain. Ive also never seen a substrain taken this way not fruit.

This process is also faster than starting spores on agar. I see many advantages to getting a good isolate this way. Do you see any drawbacks Rodger?
"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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Python (Python)
Senior Member
Username: Python

Post Number: 362
Registered: 04-2004
Posted on Sunday, June 20, 2004 - 06:26 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

absolutely cool.........
I used to do a little but a little was to little, so now i do a little more........
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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1403
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 06:28 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

it's kinda the poor mans isolate tek, ya know... like HF said.. that one dominate strain is your isolate whether you want it or not.. it's just that you don't need agar, flowhoods, or gloveboxes to do it... like HF stated.. you are going to want to make several 1/2 pints to get different substrains. You said that you get one substrain with multispore.. I think this is wrong.. esp. if you shake the jar vigorously after innoc'ing.. almost all my kernels of corn have growth after germination, leaving at least a dozen substrains.. not to mention when you break up the kernels to get them out of the jar.. that mixes all the substrains once again. i thought this is the reason that isolated mycelium gets better yields than multispore. follow? or am i missing something? :-)

fahtster
Yo. :-)
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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1404
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 06:34 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

it's like breakable agar, rodger. lol instead of doing a swipe like you would on agar.. i just put one drop in the center of the corn. If you let one of your plates go long enough, you end up with one strain on your plate, right? same theroy. :-)

fahtster
Yo. :-)
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joe (Philemon)
Senior Member
Username: Philemon

Post Number: 189
Registered: 04-2004
Posted on Sunday, June 20, 2004 - 11:24 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

okay.. so where do i get hydrated grain? and why can't i use popcorn?
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Imok Urok2 (Imok)
Moderator
Username: Imok

Post Number: 1566
Registered: 07-2002
Posted on Sunday, June 20, 2004 - 01:53 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

LOL popcorn is one type of "hydrated grain" after you
add water and cook/soak it
He was using popcorn as a demonstration.
Wish I had high speed dial/connection so I could look
at the pics, sounds good just from the text
archive material to spores/cloning
Hope this helps :-)
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rodger rabbit (Skyypilot)
Moderator
Username: Skyypilot

Post Number: 2729
Registered: 02-2003
Posted on Sunday, June 20, 2004 - 02:31 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I understand what you're doing. I certainly can't argue with success, as your cakes are the best I've ever seen.
I see you're getting an isolate, even though it consists of many different strains that have been eaten(assimilated?) by the most dominant one.
Cool.

"Whatever it is, that girl put a spell on me". . .jimi hendrix
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Yogi (Yogi)
Senior Member
Username: Yogi

Post Number: 358
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 05:20 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

What I like about this method is that even without a flowhood you can inoculate grains with myc rather than spores. This should speed up the colonization process by about a week which is good enough for me!I realize that it takes time to colonise the small jars but that is easy and it is like growing g2g masters in a way. Now I have never done g2g so this is exciting as I don't have a sterile glovebox or flowhood to use

(Message edited by yogi on June 20, 2004)
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I_am_me (I_am_me)
Senior Member
Username: I_am_me

Post Number: 2006
Registered: 10-2003
Posted on Sunday, June 20, 2004 - 06:10 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

It seems to me the easiest way to "isolate" a good fruiting substrain without agar or flow hoods or anything would be to knock up 10 half pints with multispore and just clone the best fruiting substrain that develops in a liquid solution. Then you would have a huge liquid culture and master grain jar to last you forever.
"Emancipate yourself from mental slavery, none but ourselves can free our minds."
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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1408
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 06:34 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

do you clone in open air I_am_me? granted, I've never tried to clone in open air. Always been a bit afraid of losing my work. but if you have a good open air cloning tek, i would like to hear it. :-)

fahtster
Yo. :-)
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Imok Urok2 (Imok)
Moderator
Username: Imok

Post Number: 1568
Registered: 07-2002
Posted on Sunday, June 20, 2004 - 07:55 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)


quote:

Now poke thru the center of the tyvek with a spore syringe



Shooting a liquid culture jar with a multispore syringe
(like the syringe you are using) instead of a grain jar
is what I_am_me is talking about I believe.

Hope this helps :-)
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Sweetness (Sweetness)
Moderator
Username: Sweetness

Post Number: 2377
Registered: 08-2003
Posted on Sunday, June 20, 2004 - 08:19 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Well done Faht! Thank you

Archive Material

"We do not grasp the playing together of opposite forces. Look at the bow and the harp. The invisible meaning of things is more hamronic than the thing been seen." Heraklit
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Hippie3 (Admin)
Board Administrator
Username: Admin

Post Number: 21511
Registered: 02-2001
Posted on Sunday, June 20, 2004 - 08:45 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

interesting approach.
and if it works,
who could ask for more ?


Namaste


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Imok Urok2 (Imok)
Moderator
Username: Imok

Post Number: 1572
Registered: 07-2002
Posted on Sunday, June 20, 2004 - 09:46 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Sweetness, think I already said that
Great minds think alike
Hope this helps :-)
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I_am_me (I_am_me)
Senior Member
Username: I_am_me

Post Number: 2007
Registered: 10-2003
Posted on Sunday, June 20, 2004 - 10:21 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)


quote:

is what I_am_me is talking about I believe.




Nah I was just saying grow out 5 to 10 pf jars from multispore and clone the best fruiting strain that develops.

To clone "in open air" I just make up about 5-10 liquid culture jars, clone from the center of a fruit body and work quickly and steadily to open the jar and plop in the tissue. I do this in the bathroom after a little lysol and letting the air settle. Seems to work quite well and with 5 to 10 jars you'll pretty much always get a couple to work out. Two half pint liquid cultures can go along way. :-)
"Emancipate yourself from mental slavery, none but ourselves can free our minds."
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faht (Fahtphish)
Moderator
Username: Fahtphish

Post Number: 1410
Registered: 01-2003
Posted on Sunday, June 20, 2004 - 10:49 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

kewl, thanx for clarifying. :-)

fahtster
Yo. :-)
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alpiner (Alpiner)
Intermediate Member
Username: Alpiner

Post Number: 52
Registered: 05-2004
Posted on Monday, June 21, 2004 - 04:34 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

this is great I have done this with rye but not with getting a isolate in mind just for the live culture now I feel even better about the way I do things what I like is its less of a pain than karo and I just PC them at the same time as my big jars
its nice to know that others are on similar wavelenght and I am not just a kook