fahtster - Mycotopia

Transcription

Mycotopia Web Forums - -Fahtty Isolation/Mycelium syringe tek- *RE...
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http://forums.mycotopia.net/liquid-culture-karo-honey-dextrose-q/9625-fa...
Mycotopia Web Forums (http://forums.mycotopia.net/)
- Liquid Culture: Karo/Honey/Dextrose Q&A
(http://forums.mycotopia.net/liquid-culture-karo-honey-dextrose-q/)
- - -Fahtty Isolation/Mycelium syringe tek- *REVISED*
(http://forums.mycotopia.net/liquid-culture-karo-honey-dextrose-q/9625-fahtty-isolation-mycelium-syringe-tek-revised.html)
fahtster
04-08-06 13:46
-Fahtty Isolation/Mycelium syringe tek- *REVISED*
14 Attachment(s)
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
ok.... on with the show.
-Fahtty Isolation/Mycelium syringe tek1. 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 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...
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Now clean off an injection spot on the tyvek with Rubbing alcohol...
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Now take your one syringe full of water and flame the needle. then 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. :-)
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
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motograter
04-08-06 15:22
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!
freakachino
04-08-06 22:35
:bow:
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
04-08-06 23:34
Yeah cool tek.
fahtster
04-09-06 02:50
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
04-09-06 08:06
Hooray, thank ya again faht
Elf
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reverend trips
04-09-06 09:44
:cool:
Hippie3
04-09-06 09:48
it's great to see you're active again, faht.
:love:
fahtster
04-09-06 17:53
:love:
fahtster
Soliver
04-10-06 16:39
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
dial8
04-10-06 17:11
:heartbeat
golly
04-10-06 22:05
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....
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No wait cloning
I'm putting together a complete 'how I grow' thread and figured it would be best to do it in
segments at first so it's not too much of a boring read and maybe make it a little easier for those
interested to digest.
For the purpose of picture taking I will do this out of the glovebox.
Cloning:
Through my own experience I have found that if I clone a nice fruit that was growing well off a
substrate in the growing condition I can supply and maintain, then it will most certainly improve
my yield if I grow that clone out on the same substrate in the same conditions that I can supply
and maintain.
For this reason, I will grow a small multispore project on the same substrate that I will use this
clone on for a later run on a much larger project using the same substrate and growing
parameters.
If you are careful with your sterile technique, you can inoculate your grain for the next larger
project minutes after you take the clone.
For this to work, you need to prepare your grains, or whatever you plan on for using spawn , and
have it ready to be inoculated with your new clone , before you get to the cloning.
This is what I do:
Take a half pint jar and poke a hole in the top and cover with electrical tape.
In the jar, I put in pieces of broken glass and about 100 cc's of water.
With the lid on loose, and covered with tinfoil PC for 20 minutes at 15 psi.
(you'll loose some of the water when pc'ing but 100 cc's is more you'll need)
Also do the same to a jar using an LC tek and an airport so you will be able to grow out and
store your clone.
Let cool and load into a cleaned glovebox the non-LC jar along with a small dish of isopropyl,
paper towels, the fruit you plan on cloning,and sterile tool for cutting out cloning tissue.
This is what I use(found in the nail stuff at the pharmacy)
I like to take a clone from a fruit when it is still young and vigorously growing. So, wearing
latex gloves I feel all the nice looking fruits and try to find the most dense fruit in the bunch.
Some people advise to take the biggest one from a nice cluster, but IME you can take a single
fruit and it will make nice clusters down the road.
Once everything is in the glovebox unscrew the lid of the jar that has the water and broken glass
in it, keep the lid on but have it easy to get into when you are ready to put the tissue in.
With both hands, grab the fruit at the base and rip it open, exposing the inside of the fruit.
I like to wipe the jar and tools with isopropyl as an extra precation.
Carefully not touching the outside of the mushroom with whatever you are using, cut out a piece
of the inside of the stem
and carefuly get it into the jar and put the lid back on.
Unload the glovebox and shake the shit out of that jar, shake it untill you are exhausted and then
shake it some more.
The broken glass in the jar with shred up the chunk of tissue into tiny little pieces.
Now you clean the glovebox again and put that jar back in, along with your LC jar, 3 sterile
empty syringes, and the dish of isopropyl.
Wipe the outside of both jars and the outside of the syringes with isopropyl.
Lift the tape on the jar with the clone give it a good swirl and draw the contents into the empty
syringes.
Squirt a couple cc's into you LC jar and cap the syringes.
Incubate the LC jar.
You now have 3 syringes full of clone material ready to knock up your spawn you have ready
for the next (larger) project.
Knock up your grain or whatever you plan on using for spawn immediately and incubate.
Some of the pics I took didn't come out as nice as I'd like, but you get the idea
The white shit on my hands is from wearing latex gloves.
More to come....
rev.
Cloning with liquid culture, the easy way.
This is a tek on how to clone without the need for advanced methods, agar, gloveboxes, flowhoods, or
even a pressure cooker. Its simple enough for a beginning pf-tek'er to complete. You'll probably also find
some good LC tips here if you've been struggling with those. As always, comments, questions,
suggestions, and kudos appreciated! I apologize if this is a little wordy, but I wanted to cover all the
bases. I've been answering newbie questions long enough to anticipate them before they've even been
asked, so hopefully I got everything covered.
Materials needed:
Step 1. Prepare a small liquid culture.
For this, I am of the thinking that SIMPLE IS BEST. My liquid culture containers are simple jars, with a
regular old metal lid and ring, with a single hole punched at the top. The hole should be big enough to
accomodate your needle, with a little room to spare so that a vaccum is not created later when
aspirating solution. The hole is covered with a piece of micropore tape, and then covered again either
with a coffee filter and rubber band, or a piece of aluminum foil loosely crumpled, to act as a dust cover.
Jar with just a hole
Recipe: Recipe is the same as any liquid culture; 4% media by weight. You may use any of the standard
LC media, honey, caro, dex/malt will all work. Water quality is important to LC. I find that distilled works
best, and gives the clearest solution. You may also use bottled spring water, or tap water filtered with a
Brita pitcher or the like. (Your mileage may vary depending on the quality of your locality's tap water. In
my area, I avoid it.) Honey quality also varies widely - I've found this particular variety works fantastic for
me, and doesn't make any sediment in my LC's even without filtering. You may need to experiment with
brands and types of honey to find one that works. For a sure thing, you can definitely use karo, and if
you're industrious and willing to hunt down dextrose/malt, by all means use that. The honey I've got
seems to outperform karo, so it works for me.
Measure 4 grams of media per 100ml of water
When cloning, I generally start with a small half pint container. You'll see I've measured exactly 4 grams
of honey. Water weighs 1 gram per 1ml, so I'll fill it to the 100ml point. I nuke this in the microwave for
about 30 seconds, just to warm the water, allowing the honey to dissolve easier, and I stir it up.
Obviously, when making larger LC's, just do the math to stay at that 4% mark.
This will be my "master" culture. From this culture, small samples are taken to start larger liquid cultures
in quart jars that are my "working" culture that I use for inoculation. The remainder of the master
culture can be refrigerated, where it will keep for several months. When I run out of working culture, it
only takes a few drops of the master to start a whole quart of working culture all over again. Its
important to take from the master each time, rather than go from working culture to working culture, so
that each of your working cultures are "second generation."
Step 2. Sterilization.
In my opinion there's only two acceptable ways to sterilize LC for consistent results: steaming, and
pressure cooking. This gives those an option who don't own a PC. Microwaving works for some, but I
don't trust it. If it works for you, fantastic, go with it.
In both cases, wrap the top of your jar in tinfoil as usual, and take a syringe about half full of water, and
also wrap it in tinfoil. We'll sterilize them both at the same time.
Syringe with some water; wrapped in foil and ready to sterilize
Steam sterilization:
Place a cloth at the bottom of a pot with a tight fitting lid. Use an inch or two of water. Place your jar
and syringe inside. Bring to a slow rolling boil. Once a boil has been achieved, start your timer for 30
minutes. At the end of 30 minutes, turn off the heat, walk away and don't even think about it for a few
hours. Let it cool.
PC sterilization:
Use your PC as per its instructions with something to keep the items off the bottom of the pot (like a
cloth, or a rack if you got one.) Slowly bring up to pressure. 10psi is plenty if that's all you got. 15psi is
fine too. Do not exceed 15psi. Once at desired pressure, set your timer for 15 minutes. DO NOT exceed
15 minutes. When the time is up, cut your heat and walk away until its cooled.
Step 3. Select specimen.
Find a nice looking fruit! We want to clone desirable qualities. Many have different ideas as to which
fruit to clone; ie. choosing the first one up might give you a speedy isolate to work with later, choosing a
big one might give you a genetic predisposition towards large fruits; and so on. I believe that there's just
so much variation in growing methods, even though we are taking a clone, we have no idea how closely
its going to resemble its "parent" when grown out later - so I take a nice looking fruit and hope for the
best. You'll find out in time if it was a good choice or not when you get your first crop of clones in, and
can assess how long they took to grow, the yields you got, and of course, the potency after sampling
some. If it didn't turn out as good as you were hoping, don't fret, you can try again from a different
specimen from a different multispore grow. If it did turn out for you, treat that master culture like gold!
Step 4. The biopsy
Here's where you'll want to practice your sterile procedures. No need to go overboard, all I do is ensure
there's no drafts, clean my work surface, wash my hands with antibacterial soap, and hit them a second
time with some hand sanitizer. If spraying Oust into the air and washing everything with bleach or
whatever makes you happy, knock yourself out.
Take your fruit, and clean the stem thoroughly with an isopropyl soaked cotton swab. Clean it all the
way around. Do not touch any areas with your hand near the site of the biopsy. Quickly wipe your
needle with iso (flame if desired, I don't bother - we did just sterilize it after all!) and stab it straight
through the stem to the other side.
Swabbing the stem; taking the biopsy. (This is where 3 hands would come in handy, somebody had to
work the camera! You'll obviously want to hold the mushroom near the top and swab the bottom, and
when taking a biopsy not allowing the mushroom to touch any surfaces.)
Remove the foil from your prepared LC jar. Using a dry cotton swab, clean up any
condensation/moisture on the lid. Then using an alcohol soaked one, clean the lid again. Give the needle
another wipe, insert into hole, and push the plunger. The water in the syringe will force the cross section
of the stem out into your LC. Quickly cover the hole with a piece of micropore tape (if your lid is still wet
with alcohol, that's fine, the tape will stick just fine once it dries) and cover with a dust cover of your
choice.
Knock 'er up! You might need to push a little hard, but the liquid will force the piece of stem out.
You can sort of make out the piece of stem in there. My solution really was clear I promise, this was
without a flash to reduce glare and hopefully get a picture of the piece inside. On the right, foil loosely
back on as a dust cover, don't forget to label!
She's looking a little beat up after being stabbed!
Incubate, and wait.
7-10 days later you should have a nice looking cloud of mycelium.
As they do on the cooking show.... "here's one we've prepared earlier!" When doing all the above steps,
you'll want to work quickly to avoid contamination. I obviously stopped to photograph everything so
who knows if we got a clean sample here.
Here's a "master" culture, about 10 days colonizing. You'll notice some is already missing, as I've used
some of the solution to inoculate larger LCs.
The master culture. Notice its very thick! Obviously my little micropore'd hole provides all the gas
exchange you need. You'll also note a few dark specs, that's leftover pieces of the stem. It kind of
dissolves and breaks up to an extent. It turns dark blue/grey, because you bruised it for sure when poking
it with a needle!
Here's a working culture, this is 5 days old. It was started with about 4cc of solution from a master, and
then constantly agitated on a magnetic stir plate. Use it (or make it) if you got it, but its not necessary.
They'll colonize great without, it just takes a little longer. LC to LC is FAST no matter what.
Enough LC to kill a small army, from a few CC's of the master.
Some additional LC tips:
-Follow the measurements carefully. You'll get no growth or poor growth if you use too much media.
When in doubt, use a little less than 4%.
-If your myc cloud is thick and can't be sucked up easily, suck up what you can, and squirt it back down
into the cloud. It will break right up.
-To aspirate from my single-hole jars, I just tilt them on their sides, being very careful not to spill any out
the hole. The needle reaches in just fine. I gently swirl it around and capture as much mycelium in the
syringe as possible. The thicker the better!
-People often ask, "when is my LC done?" Well, its done when you want to use it! If there's enough
mycelium floating around that you can capture a bunch in your syringe, go for it. The rest will continue
to grow until the nutrients have run out. When it doesn't seem to be growing any more, you can
refrigerate it, and it lasts many months. (6 or more.)
-When I aspirate, I take an alcohol soaked cotton swab, and swab right over the piece of micropore tape.
I then stick the needle right through the tape, and fill up my syringe. I quickly cap the syringe, and
quickly replace the soggy piece of micropore tape with a fresh piece. You can certainly use any number
of the fancy LC container teks out there, but it doesn't get much simpler than a hole with a piece of tape
on it kids.
-LABEL EVERYTHING. Keep track of dates, strains, generation number, anything you feel relevant.
-A perfectly prepared karo or honey LC should be nearly crystal clear. Dex/malt will produce a yellowish
solution. If your karo or honey solution turns yellow, you may have overcooked. Did you follow my
sterilization directions to the T? Either way, a little overcooked will still work, its not the end of the
world. Likewise, sediment from honey, or from malt/dex will not harm a thing - it just makes spotting
possible contamination a little more difficult. Some take some pretty extreme measures to filter their
solutions before sterilizing to get the sediments out, I'll leave it up to you.
-Unless you want cracked jars, heed my advice and allow them to cool gradually and naturally.
Quick Cloning FAQ:
Q: Why clone?
A: Cloning ensures the most efficient use of your substrate, the best possible yields, and once you've
found a clone that has a desired potency, the most consistent potency! See, with a multispore
inoculation, you end up with many many strains (each dikaryotic spore mating is technically its own
strain). These strains sometimes grow side by side, and sometimes mate a second time by a process
known as anastomosis. So in the end, you've got a cournicopia of genetic materials and strains. Not all of
these are capable of fruiting! I think of these strains as "guy on the couch" strains - they'll gladly eat your
food and drink your water, but never produce any fruits for you. By cloning an actual mushroom, we're
dealing with one single isolated strain that's already proven itself as being able to fruit. Another benefit
to cloning is nice even pinsets. When you're dealing with all the mixed strains of an MS inoculation,
some parts of your substrate are going to be ready to pin before others. With clones, they're all going to
be ready at nearly the same time, resulting in even, consistent pinsets, and more defined flushes.
Q: If I clone a large fruit, will the resulting fruits grown from it be large?
A: Maybe! If you have identical twin humans and one eats nothing but cheeseburgers and plays
Nintendo all day, while the other eats a balanced diet and exercises regularly, are they going to be the
same shape and size? Of course not. There's genetic predisposition of course, a large fruit obviously has
the genetics needed to be capable of producing more large fruits - but conditions have to be right. Its
nature AND nurture. Nurture is very, very important, so take care of your babies.
Q: If I clone a mutant, will the resulting fruits be mutants?
A: Again, maybe! It depends on why the mutation occurred. If there was a genetic problem that caused
the mutation, then sure, it will carry over. You might not end up with the same exact mutations later,
but you'll end up with some funky fruits. If the mutation occurred because the fruit came in contact with
a harmful chemical while growing, that's not going to carry over - the result may very well be a healthy
clone. Nature versus nurture again. Using the human analogy, if I cloned a person with 6 toes (yeah it
happens), the resulting clone will be born with 6 toes. If I clone a human who broke their leg, of course
the baby isn't going to be born with a broken leg! Got it?
Q: Where should I take my biopsy?
A: I've found that near the base of the stem works best. I can't tell you why, I don't know, just my
experience.
Q: How mature should the mushroom be before cloning?
A: As the viel breaks, or just before. If it starts to sporulate, it will drop spores on the stem. You're likely
to get spores mixed in with your biopsy, and guess what - its no longer going to be a clone! You want to
avoid spores.
That's all for now, if anybody has any ideas for the little mini clone FAQ above, ask away, and I'll answer
to the best of my knowledge (or maybe others can chime in and help out.) The only way I can make this
any simpler is if I were to come to your house and do it for you - and unless you got boobies, and plan to
pay travel expenses, it ain't happening. Thanks for reading, and good luck.

fahtster - Mycotopia

Transcription

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