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EZ Jar Clone Tek - 1
Pure Culture - 1
Cloning questionJose Cruzhippie312 2 01-03-02  04:37 pm

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Nanook of the North (Nanook)
Posted on Wednesday, October 10, 2001 - 01:57 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Please review 9er Tek

Cloning requires a Glove Box. 9er tek cloning works like this: get a pint or 1/2 pint canning jar that will screw into a Blender Base. Fill the jar about 1/3 full with Purified Water.

Set the blender blade assembly and gasket on the mouth of the canning jar. You do not need the threaded ring at this point, just the blade and gasket. Cover the jar with foil and sterilize.

In the glove box remove a tissue sample from a mature shroom. Tissue can be removed from the stipe or the cap, I usually use the stipe (stem):

Remove the foil cover from the jar/blade assembly. Remove the cap from the shroom. With sterile tweezers peel back the surface layer of the stipe, like peeling a banana.

Set the exposed peeled stipe on the sterile top of the jar/blade, it makes a nice work surface. Cut off the exposed tip of the stipe (where the cap was removed) and discard. Use a sterile exacto to make these cuts.
Next cut off a clean section of the peeled stipe with a sterile exacto, slide the blade/gasket off the mouth of the canning jar and drop this piece of sterile tissue into the jar. Center the blade/gasket on the mouth of the jar and remove from the glovebox.

Screw down the retainer ring from the blender over the mouth of the jar and blend at high speed to macerate the tissue sample.

Return the jar back to the glovebox. Liquid from the jar can be drawn up into syringes and used to shoot PF jars directly. This inoculum does not keep however, perhaps only a few days in the fridge. I keep a Sterile jar of Dextrose Solution handy and shoot dextrose with the mycelial solution... Dextrose will grow out quickly and the solution is storable in the fridge for months.

Nan
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Nanook of the North (Nanook)
Posted on Wednesday, October 10, 2001 - 08:19 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Another way to clone is to take a small tissue sample from a cap or stem... Stem samples were covered so take a cap...

You mix 10-15 drops of Betadine, (generic = Povidone-Iodine 10%, available in any drugstore) into about 1/2 cup of purified water and wash the cap (just the crown, not the gills) down with a Q-Tip and place into the Glovebox to dry.

Once dry, remove the skin from the peak of the cap with a razor blade. All instruments must be be boiled, or preferably wrapped in foil packs and Pressure Cooked. Flame the blade or get open another from your pre-prepared foil pack of sterile instruments... And remove a small tissue sample from within the boundries of your peeled area. If you take any skin it will contam...

Place the tissue sample on a sterile plate, and Macerate* it with a blade. Then use the blade to scoop up the macerated tissue and drop into a sterilized Dextrose or Honey Tek jar to grow out...

Shroom Glossary*
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Black Star (Mr_Bug)
Posted on Thursday, October 11, 2001 - 01:09 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

What are the advantages to cloning vs. sporulating?
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Nanook of the North (Nanook)
Posted on Thursday, October 11, 2001 - 04:15 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

With cloned mycelia you eliminate the "roll of the dice" you get when shooting cakes with spores, you always grow out and fruit a known strain. An albino shroom or a giant can be grown repeatedly by cloning. People use cloned strains to spawn straw beds to get higher yields and bigger shrooms: I started spawning with spores and found some beds produced better than others, then I cloned a giant shroom from my best bed... Now all of my beds produce this same shroom.

******************

In starting a culture from a spore print, factors controlling sexual reproduction must be recognized. In a tetrapolar fungus, recall that only one fourth of the spores from any one mushroom are fully compatible with any random spore from that same strain. Two of the four pairings will form illegitimate pairings and make clamp connections, but cannot fruit. Thus, two thirds of the random dikaryons formed will be of the illegitimate type. These illegitimate pairings cannot be recognized in culture. One way around this problem is to
inoculate with a large number of spores and take a tissue culture of the best mushroom that appears. This is the simplest method but the strains produced in this manner still must be evaluated thoroughly.
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Nozza (Nozza)
Posted on Thursday, October 11, 2001 - 04:36 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

You can make the task a lot easier by using Peroxidated Agar a la Rush Wayne.

Just add 10ml of 3% H202 per litre of agar when it has cooled to below 50C - when the bottle is only warm to the touch. You can then pour your Plates in the open without fear of contamination and you are unlikely to contaminate when you add your chunk of shroom to clone from. I tried the experiment Rush desribes in in the peroxide guide - leaving the plate out open in the kitchen for a day and still got no contamination.

It makes the whole thing well easy. The other plus point is that mycelium grown on H2O2 gets used to it which means that you can peroxidate your Grain too and do agar-to-rye transfer with little fear of contamination.

For me, it's revolutionised my rye technique. No Glove Box, no HEPA, dirty old kitchen and no contamination. Quality!
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Nanook of the North (Nanook)
Posted on Thursday, October 11, 2001 - 05:20 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

H2O2 Kills Spores : Peroxidated Agar works fine for cloning

But I have done Agar work and Liquid Culture smokes. Take your macerated* tissue and drop (or Shoot with 9er Tek) into a jar of Dextrose or Karo Tek. Let grow out 3-4 days at 86*F, Draw up Syringes, and shoot whatever you want. No H2O2 required. No cutting up agar blocks. You shoot liquid inoculum which spreads into the substrate. It simply is the best tek out there for cloning It's Hot!

It is killer for shooting PF Jars, which you simply cannot do with agar. Most of the growers here use PF jars at one point or another.

Shroom Glossary*
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ion ewe (Ion)
Posted on Saturday, October 06, 2001 - 05:36 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Each spore germinates to form a monokaryotic mycelium (hyphae). These little fingers grow until they touch another compatible mating type of finger from another spore. This forms the first dikaryotic mycelia by somatogamy. Somatogamy is the process by which the somatic cells of two compatible monokaryotic hyphae fuse, but the nuclei do not. The result is a cell that contains two separate nuclei! These dikaryotic cells replicate (each new cell still having two nuclei) and grow vegetatively. They consume energy and produce it. This is the body (thallus) of the fungus. The thing most people do not realize is that this stage of the fungus's life can go on indefinitely! As long as the life requirements are met and fruiting is not induced, the mycelia can grow "forever". Anyway, when appropriate conditions are met (in this case: lower temps, light, and greater oxygen content of the air) the dikaryotic mycelium knots itself up in spots and begins to form the differentiated masses we know as mushrooms (basidiocarps). These push up from under the soil (casing) by incorporating more mycelium and expanding by the absorption of water. After the gills form under the cap, something really weird happens very rapidly. Things called basidia form on the underside of the gills. This is when the fusing of the nuclei occurs (karyogamy). This state (diploid) is very brief as meiosis (a form of cell division where the nucleus separates into 4, each containing half the chromosomes {one, unpaired set}) immediately follows. Now we have four haploid (half chromes, whole structure) nuclei in the basidium. These nuclei are then pushed out of the basidium and form hard sheaths of protein around them. These are the basidiospores ("spores", for short).

Now the words get really long... Tetrapolar Heterothallism. This is the "sexual orientation" of this mushroom... sort of. Basically, if there is to be a thallus (mycelial mass) that is capable of reproducing sexually (by making mushrooms), somatogamy must occur between two monokaryotic mycelia that differ in both sets of factors ( A and B). Like this: mono-myc #1 is "A-b". mono-myc #2 is "A-B". #3 is "a-b". #4 is "a-B". Only mono-mycs with different A's *and* B's can mate. AB with ab, and Ab with aB.
Confused yet? Anyway, this is where the "Tetra..." comes in. A and B (a and b) are both *sets* of pretty complex factors, and the four types (above) come from each mushroom as 4 separate spores. The number of actual mating types is in the hundreds! Suffice to say that they can not be labeled as male and female... the heterothallic part just means that the body of the mycelium is made up of two *different* creatures (types).

Whew! I'm sure that in the time it took me to write all this (and organize it in my head), someone else has already posted more questions! Hope this answers them...

-ion

Shroom Glossary
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Mr. Tambourine Man (Tambourine_Man)
Posted on Sunday, December 02, 2001 - 07:47 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

So lemme see if I have this right.... You could take a huge shroom, cut some flesh from it, drop it in a honey jar, mycelium somehow grows from the flesh, the mycelium is used to innoculate jars, and the resulting mushrooms have the benefit of the big shroom's genes? Will this technique actually yield bigger shrooms?
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Brad (Raze)
Posted on Sunday, December 02, 2001 - 07:55 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

If you clone the right shroom, yes
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Mr. Tambourine Man (Tambourine_Man)
Posted on Sunday, December 02, 2001 - 08:01 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Does right=big?
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Brad (Raze)
Posted on Sunday, December 02, 2001 - 08:05 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Well, cloning a small shroom wouldnt result in a large shroom...
Take a primo shroom, and clone it. And you dont use pure honey. Like 4% honey, 96% water. You can also use Dextrose, Agar, and various other sources of nutrients.
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Cbee (Cbee)
Posted on Sunday, December 02, 2001 - 09:56 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

does this raise the flush size significantly? will i be bale to tell a difference if I used cloned innoculant rather then a spore syringe?
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Nan (Nanook)
Posted on Sunday, December 02, 2001 - 09:58 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

If you pick the right shroom from an excellent cake... Yes
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hippie3 (Hippie)
Posted on Monday, December 03, 2001 - 02:33 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

yes, it will make a big difference, it's well worth your while.
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Mrt (Mrt)
Posted on Tuesday, December 04, 2001 - 11:31 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Cloning will generally result in less aborts, and more consistant and uniform flushes. That's what I have experienced anyways. I like to take the fastest and largest growing shroom from a brf cake, toss some of it in honey water and then innocculate whole rye grain. I have found that the cloned tissue will be ready for innoculation 7 to 10 days after it has been placed in the honey water. What have the rest of you found to be an average time?
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hippie3 (Hippie)
Posted on Tuesday, December 04, 2001 - 01:55 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

sounds about right, 1-2 weeks.
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hippie3 (Hippie)
Posted on Tuesday, December 04, 2001 - 06:12 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Cloning.
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Joe Mamma (Madscientist)
Posted on Saturday, December 22, 2001 - 08:07 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

With cloning do I need to have a extremly new shroom. My shroom is two days old. It is very fresh. I have it sitting out on top of my terrarium. It is very sterile in the room. I have a HEPA room filter going 24/7. But I have not kept in a sealed bag. Do you think that I can use this shroom or should I wait for the next time around?
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Nan (Nanook)
Posted on Saturday, December 22, 2001 - 08:14 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Give it a try. If it does not work you count it as practice
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hippie3 (Hippie)
Posted on Sunday, December 23, 2001 - 03:36 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

use tissue from deep inside and you should still be ok.