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Organism (Organism)
Posted on Monday, April 28, 2003 - 10:46 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I cloned tissue from a big PF fruit by working as carefully as possible and peeling the outer layer off the stipe while it was still attached to my cake in the grow box.

I then grabbed some core tissue from the stipe with my sterile tweezers and carefully droped it into a jar with pressure cooked distilled water and glass shards.

Inside the 1/2 pint jar I had pieces of glass from another broken jar I was no longer using.
Everything was pressure cooked. Lid, band, jar, distilled H2O, glass pieces.

It was all cool at room temp when I droped in the core tissue sample.

Then I just shook the hell out of it and then I saw the most beutiful thing.. clean macerated tissue.

I quickly drew it up into 10 new sterile syringes and injected 100 pre-pressure cooked PF jars.

They all are doing fantastic.
All without the use of a blender or glovebox!
SWEEET
I can't remember who I got the idea from, but it was off this site. They were using it to grind up colonized mycelium, but I think it works better with tissue. (more clean)
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Hippie3 (Admin)
Posted on Tuesday, April 29, 2003 - 12:37 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

congrats!
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rodger rabbit (Skyypilot)
Posted on Tuesday, April 29, 2003 - 01:56 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Very nice!
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Bionic Grandma (Bionicgrandma)
Posted on Tuesday, April 29, 2003 - 03:38 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Very cool. Few questions here...

1) Stipe = stem? You used tissue from the inside of the stem?

2) Could you also tear away the veil and grab a tweezer-full of spores?

3) Is the broken glass necessary? Will it break things up much more than will vigorous shaking? And is any advantage offset by tissue sticking to the shards? Or perhaps the point is to get the larger pieces of tissue to stick to the shards...
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Organism (Organism)
Posted on Tuesday, April 29, 2003 - 04:56 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

stipe is stem

I would not recomend trying to "grab" spores from the cap or veil as that portion may run high in possible contaminants. This is why the outer layer of stem is peeled away. It has had contact with air.

The broken glass is very good at shredding the tissue much like a blender does.

the tissue does not stick to the glass shards, it is suspended in the water you draw up.

Hope that helps
peace
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Bionic Grandma (Bionicgrandma)
Posted on Tuesday, April 29, 2003 - 06:18 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Thanks a bunch for replying so fast. More questions, if you would be so kind...

1. How much core tissue did you use?

2. Is distilled water necessary?

3. How long would these syringes last in the fridge? Shorter, longer, or same as a regular spore syringe?

4. Sterility-wise, this seems to be as easy/hard as making syringes from a print. And you don't have to bother making or buying a print. Sounds great. Why don't more people do this? Or do they? Any thoughts on the difficulty?

Thanks so much!
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Hippie3 (Admin)
Posted on Tuesday, April 29, 2003 - 01:18 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

1. a match-head sized chunk is sufficient.
2. desirable but any will do in a pinch.
3. not long, much shorter shelf-life than spores, best used within a few days of preparation.
4. this tek is no substitute for printing, it's cloning and the genetics will be damaged as clones grow out and get cloned again.
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Organism (Organism)
Posted on Tuesday, April 29, 2003 - 03:05 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

yea, what hippie said

I don't save them or try to store them at all

I use the liquid innoculant as soon as it is prepared.

The more jars planned on innoculating the more tissue you would want to use.

Cloning is bad, but cool to reproduce big fruits I hear.
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rodger rabbit (Skyypilot)
Posted on Tuesday, April 29, 2003 - 04:44 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

3. not long, much shorter shelf-life than spores, best used within a few days of preparation.

Bingo hip. I've been growing off my own prints, but had a few syringes left over from when I was just getting started this time around, and decided to use them. Those are the logs and laundry baskets that contamed. Ya think it was the syringes? I didn't notice any contams until they were just about to the fruiting stage, then they ALL contamed. :( None of them was over 5 months old. You may have just diagnosed what went wrong with my projects.(I've been blaming the corn) I've read here that folks use syringes that are much older than that. Perhaps they just got lucky?
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Bionic Grandma (Bionicgrandma)
Posted on Tuesday, April 29, 2003 - 06:57 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Thanks everyone - good thread. So cloning seems okay if it's one-shot deal, but shouldn't be repeated? I'm gonna search the archives and the web in general to educate myself on the relevant biology...
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Micro (Micro)
Posted on Tuesday, April 29, 2003 - 07:01 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

It usually works ok for about three generations, IMO.

--
Micro
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Shroomzhilla (Shroomzhilla)
Posted on Wednesday, April 30, 2003 - 01:43 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

from past experience it seems REAL easy but as with all things I manage to find a way to screw it up. count on a 60 ml syringe to tottaly hose your cake with a ton of water. I had two dozen jars turn to mud a couple days after the mycelium spots started to grow not much you can do with them once that happens except useing to spawn your compost pile.
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Organism (Organism)
Posted on Wednesday, April 30, 2003 - 03:32 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

When I have time I will prepare some pics that document the process.

My plan is to use the clones for 2 generations while constantly keeping a fesh gene line going from a spore print of an uncloned line.

One question I would like to ask anyone who knows for sure,

Can a viable spore print be taken from a cloned fruit body?

Not that I would wnat to do it because it would be a less quality dupicate of the origional fruit bodies reproductive "seed", but would it work?
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Microfile (Microfile)
Posted on Wednesday, April 30, 2003 - 03:58 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

"Can a viable spore print be taken from a cloned fruit body? "

Usually, yes. If it is first generation almost definatly.

It won't change that much over the first two generations; it's only if you keep doing it that it matters.

--
Micro
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Scientician (Scientician)
Posted on Wednesday, April 30, 2003 - 05:24 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

As long as the fungi is still healthy, I wouldn't expect it be a problem.

The only problem with cloning is that an organism can only reproduce its cell a certain number of times.

Every time that a cell divides, the DNA contained within the cell unravels and copies itself (simplified explanation). After you've grown this out long enough the cells have divided millions and millions of times. Eventually a small mistake will happen (eg one amino acid copies incorrectly). From that point, if the cell survives, it will continue copying the mistake, and affecting the resulting mycelium/spore print.

I would say, stick with what everyone is saying about 2 generation.
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FarmerWop (Croppinsloppy)
Posted on Wednesday, April 30, 2003 - 09:08 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I like to do something similar since I have no eberbach.....

I have a jar full of glassy-water, and a solid lid with a hole drilled for a septum plug to fit in. A septum plug is one of those rubber self-healing injection ports used in hospital vials.

I use those because usually when I try to shake jars full of water they leak from somewhere....usually the taped hole in the lid.

all it takes is a little chunk of a good isolate and you got several nice myc syringes of pure juice!
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Martaxus (Martaxus)
Posted on Thursday, May 01, 2003 - 02:24 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Interesting... Organism, where did you do the core sample & transfer to jar, in the open air? Do I just have to bleach-wipe all sufaces, lysol the air 5-10 minutes before working, and wipe all utensils with rubbing alcohol?

A chunk the size of a matchhead is enough for 5-10 syringes?

How is the broken-glass jar prepared prior to PC'ing? Do you take the regular jar, add broken glass, add distilled water, add lid upside down, then band plus tinfoil? If the jar lid has holes in it, can they simply be taped over with high-temp tolerant electrical tape, or should one use a whole lid?

Mad scientist instinct rising..!
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Putney Swope (Odin13)
Posted on Thursday, May 01, 2003 - 05:19 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

In lieu of the expensive eberbrach setup, the small jelly jar canners have the same threads as a Hamilton Beach blender. You can screw the cutting mechanism on the jar with the blender bottom and some water in the jar. PC this for the std. 15 mins. Allow to cool to room temp. Take a goodly amount of mycelium and add it to the jar in your glove box or in the flow of your laminar hood. Pulse it for 5 to 10 seconds max and voila, mycelium water as good as that there eberbrach! I have this work 90% of the time, but when it fails, it fails big time!
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Organism (Organism)
Posted on Thursday, May 01, 2003 - 01:24 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I did the transfer right inside my grow box.

It was the next best area other than a glovebox, I thought.

I actually set my jar to recieve the core tissue on top of another cake (with a piece of aluminum foil seperating the top of the cake from my nice clean jar!)

It is important to note that I used a much larger peice than a 'matchhead' More like a strip 1-1 1/2 inches and the diameter of half a pencil. I wanted it thick for 10 10cc syringes.

I wiped the walls of my grow chamber with 99% iso-alcohol before working. I took the tissue from the stem center without picking it. That way I was ensured the tissue was well alive and still growing.

On the jar preparation, Add your pre-cleaned, sharp edged glass pieces, not to big, not to small.
Add distilled H2O, set on your lid with pre-made syringe hole add band (loosly), aluminum foil to cover (from PC condensation)

25 min @ 15psi BAAMMM!
Sterlie jar of water with glass shards.

Follow sterile procedure and work carefully, No mistakes allowed, It works good. Have everything ready and prepared ahead of time and it goes well.
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Hippie3 (Admin)
Posted on Thursday, May 01, 2003 - 02:03 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

if using a blender, better to go with brief 2 second low speed bursts a few times. if you go 10 seconds on high you'll kill much of the mycellia. a proper eberbach has specially designed short stubby blades that chop the mycellia correctly, a blender does not. so go easy if you're improvising.


roger, that's possible. if you were using culture syringes that old there may well have been some degredation and contamination, hard to be sure without replicating your experiment.


as for cloning, it's fine but my rule and the best policy, imo, is to never clone a clone.
that's where problems begin, or at least, can begin.
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Organism (Organism)
Posted on Friday, May 02, 2003 - 01:24 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I'm going to see what happens with clones of clones. I know it may not turn out pretty, but I'm extemely facinated and interested.

I will DEFINATELY be sure to never get the clones and my pure PFC gene line mixed up, as this could lead to a genetic disaster.

I am extremely interested and facinated by the genetics of this priceless genetic resource.

I know that I must keep up the origional gene pool of the PFC race by spore prints, but If I can optimize by running clones off the origional gene line in synch I could discontinue clones as the pure gene line continues. Do you get me?


I want to know just how far I can push the envelope!
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Hippie3 (Admin)
Posted on Friday, May 02, 2003 - 06:07 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

it to a great extent depends on luck.
you might go 5 or 6 replications without harm or it could go wrong much quicker, you never know what turn a mutation like that will take.

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