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OfflineXxMuSHRooMHeaDxX
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Methods of storeing a sterile tissue sample
    #492465 - 12/16/01 01:41 AM (14 years, 11 months ago)

looking for good and sterile teks for storing the sample for a period of time ? anybody ? thx , peace

Xx


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Invisibledimitri211
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #492469 - 12/16/01 01:44 AM (14 years, 11 months ago)

MEA and then into a cool dark place 40 Degrees F or so.


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OfflineI_AM_THE_WALRUS
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #492470 - 12/16/01 01:45 AM (14 years, 11 months ago)

formaldaheid(sp)...rubbing alcohol?


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: dimitri211]
    #492479 - 12/16/01 01:58 AM (14 years, 11 months ago)

Im sorry but im confusing you guys.
the meaning was non agar methods ,sorry 4 the mix up.
Like some sort of sterile solution that could be stored dormnent ,.


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Anonymous

Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #492530 - 12/16/01 03:01 AM (14 years, 11 months ago)

mycyl sryinge with water will last months in the fridge


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OfflineAzure
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #492759 - 12/16/01 12:38 PM (14 years, 11 months ago)

When storing mushroom cells for a prolonged period of time, you have to get the cells to differentiate back to mycelium. When the cells are in the form of the mushroom, they're programmed to die.
As mentioned earlier, the best way to keep a sterile piece of tissue without is to make a liquid culture of it with malt and water or honey water, and when there's plenty of mycelium, place that in a cool environment.


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Invisibledimitri211
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Re: Methods of storeing a sterile tissue sample [Re: Azure]
    #492766 - 12/16/01 12:47 PM (14 years, 11 months ago)

Very nicely put.
I like when people answer questions, complete and non-confusing


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OfflineSuntzu
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Re: Methods of storeing a sterile tissue sample [Re: dimitri211]
    #492965 - 12/16/01 04:41 PM (14 years, 11 months ago)

Anno posted a link awhile back showing some work that had been done on strain storage; The longest, and presumably best way was straight mycelium in distilled water in the fridge. What made this interesting to me, is that it was optimal to put no agar or other nutritional sources in the water, just straight mycelium. This is tough with some agar growth that totally sticks, but others can be swabbed off with a sterile tool [don't need much]. I pulled a 16 month culture awhile back that was stored like this and it was perfect! Anno's link claimed something like FIVE YEARS using this technique. Seemed to blow away slant storage/mineral oil, and is much simpler.

If you're talking about actual fruitbody tissue, though, I'd be wary. . .grow it out some; this will get it back into vegetative mode and weed out any 'weeds'. Assume all fruits are contaminated to some degree.


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: Azure]
    #493121 - 12/16/01 07:40 PM (14 years, 11 months ago)

Would it be best to take a smaple of mush tissue, or of myc.? How?
What is the best/sterile/healthyist tek for doing the honey water? How?
How long should the myc, be left to develop b4 cold storage?
What about just letting the solution continue to colonise till needed?
Is it easily comnatm'd?
What would be the best way to tkae a sample? and what would be s sutible subject?
Is the Myc syringe w/ water a syringe drawn up w/ liquid myc formed via: honey water/agar ?

Thx for the help guys , peace.

XX


--------------------
"Put the past at rest , Live your life to please"
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Edited by XxMuSHRooMHeaDxX (12/16/01 07:42 PM)


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OfflineI_AM_THE_WALRUS
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #493127 - 12/16/01 07:56 PM (14 years, 11 months ago)

i thought you meant a severed thumb or something...hahahaha


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Invisiblemycofile
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #493417 - 12/17/01 01:51 AM (14 years, 11 months ago)

best way would be to either clone a shroom onto peroxidated agar (skip this step if you already have a sample on peroxidated agar).

Let the sample grow until it is growing up the sides of the peroxidated agar jar, or petri dish depending on what your growing in.

Sterilize some distilled water, wait to cool.

Scrape off the strands of mycelium that have grown up the sides of the agar jar, so that you don't get any agar. Put these in the water. Seal jar. Seal jar in bag. seal jar in bag in bag. etc etc. Place in fridge.

Reuse years later.

If your sample doesn't want to grow up the sides of the jar, then peel the mycelium off of the agar, but the less agar the better. Making an extra firm agar by using slightly more agar agar is helpful. So is letting the jars/plates set for a while in front of the flow hood. Mycelium peels right off of a firm agar.


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: mycofile]
    #493613 - 12/17/01 09:22 AM (14 years, 11 months ago)

Thx for the informal posts, but was looking for nonagar methods, read in the ultimate myco directory last night that you could store a viable tissue sample in regular h2o for prolonged periods of time, nonmineral water of course, it was testing done on the 2 and the h20 reg was the control , the mineral water produced sterile fruits and showed signs of degeneration , while the water stored and produeced nice fruits for i think it was like 6moths - 1 year? somethign like that.

Anyone ever heard of this , and do you think that this could be a sterile/healthy way to store a tissue sample ?
should one do a h2o2 bath/dunk of center stem sample b4 transfer to storage jar ?
After storing via h20 should a tissue piece just be transfered to an agar dish and follow reg agar procudures after?
Thx for the post guys, peace.


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Invisiblemycofile
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #493969 - 12/17/01 06:52 PM (14 years, 11 months ago)

That's pretty much exactly what I posted. I just get my viable tissue sample from the edges of petri dishes. A stem isn't going to last long, a week, yeah, 6 months? nope.

Don't use agar if you don't want to, but what you want is mycelium with no substrate attached, then submerge the mycelium in sterile distilled water in the fridge. As I said, this will last for years.


--------------------
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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: mycofile]
    #494244 - 12/17/01 11:01 PM (14 years, 11 months ago)

>>>>>A stem isn't going to last long, a week, yeah, 6 months? nope.
why wouldnt it last long?
but wouldnt taking a nontissue sample , defeat the purose of cloneing?
SO Theres no way to store a sterile tissue sample???

aside from cloning would it be better to take a mycelum sample from say a cakes outward growth or like a myc knot?
and for tissue/myc sample is it nessisary to h2o2 bath b4 transfering to containment?(even if its a stem center?)
Thx just trying to make shure everythings seen clearly.peace


--------------------
"Put the past at rest , Live your life to please"
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Free Spore Ring
Myco-Elite
All above are lie's.


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Invisiblemycofile
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #495040 - 12/18/01 06:21 PM (14 years, 11 months ago)

Why don't stems last long? I don't know. The longest I've seen one last is almost a month. It was stored in sterile distilled water after being dunked in h202. I've tried longer periods, but no sample from a fruit body that was stored over 5 weeks ever regenerated for me.

"but wouldnt taking a nontissue sample , defeat the purose of cloneing? "
Taking a tissue sample, be it from a mushroom, or from cultured mycelium IS cloning. I don't see how it can defeat it's own purpose. A nontissue sample would be something other than the fungus we are growing. I don't no what a nontissue sample means.

"SO Theres no way to store a sterile tissue sample??? "
Sterile tissue samples can be stored in sterile distilled water in the refrigerator. If that tissue sample came from a fruitbody, it will not remain viable for longer than a month IME. If that sample came from a culture, then it will last longer than you will keep it around, IME.

"aside from cloning would it be better to take a mycelum sample from say a cakes outward growth or like a myc knot"
Either one will work. The mycelial knot is basically a very immature fruitbody, so you may end up with the shelflife of a sample taken from a fruitbody. To be sure, I would use the mycelium. It's still the same organism, and your still cloning, your just cloning a differnt part of it. On the other hand, you could transfer the myc knot to media (agar/brf paste/whatever) and grow it out, therefore cloning it, and save the resulting mycelium

"and for tissue/myc sample is it nessisary to h2o2 bath b4 transfering to containment?(even if its a stem center?)"
H2O2 is never necesary. If you aren't sterile, it's a good idea. Taking a stem center in a sterile environment doesn't usually call for h2o2, but it won't hurt it.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: mycofile]
    #495381 - 12/18/01 11:53 PM (14 years, 11 months ago)

first , when speaking of a tissue sample , was meaning a sample from the actual mushroom flesh, nontissue was meaning like myc , sorry if it was confusing

>>>>Taking a tissue sample, be it from a mushroom, or from cultured mycelium IS cloning. I don't see how it can defeat it's own purpose. A nontissue sample would be something other than the fungus we are growing.

When reading about this subject , came across somewhere that stated when cloning the flesh from a mushroom fruit body , it was surpassing the odds the mycelum surface layer had to pass in order to result in a fruit developing , so cloning from a mushroom sample (obviously passed the odds and is able to grow a fruit body) would result in a myc culture with a higher expantacy to put out fruits , and fruits with same/like genetic code. Also form other sources , have stated when culturing/inoculating from a sample that was cloned from a fruit body , that the result in casings proved to be much more even flushes.
With that said isnt there a differance when cloning from either a fruit/mycelum ?
What are your thoughts about the above?
Is the above true when speaking of the clone would be the same as parent sampled? as in genetics/growth traits?

aside from all this , u think it would be possible to store the tissue sample frozen suspended in the containg solution? wouldnt this prevent aging and deterieration?
Thx , peace


--------------------
"Put the past at rest , Live your life to please"
Mushroomheads Official Site
Free Spore Ring
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Invisiblemycofile
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #495887 - 12/19/01 01:45 PM (14 years, 11 months ago)

Oh, I see the nontissue thing now.

Cloning from a fruit body will give mycelium that is 100% identical to source fruitbody. This means that it is able to fruit, and often will give very even flushes etc. This culture is now a single organism. It can be transferred to other media, fruited and cloned again, or stored. In all cases, the resulting culture will always remain 100% identical to the original fruitbody, and retain it's characteristics.

There is no difference when cloning from a fruit or mycelium of the same organism (isolated strain). There is a big difference in taking a mycelium sample from a multi-spore innoculation and cloning a fruitbody. The sample from the spore innoculation is a crapshoot. You may be getting more than one isolated strain. The strain you isolate may be a poor/non-fruiter. With the clone from the fruitbody, it has already proven that it can fruit, and that it can produce decent shrooms.

Clones are the same genetically as their parents. However, they can differ phenotypically due to their environment. The same isolated strain (clone) is going to look very different from it's parent if the parent was grown on compost and the clone is grown on pf cakes.

I don't know about freezing. In general, I would say no, because it will cause the cells of the sample to burst as the water in them crystalizes. I have however heard of people storing samples in the freezer in a glycerin solution. I don't have any information on that technique though.

I never really neede to play around with it, because a sample can be kept in sterile distilled water in the refrigerator for years.

Also, don't forget that over time (years) of repeated cloning, eventually genetic mutations will show up which change the strain, almost always for the worst. You will eventually have to go back to a younger culture (one that hasn't been cloned so many times, eg the original mycelium sample, stored in water, or an early clone of the clone). or you will have to make spore prints, and start over.


--------------------
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-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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OfflineXxMuSHRooMHeaDxX
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Re: Methods of storeing a sterile tissue sample [Re: mycofile]
    #496547 - 12/20/01 01:29 AM (14 years, 11 months ago)

first off gotta say man that was probally the most informal thing ive read about cloning to date, thx so much

>>>I don't know about freezing. In general, I would say no, because it will cause the cells of the sample to burst as the water in them crystalizes
so if the cells burst is it then dead? nonable to reproduce?

>>>I have however heard of people storing samples in the freezer in a glycerin solution. I don't have any information on that technique though.

wouldnt it be the level of the temp that eould rupture the cells? so then how would/could this work if the temps at at similar settings in the freezor?? they would both stilll be at the same temps right??

So do you think/know of or heard of any way of storing a rissue sample in a sterile viable stasis untill later use?

Thx for all the great info so far again man.

To everbody else : dont be shy even if you have any kind of ideas id love to hear em , plz share what you can , this is a great learning experience for all .
Peace



--------------------
"Put the past at rest , Live your life to please"
Mushroomheads Official Site
Free Spore Ring
Myco-Elite
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OfflineabsoluteZero
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Re: Methods of storeing a sterile tissue sample [Re: XxMuSHRooMHeaDxX]
    #496574 - 12/20/01 01:58 AM (14 years, 11 months ago)

I saw a show on cryogenics once, ya know, when they freeze a person or animal, then thaw them out. Actually they were using frogs. See many frogs have the ability to be frozen in the ground and then thaw out and come back to life in the spring. What they do is push all the water out of their cells. If they didn't, the sharp points of the ice crystals that form would destroy the soft membrane of the cell walls. I assume that this is what destroys mushroom cells as well, not so much the temperature, but the effects of ice crystals.

-zero


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Invisiblemaxxjones
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Re: Methods of storeing a sterile tissue sample [Re: mycofile]
    #499888 - 12/24/01 04:53 AM (14 years, 11 months ago)

mycofile, you state thngs well. Agar cultures are interesting. Here is a pic of some first-time cubensis agar attempts - Puerto Rican, Equadoran, and one Pan. Tropicalis (lower right).



The plates include (multi-spore) mycellium cultures as well as spore-water and cap and stem clones. It's very cool. The mycrobiology is almost more interesting than the substances themselves!

Go agar!


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