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OfflinePimpDizzleNerd
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A question to the expirenced guys
    #4707347 - 09/24/05 11:48 PM (11 years, 2 months ago)

Actually, this isnt too hard of a question to answer, but I couldnt find it in any of the info pages. If I have a excellent mushroom growing in my cake and want to clone it via agar culture, and after wards use it to inoculate a few cakes, then clone from them again will i lose potency. I know that I cant use the spores because they will be multi-line again, so I wonder if there is a loss in potency through clonings


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InvisibleIntelligentMind
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Re: A question to the expirenced guys [Re: PimpDizzleNerd]
    #4708132 - 09/25/05 03:28 AM (11 years, 2 months ago)

Just make sure you don't keep using the same agar for generation after generation.. Try using PDA if you've been using MEA, and vice versa..


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Invisiblemycofile
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Re: A question to the expirenced guys [Re: PimpDizzleNerd]
    #4708170 - 09/25/05 03:39 AM (11 years, 2 months ago)

Quote:

If I have a excellent mushroom growing in my cake and want to clone it via agar culture, and after wards use it to inoculate a few cakes, then clone from them again will i lose potency



You will not loose potency, but you will loose health. It's called senescence and you will run into eventually if you continually clone. You get around it by saving some mycelium from the first time you clone it, and use this to start all subsequent generations rather than cloning again.


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Invisiblenoxy
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Re: A question to the expirenced guys [Re: mycofile]
    #4730318 - 09/29/05 10:21 AM (11 years, 2 months ago)

even myc in cold storage will eventually senesce
the bigist problem you will likly have is keeping a pure strain free from contaminants

start a stock culture with several slants
incubate for a week at 75 deg f
store the resulting pure cultures at about 37 degrees
your good for maybe 2 - 2 1/2 years at least without oil immersion

grow from these stock cultures as you need to

sterile oil immersion kept just above freezing will keep a strain for several years
after a point though vitallity fades away


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InvisibleZen Peddler
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Re: A question to the expirenced guys [Re: PimpDizzleNerd]
    #4734984 - 09/30/05 04:10 AM (11 years, 2 months ago)

phenotype to the original mushroom.


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Edited by Zen Peddler (02/02/07 05:56 AM)


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OfflineBlue Helix
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Re: A question to the expirenced guys [Re: mycofile]
    #4758509 - 10/05/05 12:45 PM (11 years, 2 months ago)

mycofile, have you run into this senescence stuff? I know it's widely believed, yet there are mycelium networks on the order of square acres. In fact a living mushroom is the biggest organism in the world. Why didn't it malfunction from senescence by now (it's hundreds of years old)? I wonder if there is more to it.


Edited by Blue Helix (10/05/05 12:47 PM)


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Offlinemattymonkey
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Re: A question to the expirenced guys [Re: Blue Helix]
    #4758567 - 10/05/05 12:57 PM (11 years, 2 months ago)

I think I cam experiencing sensecnense(sp) on a culture right now. It grew out free of contams then just sat there... for awhile... then eventually contam'd.. did quite a few bins.. maybe 2 of them fruited, but the fruits never matured... i just checked a petri out and its 1/4 cottony, the rest is rhizo... i guess it does happen =\


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Invisiblemycofile
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Re: A question to the expirenced guys [Re: Blue Helix]
    #4762910 - 10/06/05 10:09 AM (11 years, 2 months ago)

Hey blue, yeah, I've run into it (or what I attributed to it) but I've also done things that conventional wisdom says you can't do. Like fruited/cloned/fruited/cloned/etc for like 5+ generations without going back to a younger culture, and without seeing any decline in vigor. Standard warnings on senescence would say that's impossible.

So, there is a more to it. I'm sure it varries with speed of growth, species, strain, other environmental stresses etc.

I always think of the potential of a culture before I even think if I've run into senescense. You can go from a 1 cm^2 slice of agar, to LC, to 100 times as much LC, to 100 grain masters, to 1000 2nd gen grain, to 10,000 3rd gen grain jars, to millions of tons of fruiting substrate. Then I think if my culture has been through anything near that number of cell divisions before I start attributing a problem to senescence.

Senescence is real and certainly exists, but little is actually known about the hows and whys of it. I feel that many hobbyists see it as much more of a problem than they are likely to run into. But it can happen.


--------------------
"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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Invisibleagar
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Re: A question to the expirenced guys [Re: mycofile]
    #4762984 - 10/06/05 10:35 AM (11 years, 2 months ago)

It was ANNO that pointed out - young vigorous mycelium does better than old. However, I have gone out 5 G2G transfer generations - without ill effect. Genetics plays a part in it. In nature - mycelium is designed to grow out & fruit in one season. You can FOOL with mother nature - but extending it to far out - usually brings on some mutation.


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InvisiblePinback
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Re: A question to the expirenced guys [Re: agar]
    #4765262 - 10/06/05 07:56 PM (11 years, 2 months ago)

Has anyone seen any reference to cell division related senescence that does not originate from Stamets books? I tried some fast searches but didn't find anything of use. It would be interesting to see where he got his information from.


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OfflineJohnHolliday
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Re: A question to the expirenced guys [Re: Pinback]
    #4766168 - 10/06/05 11:16 PM (11 years, 2 months ago)

senescence: I run a commercial mushroom company and we have produced over 55 million pounds of mushrooms since 1971. We have some strains of mushrooms which we have cultivated continuously since the early 70's. About 400 generations so far. We carefully track each plate, and the yeilds, etc, and mix generations of spawn for each fruiting. That is really important for good fruiting, contrary to what Stamets books suggest. If you use all young generations, or all old generations you will have even more of an unnatural condition then you have anyway just through monocropping, which in itself is against all the laws of nature. OK, so what are the tricks to lack of senecence? First, always challenge your cultures. DO NOT grow them repeatedly on the same agar. They develope a specific set of enzymes that way, and quickly lose the flexibility that you typically find in younger cultures. So transfer them from MEA, to PDY, to MYA, to Catfood agar, to dogfood agar, to ovaltine agar, etc. Always make lots of different agars and keep crossing them back and forth. Don't feel like you need to follow some book recipe. Dig through the cupboard and see what you find. Make some agar. Easy. The rule: Use around 20 g per liter of a starch or sugar. It is not too crucial what you try. Some agars will be total failures, for sure. But most will grow fungi just fine. Try some stuff like, oh I don't know, how about strawberry jam? 20g in a litre of water along with 22-24g agar (I like my agar hard, my assistant likes his softer and uses 18-20g/L) Strawberry jam grows fungus just fine. Last week I bought 50 crickets at the pet store and blended them into a smoothy, then added 22g agar and it worked just fine. So there is a challenge for the fungus. By forcing them to digest crickets, they are utilizing so much more of their genome then they would just on MEA or PDY. So maybe they grow a bit slowly, so what. You can transfer them in a couple of weeks back to MEA and see what renewed vigour they have! Once you see that, you will realize the fear of senescence is overated! It should go without saying that you should keep a couple of copies of each generation, because you will find that sometimes, a plate will just go hog-wild for some reason and produce all out of proportion to what the generations either side of it did. Keep really good records. And always keep your early generations. The idea that a culture has to be frozen in liquid O2, or covered with oil, or some other such trick is nonesense. We have lots of culture that have been stored for 20-30 years and they are as viable today as they were then. We do transfer them from time to time, but not all that often. Every couple years or so. Don't go back to spore prints if you can avoid it. If you go to spores, you are rolling the dice. Spore are different genes each time around, and there is no way to predict what you will get. In commercial spawn production spores are never used. The transfer of tissue is what you need to do. If you want to run out your P5 generation for evermore, no problem. Just start by making 50 copies of it. Then make 50 copies of each of those, (OK, so they are P6 now, but still, you are very close in age to where you wanted to be.) Then you have 50 x 50 (2500 cultures) plates of P5/6, so that should allow you plenty of that generation even if you never take the cultures further. Every time you cultivate a new crop, you use one of your plate of P5 or P6, then your crops are always identical. Provided of course that all the other parameters are the same. The "risk" of senescence is far over-rated and really is never an issue in the commercial realm.


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InvisibleRoadkill
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Re: A question to the expirenced guys [Re: JohnHolliday]
    #4767803 - 10/07/05 09:38 AM (11 years, 2 months ago)

Welcome to the Shroomery John!~

Very nice post!~

It's nice to see a commercial mycologist such as yourself posting here...most don't want to talk about actives or hang out at the Shroomery.
So mad props to you!~
:smile:

I have a question for you...about something you said.

Quote:

The idea that a culture has to be frozen in liquid O2, or covered with oil, or some other such trick is nonesense. We have lots of culture that have been stored for 20-30 years and they are as viable today as they were then.
We do transfer them from time to time, but not all that often. Every couple years or so.




would you please explain how you store your cultures for us.

and are these cultures actually 20 to 30 years old?
since you said that they are transfered from time to time...every couple years.

I know that mycelium stored in sterile water...will last in the frig for well over 10 years.

I'm very interested in storing mine as long as possible.

thanks!~

tc


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You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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Offlineblackout
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Re: A question to the expirenced guys [Re: Roadkill]
    #4767885 - 10/07/05 10:13 AM (11 years, 2 months ago)

Welcome, another knowledgeable Mushroom John!
I am intersted in your storage technique too. Some here have talked of storing myc in distilled water for years. I don't do agar work and was thinking I could just "core" a mushroom, i.e. stick a syringe through a stem to cut a section out, then inject it into a small jar of sterile water. Do you think this is viable? I have some sclerotia cored in a jar that I will try and revive soon.

Do you ever freeze myc or spores in a regular freezer?

I see your business is in medicinal mushrooms, do you have any techniques or additives to increase the active compounds in the mushrooms?

p.s. When making long posts like that I would recommend highlighting it all and copying it to your clipboard before pressing continue. Sometimes you can lose a post due to errors, especially if it takes a long time to write it.


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Edited by blackout (10/07/05 10:18 AM)


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OfflineJohnHolliday
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Re: A question to the expirenced guys [Re: blackout]
    #4768385 - 10/07/05 12:25 PM (11 years, 2 months ago)

Regarding stored cultures: The answer to how long cultures can be stored is very much strain related. I know of Morels that were frozen whole and kept for extended periods, and when thawed out were viable. Others cultures, like Volvariella, tend to die no matter what you do to them. The way we store cultures is in test tubes or petri dishes, on agar, well para-filmed, in a refrigerated container that maintains at about 36-38 degrees F. The same reefer stores large qty's of spawn, with the resulting side effect that the O2 content is about 50% of atmospheric (remainder N, CO and CO2). Some of the cultures are indeed 30 yrs old. Another trick I like is to get the wooden stir sticks that are used for coffee, cut them to the right length, and put one into the test tube before you pour slants. They should stick up well above the agar surface. That way, the fungus will colonize the wood, and even after the agar culture is long dead, the wood is still viable. Just take a scraping, or stick the whole thing into another dish of agar, or even directly into a bag of grain as in the old Amycell spawn technique. As far as sterile water storage I tried that with some strains a few years ago and as I recall it worked out OK. I don't normally try new stuff because what we are doing seems to work OK. As for spore storage, most spores will store for long periods of time, like longer than you and I will be around. A note on collecting spores: Make it easy on yourself and collect sterilly. As in, sterilize the collection surface, filter paper or glass, etc, or use a pre-sterile petri dish for collecting. You can put a cleaned cap in a dish and close it up, leave in the hood for 24 hrs, then remove the cap, parafilm and store away. Always put plates inside of plastic bags, like zip-locks. The parafilm is not always dependable. And always wrap at least two layers of parafilm over the joint. If collecting on paper, sterilize the paper first in a clean gallon jar with a filter disc on the lid, then open in the hood and place the cap inside the jar, re-close, and leave in hood for 24 hrs. Then you have a sterile spore print in the jar. Keep in mind always, sterile techniques. You cannot be Too Sterile. It is like being too rich or too beautiful, just doesn't happen. Another good collection surface for spore prints is glass plates, then place two plates together and tape the edges all around. Stored this way, spore prints are good for a LONG time. See the photos at: http://fruit.naro.affrc.go.jp/kajunoheya/epfdb/method/spore1.htm


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Offlineblackout
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Re: A question to the expirenced guys [Re: JohnHolliday]
    #4791450 - 10/12/05 04:55 AM (11 years, 2 months ago)

Quote:

JohnHolliday said:
They should stick up well above the agar surface. That way, the fungus will colonize the wood, and even after the agar culture is long dead, the wood is still viable.



Thanks for the great info John, do you use liquid cultures for storage or inoculation at all?
I didnt know a agar culture would "die", I would have thought you could scrape some off years later and revive it. The wooden sticks sounds like a very simple method, easy to handle with tweezers, if you got a bunch of cocktail sticks it would be a very easy method of trading, I wonder if floating them in a LC would be enough. Does anybody know if cubensis would take to wood well, I guess it would.

Has anybody here tried reviving frozen cubensis, either dried of fresh frozen.


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Edited by blackout (10/12/05 04:59 AM)


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InvisibleBlimeyGrimey
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Re: A question to the expirenced guys [Re: blackout]
    #4791515 - 10/12/05 05:29 AM (11 years, 2 months ago)

hey blackout
a few weeks ago i stumbled upon a site that had a "wood loving" cubensis strain.
if i ever find that site again i'll send ya the link.


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InvisibleBlimeyGrimey
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Re: A question to the expirenced guys [Re: BlimeyGrimey]
    #4791525 - 10/12/05 05:35 AM (11 years, 2 months ago)

well i found the link and the name of the strain.

the name of the mushroom is Psilocybe cubensis var. azurligncola

i'll send the link in a pm because they arent a sponsor.
its #226 on the page.

hope this is useful info.


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Offlinebiomyc
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Re: A question to the expirenced guys [Re: BlimeyGrimey]
    #4791541 - 10/12/05 06:06 AM (11 years, 2 months ago)

np... every cubensis should do it on wood...


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Offlineaaa
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Re: A question to the expirenced guys [Re: biomyc]
    #4791574 - 10/12/05 07:01 AM (11 years, 2 months ago)

Anyone tried to vary grainsort(between generations) when doing G2G to get more generations?


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OfflineKayTron
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Re: A question to the expirenced guys [Re: PimpDizzleNerd]
    #4901166 - 11/06/05 07:14 PM (11 years, 1 month ago)

I've gotten away with first, getting the mushroom you wish to clone into a sterile environment asap, and with sterile equip, etc, hack off the bottom knob, and from the inside of that region, took samples, say 1mmx1mmx1mm, tiny samples. I put about 4-7 of those little units into sterile bird seed (finch select.) Few days passes, and they're bluing in the jars, I'm convinced they'll fail, then day 5 out of nowhere, good rhizomorphs, and this clonee muchroom was a fat little umbrella. About a 50% success rate later, donated some jars to cases(which yeilded a good mix of fatties and good strong and all ones), and others to reproduction grain to grain-wise, good results. 3rd cycle I demanded out of it just wouldn't take. 0% success. Not considering this from the start, I lost the genes, but no biggie, there will be others. Tazmanian strain.

And maybe it demands a fresh post, but what about mutation?
After genetic breakdown, I've seen some weird stuff happen, funky fruits.


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