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OfflineMurit
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caring for a master culture
    #5845947 - 07/11/06 06:41 AM (17 years, 6 months ago)

okay, for the past several hours I've been spending my time researching substrain masterculture creation as well as substrain isolation itself...but the one main question I'm left with after doing this is...how on earth do you keep the master culture going?
I mean...I saw one guy claiming that liquid nitrogen was the way to go as far as preserving an MC...but um...yeah...that sounds a bit extreme (not to mention FUCKING DANGEROUS) to me...what method do you old hands suggest I use?


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..in those who have never tried them."
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Invisiblecreamcorn
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Re: caring for a master culture [Re: Murit]
    #5846043 - 07/11/06 07:58 AM (17 years, 6 months ago)

refrigeration is most practical. you can keep LC, grain jars, or agar plates/slants under refrigeration (but above freezing) for many many months (6 or more). you can make a new master from the master a couple times before losing vigor, so "re-newing" your master every 6 months or so in this manner you can keep the same isolate around a couple years. doing this with agar and rotating the media type (i.e., using PDA one round, then MEA next round, etc) will help keep up the strain's vigor for even more transfers.

TMC speaks of actually covering a colonized agar plate/slant in mineral oil, refrigerating, then draining off the oil when its time to use - the oil is harmless to myc, but prevents air from getting at it at all, stopping its growth 100% and you can keep a culture for many many years in this manner. liquid nitrogen will work as well for a slant or plate and is used on occasion in commercial growing (cultures are stored off-site for long term in a special facility, kind of like a computer data back-up in a way since commercial growers may rely heavily on a particular isolate to keep their product very consistent over long term), but yeah, its expensive and probably dangerous like you say. refrigeration is probably all you'll need...

there's always the possibility of growing mycelium from dried mushroom tissue (can be difficult but is possible), so fully drying a mushroom grown from your isolate and vacuum sealing it may be another way to save an isolate indefinitely (assuming you get it back to life later!)


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OfflineMurit
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Re: caring for a master culture [Re: creamcorn]
    #5846062 - 07/11/06 08:08 AM (17 years, 6 months ago)

awesome, cream. Thanks! Though, I do have another question, if I may.
How do I decide which isolate to keep out of the multitudes that I'm probably going to end up with? Is there any way aside from simply taking a sample of each, colonizing it and testing? or will it just take a long hard process of experimentation on my part?

(Woot! my 100th post is a question! and not a stupid one! WOOT! hehe)


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"Halucinogens produce severe
and violent reactions..
..in those who have never tried them."
- Terence McKenna


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Offlineroyer
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Re: caring for a master culture [Re: Murit]
    #5846084 - 07/11/06 08:22 AM (17 years, 6 months ago)

i took a 2 year old bag of dried mushrooms and took a dried cap and made a slurry out of it and now i have 2 fully contam free cakes that are ready and i am going to take a fresh mushie print it and then start with my agar and MC it . i didnt think it would work but it did i kept in a dry shoe box in a baggie.


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InvisibleRoadkillM
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Re: caring for a master culture [Re: Murit]
    #5846138 - 07/11/06 08:48 AM (17 years, 6 months ago)

mycelium in sterile water will last for over 10 years in the frig!~


and then there is a few other methods...

but I'll wait and let RR talk about those.



tc


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Laterz, Road

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Invisiblecreamcorn
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Re: caring for a master culture [Re: Murit]
    #5846188 - 07/11/06 09:12 AM (17 years, 6 months ago)

Quote:

Murit said:
awesome, cream. Thanks! Though, I do have another question, if I may.
How do I decide which isolate to keep out of the multitudes that I'm probably going to end up with? Is there any way aside from simply taking a sample of each, colonizing it and testing? or will it just take a long hard process of experimentation on my part?

(Woot! my 100th post is a question! and not a stupid one! WOOT! hehe)




well... when growing out on agar you can isolate from multispore the most rhizomorphic looking mycelium... this is a good indicator it will be a good fruiter, but certainly not a magic bullet, its just useful for selecting candidates at an early stage.  tiny pins will form on agar if left long enough, so you can certainly wait that long and save yourself the trouble of a whole grow... the only way to know for certain how its going to perform with your "growing style" is to put it through a grow (and sample the end results to make sure the magic is what you want  :wink:)... so it is time consuming, tedious, and make sure to label and track everything.  (now you see the reason for long term storage once you got it? :smile:) i keep a log book myself for just that sort of reason to track things, its an ongoing process. 

of course if you wait until you have a desirable fruit and simply clone it, you've got most of the battle over with at that point, you know you've got a fruiting strain that will resemble that particular fruit.  of course there's nature AND nurture involved, so there will be variance in your clones depending on growing conditions so things can turn out different between grows, but you can capture that general set of characteristics by going that route.


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OfflineRogerRabbitM
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Re: caring for a master culture [Re: royer]
    #5846318 - 07/11/06 10:15 AM (17 years, 6 months ago)

Quote:

royer9864 said:
i took a 2 year old bag of dried mushrooms and took a dried cap and made a slurry out of it and now i have 2 fully contam free cakes that are ready




I seriously doubt it. I've been doing a lot of work restoring dry tissue lately and attempting to develop teks for it. With all my microscopes, antibiotics, flow hood, other gear and experience, I fail over 75% of the time. Don't fall into the trap that because you have 'white' growth that you have mushroom mycelium. The odds with your technique are stacked against you.

Roadkill has it right about the sterilized distilled water. The trick is to get pure mushroom mycelium into the sterile water without any trace of nutrients. Lightly scraping a petri dish to shave off a bit of mycelium while leaving all the agar behind is one method. Stamets himself no longer uses mineral oil to preserve cultures. There are too many other problems with using oil.

If you're isolating strains, don't save any until you fruit out each one to determine which has the qualities you are looking for. Just keep the originals on well marked/labeled petri dishes in the refrigerator until each strain(isolate) fruits out, then you can make master slants from that culture. Drop a toothpick into each test tube slant you do, with at least part of the toothpick extending up and out of the agar. The mycelium will colonize the wood, then can go dormant and last much longer than it would on plain agar. My slants easily last a year, at which time I take a small piece and transfer to another slant, allow it to colonize, then back into the refrigerator with it. I have some experiments going now where I put a couple of cc's of sterilized distilled water over the mycelium in the slant. I'll let a couple of those go for two years without touching to see if it works. The problem with this method is the test tubes have to be stored in a holder to keep the water above the culture. I have several hundred stored cultures, so this method would require a larger lab refrigerator to use on all of them.

I think workman is looking into cyrogenic storage of cultures, so perhaps he'll have some input on how that works.
RR


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OfflineMurit
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Re: caring for a master culture [Re: RogerRabbit]
    #5847172 - 07/11/06 02:18 PM (17 years, 6 months ago)

Okay, awesome. Thanks for all the great info, guys. One more quick question, though. As far as the 10 year shelflife of myc in sterile water...does this apply to sterile water/honey LC mixture? or to water ONLY?
well..actually...make that two more quick questions...
second one being, once I get the master culture set up, after taking the long hard road of isolation....heh...sounds like a band name...anyway! How do I keep it stocked from the drawing of myc over time? or rather than drawing directly from the MC should I just keep a few plates colonized to pull from instead?
...and sweet jesus that just leads me into another question...but I'm busy sterilizing right now, so I guess I'll ask it later.


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and violent reactions..
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OfflineRogerRabbitM
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Re: caring for a master culture [Re: Murit] * 1
    #5847588 - 07/11/06 04:25 PM (17 years, 6 months ago)

You'll only need to break into your master culture every six months or so. Just inoculate a petri dish and put the test tube back in the refrigerator. You can split the petri dish into two or three more in a week, and put them in the fridge to use over the next six months, then use the original to inoculate a few quart jars of grains, that you can then do g2g with.

You don't replenish the master culture, you replace it. Just take a small piece of myc and transfer it to a new test tube. Let it grow until it's mostly grown over the surface, then put it in the refrigerator. It's best to keep three or four master cultures if you get a super strain. Even better is to wrap them up well and divide them among several refrigerators in different locations to guard against thieves and/or power failures.

Store on a test tube slant or in distilled water, not LC.
RR


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OfflineMurit
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Re: caring for a master culture [Re: RogerRabbit]
    #5847978 - 07/11/06 06:18 PM (17 years, 6 months ago)

unfortunately, in my situation (being relatively poor) I cant do that...hell...cant even afford the agar plates to begin isolating yet...but I soon will! (thank god for excess financial aid moneys!)
Anyhow...first question that arises now is a very noob one.
what is G2G? I've seen it mentioned a few times, but never actually figured out what it means.
Next question that comes to mind is: when replacing the MC with myc from another sampling of it...wouldn't this cause degradation? I know that there are ways to help against this (such as switching up the culture media)...but surely it's going to happen eventually...Monstermitch described a method to me a couple days ago that he said would help prevent degradation, but I didn't really understand that part of what he was saying...due in majority to my extreme noobliness...afterall...Ive only been studying this stuff for about two weeks now, but Gods it's fascinating! Almost makes me want to jump career tracks into mycology.
...anyway, back from my segway...the final question I have about this is how to you grow the MC itself. Do you just drop a cutting of agar+myc into a test-tube/Vacutainer of sterilized distilled water and let sit at room temp untill it colonizes mostly in the container, then simply fridge it?


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and violent reactions..
..in those who have never tried them."
- Terence McKenna


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OfflineMurit
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Re: caring for a master culture [Re: royer]
    #5848019 - 07/11/06 06:30 PM (17 years, 6 months ago)

Quote:

royer9864 said:
i took a 2 year old bag of dried mushrooms....




my only question to you is...why the HELL did you have a 2 year old bag of shroomies? lol That shit woulda been SO eaten within about 2 minutes in my house.


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..in those who have never tried them."
- Terence McKenna


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Re: caring for a master culture [Re: Murit]
    #5848031 - 07/11/06 06:33 PM (17 years, 6 months ago)

G2G = Grain To Grain

> when replacing the MC with myc from another sampling of it...wouldn't this cause degradation?

You just keep subculturing the MC. Sure it'll eventually senescence. You can't keep something forever unless you freeze it in liquid nitrogen.

> but surely it's going to happen eventually...

Yes, if you do things right you don't have to worry about that for probably a decade or longer.

> the final question I have about this is how to you grow the MC itself.

You make a slant in a test tube. Then you pick a piece of myc to store and transfer it onto the slant. You let it grow until it's colonized most of the slant then refrigerate it.

You should make a number of slants depending on how frequently you want to access the culture. You save one for long term storage and another for sampling from. You can keep picking from a slant for a long time. When it gets down to almost nothing you can warm it up and let it recolonize the agar, then refrigerate it again. You can do this until you run into problems, then start a new "master slant" from your long term storage slant.

So you have one long term storage slant, another "master slant", then any active agar cultures you keep growing for more frequent access. Using this method you can keep things going for decades without problems.


-FF


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Re: caring for a master culture [Re: Murit]
    #5848052 - 07/11/06 06:35 PM (17 years, 6 months ago)



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OfflineMurit
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Re: caring for a master culture [Re: fastfred]
    #5848100 - 07/11/06 06:42 PM (17 years, 6 months ago)

hm. Alright then. I think I understand now...but let me regurgitate what I'm thinking just to clarify...in order to create more master culture...you take a sample of the first master and knock it into some grains (making DAMN sure they're contam free...cause wasting SSMC (super strain master culture...in case I just made that acronym up...) would be a damn crime in my book...anyway...I would let that knock colonize, then do a g2g...then once THAT's colonized, simply pull several samples of myc from that and kapoof? more master culture?
thanks for the link, tippin.

edit:
no...as I thought about it that simply doesn't make sense...ok...so in order to create subcultures...okay...
Step 1...Isolate the strain you want to grow
Step 2...inoculate several agar plates with the master culture (I'm personally going to do 4)
Step 3...take myc samplings from the master culture plates and make slants with the myc samples and the sterilized distilled water.
Step 4...use the agar plates for spawning LCs over time...
Step 5...use the LCs to colonize grains...
Step 6...so on...and so forth.

is this at least somewhat correct?


--------------------
"Halucinogens produce severe
and violent reactions..
..in those who have never tried them."
- Terence McKenna


Edited by Murit (07/11/06 06:54 PM)


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Re: caring for a master culture [Re: Murit]
    #5848177 - 07/11/06 06:55 PM (17 years, 6 months ago)

NO!

You have a long-term slant, a "master" or "in-use storage" slant, then any active petri dishes you are using, THEN any substrate you're colonizing.

Each level is another level of activity or usage.

For colonizing grains or substrate you use a wedge from an active petri, or if you don't have any going you use your master (in-use) slant. When your master (in-use) slant runs out you go back to your master (long-term storage) slant.

What you are trying to do is keep your master storage slant as young and as little used as possible. You should never run out of your long-term storage slant, if you do you should have used more levels or worked things a little differently.

Eventually, after a long, long time you'll have to replace your master (long term storage) slant. To do that you basically have to transfer it to substrate, fruit it, grow the spores out, then take a new culture. You're never getting your original sub-strain back, which is why you have to maximize the longevity of your storage master. Completing the sexual cycle is the only way to revive and preserve the genetics of your sub-strain, and you don't recover the exact same sub-strain, that's why you need to keep your masters as young as possible so that they'll last a long, long time.

Hope this explains things better for ya!


-FF


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OfflineMurit
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Re: caring for a master culture [Re: Murit]
    #5848231 - 07/11/06 07:06 PM (17 years, 6 months ago)

ohhhhhh I understand now. Thanks frank.

Thank you very much, everyone...your knowledge and expertise is surpassed only by your helpfulness. Thanks.


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..in those who have never tried them."
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OfflineClavo
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Re: caring for a master culture [Re: Murit]
    #21353271 - 03/02/15 06:51 PM (8 years, 10 months ago)

When doing initial master culture slants to petri dish cultures, do you need to take out the slants days in advance or can you do it right before inoculation?  I'm new to the process and after watching Let's Grow Mushrooms I wasn't sure if the slant should be at room temperature for a few days prior to culturing out the strain.  Any suggestions would be greatly appreciated!


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OfflinePussyFart
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Re: caring for a master culture [Re: Clavo]
    #21353333 - 03/02/15 07:03 PM (8 years, 10 months ago)

It doesn't matter, it will hit room temp in the dish....doing it cold is ok.....


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