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OfflineQuincunx
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practical method for long term storage of mycellium in dH2O * 1
    #2314067 - 02/08/04 01:03 PM (12 years, 9 months ago)

practical method for long term storage of mycellium in dH2O using ependorf tubes as vials



background:
This is a simple method (TEK of some kind) i use to store mushroom mycellium cultures in dH2O for prolonged time. It is far from being ideal but i thought it could come handy for some of you (read the part about mailing cultures stored like this). Feel free to improve it. I came up with it because i couldn't find any appropriate TEK at The Shroomery FAQ about dH2O storage.

primary objectives:

  • at least 50% of isolates should survive a period of 2 years
  • isolates should be stored in as small containers as possibile


secondary objectives:

  • isolates should survive occasional temperature fluctuation
  • vials should be crash resistant to survive mail shipping & handling
  • method/TEK should be simple and cheap so anyone can use it


materials used:

  • 0.5 mL PP ependorf tubes
  • dripping-tube or a pipette - pick one that will fit into neck of your flask:

  • cotton bud sticks
  • small flat bottom flask or erlenmeyer flask or even standard test tube will do
  • pure dH2O (bottled spring water is drinkable and contains minerals - i'm not sure if this is good in this case)


High sterility is a must so think in advance and sterilize everything. If possibile find presterilized cotton buds (pharmacy), ependorfs and plastic dripping tube. Otherwise make sure that materials can withstand PCing.
I've found cotton bud stick as the best tool for removing mycellium from the surface effeciently with a minimum of agar material transfered as well. Spatula and/or scalpel will work too, but they are too sharp for me - mycellium will deposit on the edge and form hard to break clumps once transfered in dH2O. Also, one false move and you can dig deep into agar. Suntzu gave me the original idea with sterilized swab (thanx :thumbup:) so i've pondered on this and came up with sticks.
Ok. Here's my method.
First i cut a stick(it's hollow) at the base and fit it at the end of the dripping-tube to construct a simple tool like this:


Then take a petri dish (colonized as needed) and roll the stick over surface:


You can gently wipe mycellium out of agar surface and even if you touch the surface with the bud tip it will feel like waxed solid floor (unlike with scalpel or spatula :biggrin:). Then transfer to flask with dH2O (constructed tool is thin and more than long enough to fit the neck of the flask):


Once in water, gently press dripping-tube nozzle to push some air through stick and shake - this will release the mycellium from the cotton bud. Do not release the nozzle again until you remove the stick from water otherwise it will suck the mycellium back on the cotton bud (sounds easy but you'll see what i mean).
Once suspention is dense enough, stir the flask some more. Now it is time to fill those little ependorfers. This can be tricky and requires still hand and patience (side note: it is a nice workout for those of you that are deep into the zen or yoga to improve mind concentration :biggrin:).
Remove the stick from the end of dripping tube and fill it with mycellium suspension. Then slowly drip the suspension into ependorfers all the way up, be carefull to remove all air bubbles (tricky thing due to capillarity) until they are full like this:

... and seal them. If they are not filled all the way up and above some air can remain inside when you'll push the plug in.



Well. This is it. After some practice you'll find the method quick and easy to perform. 0.5 mL is enough to inoculate a lot of fresh petris and more than enough to start a single 750 mL jar with grain spawn. Also, since PP ependorfers will not break easly and are small enough and mycellium stored like this can sustain a lot of temperature fluctation, they are great for discreet mailing in almost any envelope. (a hint: they will fit into any cheap pen and if X-rayed it will seem like spare ink container) The reciever does not need a pipette or anything. A sterile needle and syringe will do. Just wipe the plug of the ependorf with some alcohol and stick a needle through and pull the syringe bat like this:


Wishing you all healthy crops.



special thanx to Anno for having great patience with my picture UL. (Danke!)


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Invisiblekeyeghost
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Re: practical method for long term storage of mycellium in dH2O [Re: Quincunx]
    #2314447 - 02/08/04 02:55 PM (12 years, 9 months ago)

Nice post! My friend has done, the same, though clearly not in such an advanced labratory! =)

KI


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Offlineragadinks
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Re: practical method for long term storage of mycellium in d [Re: keyeghost]
    #2314771 - 02/08/04 04:47 PM (12 years, 9 months ago)

Nice writeup. Wouldn't that be something for the FAQ section ?


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InvisiblePrisoner#1M
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Re: practical method for long term storage of mycellium in d [Re: ragadinks]
    #2314776 - 02/08/04 04:51 PM (12 years, 9 months ago)

very ice...distilled water is one of the better methods of long term storage of mycelium cultures....


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OfflineAnnoA
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Re: practical method for long term storage of mycellium in dH2O [Re: Quincunx]
    #2314889 - 02/08/04 05:41 PM (12 years, 9 months ago)



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InvisibleMrMaddHatter
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Re: practical method for long term storage of mycellium in dH2O [Re: Quincunx]
    #2315984 - 02/08/04 11:40 PM (12 years, 9 months ago)

VERY nice write-up. :thumbup: :thumbup: I've had much success with storing mycelium in dH2O.


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Offlinezeronio
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Re: practical method for long term storage of mycellium in d [Re: Quincunx]
    #2316712 - 02/09/04 04:31 AM (12 years, 9 months ago)

Very good! :thumbup: :thumbup: :thumbup:


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Offlineragadinks
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Re: practical method for long term storage of mycellium in d [Re: MrMaddHatter]
    #2316751 - 02/09/04 05:02 AM (12 years, 9 months ago)

MrMaddHatter, how long have you stored mycelium in dH2o successfully so far ?
And what is your tek like ?


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Offlinebonsai
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Re: practical method for long term storage of mycellium in dH2O [Re: Quincunx]
    #2316764 - 02/09/04 05:21 AM (12 years, 9 months ago)

To make this complete where would one buy some of the ependorf tubes, dipping tubes (what exactly do you mean by this?) and those hollow cotton bud sticks?
Thanks.
bonsai


--------------------
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--Frank Zappa--

:laugh:


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InvisibleMrMaddHatter
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Re: practical method for long term storage of mycellium in d [Re: ragadinks]
    #2317522 - 02/09/04 01:21 PM (12 years, 9 months ago)

Longest time so far is about 9 months.

I just take a syringe full of sterile water, then open a fresh plate and gently scrape the mycelium with syringe needle while squirting out about half the syring, then I lean the plate a little sideways to pool up all the water and suck it back into the syringe. I then squirt this into small testubes and refridgerate.
I also use the water-mycelium syringes to inoc. honey water.


--------------------
I hate to advocate drugs, alcohol, violence, or insanity to anyone, but they've always worked for me.
Hunter S. Thompson

It's always tea-time, and we've no time to wash the things between whiles.

Don't be a fool, this is a war on drugs. Act like. The government sure does!


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InvisibleMrMaddHatter
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Re: practical method for long term storage of mycellium in d [Re: MrMaddHatter]
    #2317618 - 02/09/04 01:47 PM (12 years, 9 months ago)

One question I've been pondering.....Why is it that sugar based liquid cultures will not store as long? If the mycelium has used up most or all of the sugars available in the solution, then wouldn't there be mostly water left. Also, once put into the fridge, the mycelium pretty much goes dormant, right? Whats to keep it from storing for just as long as with dH2O?

On that note, I've had honey water inoculant that was still good after 7 months (and counting).


--------------------
I hate to advocate drugs, alcohol, violence, or insanity to anyone, but they've always worked for me.
Hunter S. Thompson

It's always tea-time, and we've no time to wash the things between whiles.

Don't be a fool, this is a war on drugs. Act like. The government sure does!


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OfflineQuincunx
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Re: practical method for long term storage of mycellium in dH2O [Re: bonsai]
    #2318174 - 02/09/04 04:26 PM (12 years, 9 months ago)

Thank you all for kind words. I'm really interested in preserving mycellium cultures for a *long* time. But every method i've read about so far is not fullproof. There's always a possibility to transfer some nutrients and air together with mycellium. I would like to have a 100% separation of mycellium from nutrients and air.

But i'm sure there are some of you who are experts in microbiology and maybe you have some experience in this matter. If sky is not the limit and you would have all the fancy equipment how would you do it?

I was thinking about centrifuge machines a lot lately for separating agar from mycelium cultures. I asked a friend about them, but she said they only have the one that goes in thousands of RPMs and she's not sure if it's appropriate for mushroom cultures. I left her some cube cultures to play around with, for the mean time, she showed me a niffty lab tech trick she uses with fungi. She never tried it with mushrooms but we both think it will work:

She cuts a circle from paper(coffee) filter that fits into petri and puts it on fresh agar surface then she inoculates the petri on the filter surface. Pores of filter are large enough for mycellium to grow through. After colonization is complete, she just removes the filter paper and scrapes or washes the mycellium off. Agar stays on the other side of paper filter.

Hm. Clever young girl.

I'm trying it also. And if it is successfull i'll post the results.

Quote:

bonsai said:
To make this complete where would one buy some of the ependorf tubes,



Oh. Well. I just go downtown to a labware store. Glassware is expensive as hell, but PP ependorfers like most disposable lab supplies are really cheap. But i'm sure they can be ordered over the Net. Google search might help.

Quote:

dipping tubes (what exactly do you mean by this?)



Ooops. :blush: I meant dripping-tube (stactometer). I'm so sorry. English is not my native language. I'll correct the original post.

Quote:

and those hollow cotton bud sticks?





http://www.johnsonsbaby.com/products/cotton/cotton-swabs

Hm. I see now that Suntzu meant the same with a "swab" term. But that translates into something like "cloth on a stick" in my dictionary. :biggrin:


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Offlineragadinks
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Re: practical method for long term storage of mycellium in d [Re: Quincunx]
    #2318417 - 02/09/04 05:46 PM (12 years, 9 months ago)

Quote:

She cuts a circle from paper(coffee) filter that fits into petri and puts it on fresh agar surface then she inoculates the petri on the filter surface. Pores of filter are large enough for mycellium to grow through. After colonization is completed, she just removes the filter paper and scrapes or washes the mycellium off. Agar stays on the other side of paper filter.




Sound good, but how does she sterilze the filter ?


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OfflineQuincunx
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Re: practical method for long term storage of mycellium in d [Re: ragadinks]
    #2318521 - 02/09/04 06:13 PM (12 years, 9 months ago)

It's not necessary to sterilize paper products that come directly from manufacture. They are considered clean. That's why we can make sterile spore prints on commercialy avaiable office/copier paper, cartons, alu/tin foils...


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Offlinelalala
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Re: practical method for long term storage of mycellium in d [Re: Quincunx]
    #2319679 - 02/09/04 11:28 PM (12 years, 9 months ago)

Wow, fabulous technique. I'm very impressed. 5 shrooms!


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Offlineragadinks
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Re: practical method for long term storage of mycellium in d [Re: Quincunx]
    #2320404 - 02/10/04 03:49 AM (12 years, 9 months ago)

Quote:

It's not necessary to sterilize paper products that come directly from manufacture.



If I only would cut that piece of paper with some pair of scissors it would not longer be sterile. But that might be due to the fact that I do not have a fancy lab.


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OfflineQuincunx
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Re: practical method for long term storage of mycellium in d [Re: ragadinks]
    #2320558 - 02/10/04 05:59 AM (12 years, 9 months ago)

Quote:

I do not have a fancy lab




You don't need a fancy lab. Sterilize your tool (try presterilized razor blades) and wear glowes. Cut some filter papers and put them all in an empty petri dish, cover it then nuke it real good in a microwave. That should do it.


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Offlineragadinks
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Re: practical method for long term storage of mycellium in d [Re: Quincunx]
    #2320639 - 02/10/04 08:39 AM (12 years, 9 months ago)

Oh, from your pictures above I draw the conclusion that this lab were yours.
That with the microwave sounds like a good idea to me - have to try that out.


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Offlinedisturbed
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Re: practical method for long term storage of mycellium in dH2O [Re: Quincunx]
    #2320776 - 02/10/04 10:49 AM (12 years, 9 months ago)

how about you send me a culture so i can confirm your findings? haha j/k great write up man


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OfflineQuincunx
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Re: practical method for long term storage of mycellium in d [Re: ragadinks]
    #2320808 - 02/10/04 11:06 AM (12 years, 9 months ago)

Quote:

Oh, from your pictures above I draw the conclusion that this lab were yours.




Yes, it is mine. But it is not fancy or expensive. It is simple and i just keep it clean.
The object of this thread is not to impress anyone with my lab. My mission is to help people to see that they don't need fancy equipment and money to grow Mother Nature's gifts. Growing mushrooms is like seeking The Truth - it reveals itself in simple things, not in fancy, expensive and complicated ones.  :smirk:


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