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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.




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 ) 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 ). 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 dipping-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.
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 int E7D o the zen or yoga to improve mind concentration ).
Remove the stick from the end of dipping tube and fill it with mycellium suspension. Then slowly dip 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.



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.

Check also Resurrecting a Better Method for Long-Term Storage of Mushroom Cultures .

by Quincunx

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