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OfflineMandrake Erpelmann
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longtime culture preservation by castellani's method/ hood version
    #28181190 - 02/10/23 04:32 PM (1 year, 3 months ago)

This method is about longtime preservation of cultures in distilled sterile water at roomtemperature, especially suited for isolates, to prevent/slow degeneration by continuous subculturing and to have some kind of backup.

Some perks of this method are:
-cultures store up to 5 years, in some cases up to 10-20 years
-cheap and easy
-cultures are stored at roomtemperature/no need for refrigeration etc.

Manual:
-suck up fresh LC into syringes, Flame needles, put cap back on and place them with the Tips pointing up over night to let the mycelium sediment.

-keep the needles pointing up and carefully squirt out the excessive liquid, flame needles and inject the "concentrated LC" into a jar with sterile distilled water, its works well, when the jar is rather narrow. Let it stand over night, to let the mycelium sediment.

- now take a fresh sterile syringe and gently suck up the excessive water, this can take a While, instead of going in and out with the syringe all the time, you can aswell attach a sterile syringefilter to a sterile Needle, and Stick it through the injectionport/ PP wool plug, and keep it inside. Take syringe with no needles, Dock, fill, undock, squirt out repeat, without the need of flaming inbetween the steps. Do until most of the water is removed.

- suck up the washed and concentrated mycelium with a fresh syringe and inject it into a fresh jar of sterile distilled water. Now let it stand for 2-3 days in a dark place, to let the mycelium form a cohering mat at the bottom of the jar.
Go in with a fresh syringe (with needle), and very gently swirl up the cohering mycelium mat with a chemist's flask shake stile movement. Wait until the mycelium floats by your needle and suck it up. If u are doing it well, you can suck all of the mycelium up with one draw and leave the excessive water behind. Flame the needle.

-Put a sterile syringe cap on the syringe and store, or inject it into sterile hplc vials. Hplc vials have the advantage, that they have an injectionport in the lid, and are autoclaveable. The ones i use, hold 1,5ml. When u inject into the closed vials, u have to create an underpressure first with your syringe, and then let the vial suck in you mycelium suspension.

I know this sounds a bit tedious, but u can save a lot of time this way.

Edited by Mandrake Erpelmann (02/10/23 04:58 PM)

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OfflineTryptaminerXXXIII
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Registered: 02/04/23
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Re: longtime culture preservation by castellani's method/ hood version [Re: Mandrake Erpelmann]
    #28181204 - 02/10/23 04:46 PM (1 year, 3 months ago)

"A modification of Castellani's method [3–7] is proposed for higher basidiomycetes,..."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472852/


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Invisiblebakedbeings
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Re: longtime culture preservation by castellani's method/ hood version [Re: TryptaminerXXXIII]
    #28182090 - 02/11/23 10:09 AM (1 year, 3 months ago)

no shade but this is the most complicated version of this idea ive read. why all the steps?

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OfflineTryptaminerXXXIII
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Re: longtime culture preservation by castellani's method/ hood version [Re: bakedbeings]
    #28182110 - 02/11/23 10:22 AM (1 year, 3 months ago)

Shroomery user Farm3r has a video on his YouTube where it's a little more simplified I think. He uses water agar instead of filter paper, as it's not nutritional and readily available.

It's not that complicated at all really.


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Nothing is sacred
Rest In Paradise
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OfflineMandrake Erpelmann
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Re: longtime culture preservation by castellani's method/ hood version [Re: TryptaminerXXXIII]
    #28185056 - 02/13/23 07:14 AM (1 year, 3 months ago)

Its nice to have it as a liquid cultere for me, in terms of the ease of use. You can have a lot more viable Material in one tiny vial, and directly suck it up with a syringe and put a tiny drop on to your media if u want to reactivate the strain.
Sure its way more easy to throw some wedges of agar into sterile distilled water...

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Invisiblebakedbeings
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Re: longtime culture preservation by castellani's method/ hood version [Re: Mandrake Erpelmann]
    #28185125 - 02/13/23 08:20 AM (1 year, 3 months ago)

an agar wedge in distilled water makes liquid inoculant. the mycelium sheds off into the water, so you can just dip a loop into it as with LC. but mostly i was asking why you felt the need to keep moving the inoculant from container to container so many times when you could just draw it into a syringe with water and be done

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OfflineMandrake Erpelmann
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Re: longtime culture preservation by castellani's method/ hood version [Re: bakedbeings]
    #28185380 - 02/13/23 11:48 AM (1 year, 3 months ago)

To decrease the concentration of nutrients and dissolved ions as much as possible... Have you gained much experience with suspendet agar? Did u use wedges from nutrients rich agar, and if so can u compare the results to water agar?

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Invisiblebakedbeings
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Re: longtime culture preservation by castellani's method/ hood version [Re: Mandrake Erpelmann]
    #28185456 - 02/13/23 12:51 PM (1 year, 3 months ago)

ive been playing with low nute LC further diluted in water. consensus on this site seems to be that even if there is some food left in the solution, the myc will just spend it and go into stasis after

another idea that interests me is just throwing tissue samples in water. no nutrients no extra steps

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OfflineMandrake Erpelmann
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Re: longtime culture preservation by castellani's method/ hood version [Re: bakedbeings]
    #28185721 - 02/13/23 03:50 PM (1 year, 3 months ago)

Ok, good to know!
... Cool idea, I'd be interested in the results.
Could be useful to homogenize the tissue samples for that purpose.

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