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Offlinekrypto2000
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Question on Sterile Procedure for Plating Small Specimens on Agar
    #23737257 - 10/14/16 02:05 PM (7 years, 3 months ago)

This is just a general question, not sure if it belongs in Advanced Cultivation or regular, but it seems advanced to me. I'm wonder the proper procedure for surface sterilizing small specimens.

I have successfully made tissue clones from various mushrooms before, wild or indoor grown, and usually have a clean culture on my first go. I will simply wipe the outside of the mushroom with 70% isopropyl, break it open, and then take a sample from the inner tissue. No problem.

Now my question regards smaller specimens where this is not possible. For instance ergot sclerotia or plant tissue (in the case of micro propagation). I have not tried growing sclerotia, but I have plant tissue and while I have had some root I always get bacterial and fungal contamination.

My procedure for plant tissue is to soak the tissue in 10% bleach for 10 minutes (maybe up to 20% for 20 min), and then rinse in 70% IPA. Of course I sterilize my vessels and such, but there are just so many vectors for contamination that I am not sure where my faults lie. One thing that especially makes me curious is if I need to pressure cook my bleach and IPA. For one though I would be concerned about the integrity of the solutions, especially the bleach, but I do have sealable autoclaveable bottles so maybe it won't be an issue. I don't know about bleach, but is it not true that 70% IPA can actually harbour contaminants in itself? Does anyone have access to a manual or something I could review? I have seen some before, but they never actually go so far as to say how to prepare the IPA, bleach, h202, or w/e is used.


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InvisiblebodhisattaMDiscordReddit
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Re: Question on Sterile Procedure for Plating Small Specimens on Agar [Re: krypto2000]
    #23737380 - 10/14/16 02:46 PM (7 years, 3 months ago)

I put small pins right onto agar. They're still in such vigerous growth that you can take a transfer after it grows out in two days.


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Offlinekrypto2000
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Re: Question on Sterile Procedure for Plating Small Specimens on Agar [Re: bodhisatta]
    #23737446 - 10/14/16 03:05 PM (7 years, 3 months ago)

True, that will work with active mycilium, but I'm interested in culturing things such as dormant sclerotia or plants which need a much bigger window. With plants that's not an option even since they take weeks to establish roots and then to clone them you have to basically take another cutting and start all over. With the sclerotia I'd be getting them in the wild or from bags of rye in which case I'd imagine they would take awhile to start growing, more so from dried out sclerotia in rye. This would be a great practice for me as I figure if I can get either of these to grow, and sterile of course, then I can grow anything.


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