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OfflineKevx
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Registered: 07/10/14
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spores from dry mushrooms
    #22105480 - 08/18/15 06:04 AM (8 years, 5 months ago)

Hello shroomery community,

I recently came across some one week old dry cubes and decided I'm going to practice my agar work and try to produce some mycelium from spores left in the caps.

I placed the most promising looking cap in a 30ml vial filed with sterilized distilled water... and to my surprise the cap absorbed about 5-10ml of water and expanded exposing the gills (which definitely had spores on). After a few hours the solution turned dark purple and a spore print started to form at the bottom of the vial. Now I know that this spore solution is contaminated like crazy, since I had no way of disinfecting the cap before this but I hope that with some agar work and help from antibiotics I could isolate a clean strain of mycelium.

I let the cap soak for about 10h then mixed small amounts of the spore solution with various concentrations of doxycycline and inoculated 9 home made potato dextrose agar plates.

Now after about 50h all of the plates are contaminated with the same milky-white bacteria colonies (plates that had the more concentrated antibiotic solution have less colonies, diluted ones more of them...) which was to be expected... But the surprising thing is that I see ZERO mycelium growth.

I have a control plate which was inoculated without any doxycycline and it has the most bacteria colonies but again no mycelium... so I can exclude doxycycline as being the culprit.

There are only two reasons for the lack of mycelium I can think of:
a) The bacteria is killing/suppressing mycelium growth

b) I didn't find any pure dextrose for my potato dextrose agar so I used the equivalent of Dextro-energy tablets instead. They contain: dextrose (89 %), maltodextrin, acid (citric acid), anti-caking agent (magnesium salts of fatty acids), flavouring. Could this be messing with my mycelium ?

I have enough of the spore solution left to try again but I need to figure out what I'm doing wrong first.

Any help will be greatly appreciated !


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OfflineLaughingcowwa
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Re: spores from dry mushrooms [Re: Kevx]
    #22105489 - 08/18/15 06:11 AM (8 years, 5 months ago)

Good luck I don't see this working at all. But guess its worth a try just be ready for contaminations galore.


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OfflineLoveNaborFuckHater
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Re: spores from dry mushrooms [Re: Laughingcowwa]
    #22105510 - 08/18/15 06:24 AM (8 years, 5 months ago)

Another way you could try is dropping water into the cap and soaking that or into the cap and trying for a print.


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OfflineKevx
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Re: spores from dry mushrooms [Re: LoveNaborFuckHater]
    #22107553 - 08/18/15 04:52 PM (8 years, 5 months ago)

Quote:

Laughingcowwa said:
Good luck I don't see this working at all. But guess its worth a try just be ready for contaminations galore.




I'm doing this as an experiment and to practice my agar work... so yeah, It doesn't hurt to try :laugh:

Quote:

LoveNaborFuckHater said:
Another way you could try is dropping water into the cap and soaking that or into the cap and trying for a print.




I might try that too... it should limit the contamination, but it won't eliminate it so I wonder if it will make any difference. But the problem is that I get no mycelium growth in my agar and I'm sure there are alot of spores in there. I'm going to try again today and prepare stronger antibiotic solutions.

I'm still interested in your opinion as to why there's no mycelium growth.


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OfflineLoveNaborFuckHater
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Re: spores from dry mushrooms [Re: Kevx]
    #22107631 - 08/18/15 05:10 PM (8 years, 5 months ago)

See what happens if you cut flesh from the least dry and dead fruit and cloning if you're doing it for shits


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OfflineKevx
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Re: spores from dry mushrooms [Re: LoveNaborFuckHater]
    #22168350 - 08/31/15 10:12 AM (8 years, 4 months ago)

Now after about 2 weeks of trying I'm sad to report all plates were lost to contamination and no amount of transfering helped :frown:


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OfflineLoveNaborFuckHater
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Re: spores from dry mushrooms [Re: Kevx]
    #22169846 - 08/31/15 04:08 PM (8 years, 4 months ago)

Did you try that method I mentiod? I was thinking about doing it soon cause I just got some better looking dry caps


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Offlinekeeno
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Re: spores from dry mushrooms [Re: LoveNaborFuckHater]
    #22181884 - 09/03/15 07:08 AM (8 years, 4 months ago)

could it be that the energy tablets you used had some sort of preservative/ anti-fungal element to them to stop them going off?

:shrug:


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Offlinegreencreative850
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Re: spores from dry mushrooms [Re: Kevx]
    #22801854 - 01/20/16 01:13 AM (8 years, 11 days ago)

I'm currently looking to do the same thing. I'm not sure how much of my info can help. I'm currently in CA and cannot get any spores or prints delivered, so i'm in the hunt to figure out how to do this from dried caps.

My first question would be; is it possible to sterilize the cap in a dry heat sterilizer? I have access to one. Would that damage the spores? Also, what if you tried using Bacteriostatic water? Thats normally used to mix medicine in syringes to give yourself.

would any of this help with elimination or lowering the amount of bacteria?

Just throwing thoughts out there in case anything helps.


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Offlinegreencreative850
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Re: spores from dry mushrooms [Re: Kevx]
    #22801866 - 01/20/16 01:18 AM (8 years, 11 days ago)

I forgot to mention that you can find 100% pure dextrose online on amazon. NOW FOODS dextrose natural sweetener.


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OfflineEywa_devotee
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Re: spores from dry mushrooms [Re: greencreative850]
    #22860716 - 02/03/16 06:25 PM (7 years, 11 months ago)

The anti-caking agent (magnesium salts of fatty acids) probably was the problem, as that is actually a synergist in some antifungal spray preparations. You can get very pure dextrose as corn sugar from a home brew shop. One thing to try with your spore juice if badly contaminated is instead of using agar, spray it onto something not as nutritious like a piece of wet cardboard. Incubate as usual, and transfer any rhysomorphic growth that forms to agar. Your juice is OK if it's purple and should be good so long as the dehydrating was done gently.


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Offlinemolemole
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Re: spores from dry mushrooms [Re: Eywa_devotee] * 1
    #23228446 - 05/15/16 01:48 AM (7 years, 8 months ago)

I have done what you guys are attempting. All I did was just use fine point tweezers, flame sterilize. Then pluck a small piece of the gill and drop it onto plate.Inside SAB or in front of hood. Did this with a wild picked mushroom. There was contaminants, but in a few transfers it was clean. Still have this culture from 3 years ago. Not much different than going from a wild print. If u are unsure do several plates you are bound to be succesful on one attempt.


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Invisiblewoodrow
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Re: spores from dry mushrooms [Re: Kevx]
    #23230521 - 05/15/16 03:49 PM (7 years, 8 months ago)

I have found this to be a good way to extract spores from caps. Soak the cap in water for about an hour until soft and then gently massage the cap with your fingers to release the spores. There is no need to be sterile at this point because the water is loaded with bacteria from the cap. Then filter the water through a coffee filter and into a clean container. Something tall and narrow works best. The spores pass through the filter with the water leaving anything larger like debris from the cap behind. The filter can be discarded.

Let the filtered water stand and the spores should settle out in about an hour but let them remain undisturbed for twelve hours or more. This gives the spores time to adhere to the bottom of the container. The next day, pour ALL of the water into a clean container but be careful not to disturb the spores on the bottom. If all goes well, the spores should remain adhered to the bottom of the original container and the water and bacteria will be in the second container and can be discarded.

(In case too many spores are lost in the wash, they will be in the second container and can be salvaged by letting them settle out as before.)

You should now have a “spore print” of nearly pure spores on the bottom of the container. Re-suspend the spores in sterile water and let them remain for one, two, or three days to hydrate. This helps the spores germinate quicker when planted on agar. A second or third wash should help remove even more bacteria but some bacteria adhere to the spores so no amount of washing will ever give you a pure spore suspension

When ready to inoculate the spores to agar, carefully pour off all of the water as before and work with the spores that remain at the bottom of the container.


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OfflineLoveNaborFuckHater
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Re: spores from dry mushrooms [Re: woodrow]
    #23245146 - 05/19/16 07:45 AM (7 years, 8 months ago)

I'm not going to try to do an experiment with this but for anyone wondering or maybe wanting something to try; I ate mushrooms out of a small mason jar then rinsed it and drank the remaining bits, I threw it under my bed and a few weeks later I found the jar with mycelium growth all over the jar. This may be an easy way to make a LC. If anyone tries this I'd love to hear if it worked or not.


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OfflineGteach
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Re: spores from dry mushrooms [Re: LoveNaborFuckHater]
    #23391751 - 06/28/16 05:41 PM (7 years, 6 months ago)

Ive cloned dry mush with cardboard tek. WOrks for me !


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Invisibledankington
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Re: spores from dry mushrooms [Re: Gteach] * 1
    #23411994 - 07/04/16 09:47 PM (7 years, 6 months ago)

Quote:

Gteach said:
Ive cloned dry mush with cardboard tek. WOrks for me !




:picdidnthappen:

what's all this then? you're taking spores and making prints and getting all this mycelial growth from dried mushrooms? they either weren't really dried, or someone's not telling the truth.


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Offlineleschampignons
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Re: spores from dry mushrooms [Re: dankington]
    #23412040 - 07/04/16 10:05 PM (7 years, 6 months ago)

one technique you could try is get a flame sterilized inoculation loop with a tiny bit of the contaminated spore slurry and swipe across the agar plate. then flame sterilize it again and swipe across the plate again, crossing one tiny part of the previous line you swiped onto the plate as you do so. Then sterilize it again and swipe again crossing only a tiny section of the second line you swiped on the plate.

Sorry I am explaining this kinda poorly but google streaking a bacterial culture out for a single colony on agar

here is a pic that explains better than I can





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Edited by leschampignons (07/04/16 10:07 PM)


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InvisibleInocuole
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Re: spores from dry mushrooms [Re: dankington]
    #23419023 - 07/07/16 01:53 AM (7 years, 6 months ago)

Quote:

dankington said:
Quote:

Gteach said:
Ive cloned dry mush with cardboard tek. WOrks for me !




:picdidnthappen:

what's all this then? you're taking spores and making prints and getting all this mycelial growth from dried mushrooms? they either weren't really dried, or someone's not telling the truth.




This would work with fan-dried mushrooms in the right circumstances.  Dehydrators kill them though, so nobody who's doing any serious drying of mushrooms will be performing this stunt.


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Invisibleweetsie
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Re: spores from dry mushrooms [Re: dankington]
    #23419098 - 07/07/16 03:01 AM (7 years, 6 months ago)

Quote:

dankington said:
Quote:

Gteach said:
Ive cloned dry mush with cardboard tek. WOrks for me !




:picdidnthappen:

what's all this then? you're taking spores and making prints and getting all this mycelial growth from dried mushrooms? they either weren't really dried, or someone's not telling the truth.




RR has mentioned cloning long dried mushrooms (as in years old) being possible.

The tek is basically to take a tiny piece of dried tissue and drop it in plain sterile water for a long time, 6 weeks? 6 months? can't remember. Then transfer it to agar.

Pretty simple, I'm trying it at the moment with spawn that dried out 3 years ago.

Quote:

molemole said:
pluck a small piece of the gill and drop it onto plate.




:whathesaid:

don't over complicate if all you're after is spores.


Edited by weetsie (07/07/16 03:05 AM)


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Offlineeternallove
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Re: spores from dry mushrooms [Re: weetsie]
    #23428331 - 07/10/16 02:57 AM (7 years, 6 months ago)

Long time lurker and first time poster here in a similar position. Getting prepared for a first time grow. Cant order spores directly to my state but could figure out a way to get them if need be but not my preferred method. I'm thinking of trying to clone from mushrooms that I have access to. They are not fully dry and crispy, is this beneficial? I was planning on placing both gils and pieces of stem on several agar plates under sterile conditions, and then moving the mycelium to new agar plates to avoid contam. Have also heard of the cardboard technique working for people. Any recommendations?


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