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OfflineGorguss
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Registered: 02/03/10
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Spore age working with agar.
    #26473473 - 02/07/20 08:46 AM (3 years, 11 months ago)

I'm finally working with agar to really bang out pure cultures; some day specimens unique to my grows. I'm struggling to obtain clean culture. I can't help but wonder if my spores are too old. They were bought back in 2016 so 4 years old. They have not been refrigerated or frozen, just in a zip lock baggy in a shoebox more or less. Since my experience with cleaning up culture is limited I thought it would be easier to start working with new spores.

I poured my 6th or 7th (total) batch of agar yesterday this time finally using some H2O2. I poured a bit into the flask of molten agar, swirling between plates. After they cooled for 10 minutes I pulled wedges from the only good spots I could find, on the only plates that looked clean enough.


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OfflineBilliswow
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Registered: 01/23/20
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Re: Spore age working with agar. [Re: Gorguss]
    #26473481 - 02/07/20 08:53 AM (3 years, 11 months ago)

I'm interested in this too, trying to germinate spores from a print I took 2 years ago and no growth yet after 2 weeks on agar. Are you using a spore print or syringe? Did you re-hydrate your spores in water before plating?


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OfflineGorguss
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Registered: 02/03/10
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Re: Spore age working with agar. [Re: Billiswow]
    #26473499 - 02/07/20 09:02 AM (3 years, 11 months ago)

Quote:

Billiswow said:
I'm interested in this too, trying to germinate spores from a print I took 2 years ago and no growth yet after 2 weeks on agar. Are you using a spore print or syringe? Did you re-hydrate your spores in water before plating?




I got scared to work with the print I have; which too is old. However the MS syringes I have are just in sterile water from the vendor. I have some pictures to upload to help out. I think I'm seeing some rhizomorphic growth, I'm just super skeptical of my ability to differentiate between mold and cube myc.


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OfflineGorguss
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Re: Spore age working with agar. [Re: Gorguss]
    #26473515 - 02/07/20 09:15 AM (3 years, 11 months ago)



So here is a plate that was transferred once from its MS plate. I was running low on plates so I did 3 wedges per dish, with the intent to move the leading edge of growth asap. The original plate was a 1:1 gram of Agar to LME 20g per 1000mL

The second generation, this plate, was a Cat food recipe 10g crushed sifted meow mix and 10g agar.

The transfers off of these were on to the LME recipe I previously used with a slight tweak. 2 grams shy of nutritional yeast and some blue food coloring. As well as I mixed a good squirt of H2O2 into the flask of molten agar before pouring.


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Invisibletramalot
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Registered: 12/13/19
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Re: Spore age working with agar. [Re: Gorguss]
    #26473894 - 02/07/20 12:39 PM (3 years, 11 months ago)

I'm new but I thought that the water in the syringe hydrates the spores, wakes them up and should be used in 60 days!

But I'm newwwwwb ymmv


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It's the Journey I enjoy!


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OfflineThe Mycologist
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Re: Spore age working with agar. [Re: tramalot]
    #26473925 - 02/07/20 01:04 PM (3 years, 11 months ago)

:whathesaid:

Yea that can help. I have even seen people putting pressure on the plunger with rubber bands and leaving it for a while. The pressure forces water into the spores even more.


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"That you are here—that life exists, and identity;
That the powerful play goes on, and you will contribute a verse.”
― Walt Whitman, Leaves of Grass
:acidfire::tmckenna:


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InvisibleMr.Mouse
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Re: Spore age working with agar. [Re: The Mycologist]
    #26474766 - 02/07/20 10:15 PM (3 years, 11 months ago)



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InvisibleMr.Mouse
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Re: Spore age working with agar. [Re: Gorguss]
    #26474768 - 02/07/20 10:18 PM (3 years, 11 months ago)

Quote:

Gorguss said:





That healthy looking bit at the top right looks awesome!  That ought to transfer out nicely.


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OfflineCatsLoveHouseMusic
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Re: Spore age working with agar. [Re: tramalot]
    #26474815 - 02/07/20 11:27 PM (3 years, 11 months ago)

I am using syringes (albeit from a known vendor,) that are about 15 months old atleast and they work well. I also have like 5 syringes from like 5 years ago I want to try and use, from the same vendor. The 15 month old ones are working great, so far that is.


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OfflineZackk
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Re: Spore age working with agar. [Re: CatsLoveHouseMusic] * 1
    #26479986 - 02/11/20 11:14 AM (3 years, 11 months ago)

I recently used a 12 year old syringe of B+ on agar, 2 drops per plate in a SAB and look clean and healthy. Spore syringes were stored in a desk drawer.


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OfflineElrik
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Re: Spore age working with agar. [Re: Zackk] * 1
    #26480024 - 02/11/20 11:35 AM (3 years, 11 months ago)

When I returned to growing I started with 9-10 year old spore prints.
The prints I had stored in my refrigerator all that time grew just fine by PF tek or on agar. The prints I had accidentally stored with seeds in my larder [coolest room in the house, used for storing food, medicinal herbs, and long lived seeds] were all dead. 100% dead, even on agar.
Store spores in a clean cardboard box in a ziplock bag in the back of the fridge away from the cold air inlet and they'll last a decade or two.
Most seeds can be desiccated and stored similarly.


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OfflinePrimalSoup
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Re: Spore age working with agar. [Re: Elrik]
    #26484338 - 02/13/20 08:25 PM (3 years, 11 months ago)

I've resurrected 15 year old Ps cyanescens spores by rehydration (see https://www.shroomery.org/forums/showflat.php/Number/11937464#11937464 for some pics and story), stored in the fridge.  I've recently gotten germination off a 6 year old PE swab I made and stored in the fridge, but it took a long while (see https://www.shroomery.org/forums/showflat.php/Number/26477683 ).  The older they get the more I suspect they require rehydration but some people seem to be able to avoid that.  Don't freeze them though, none of the old prints I had in the freezer would germinate over several tests.


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OfflineGorguss
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Re: Spore age working with agar. [Re: PrimalSoup]
    #26497231 - 02/21/20 03:56 PM (3 years, 11 months ago)

Thank you for all your feedback. I was able to mix up some of this activated charcoal agar. I don't keep very good records so I can't tell you if it improved spore germination or not, but some spores did end up germinating on them. I noticed that it seemed to change the mycelium growth. Seems like I was seeing lots of fuzzy and no clear lines. Upon more and more transfers, especially to different medias, it seemed to stimulate the mycelium to do different things.

Here are some plates with growth on the charcoal agar:





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OfflineCatsLoveHouseMusic
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Re: Spore age working with agar. [Re: Gorguss]
    #26497669 - 02/21/20 08:24 PM (3 years, 11 months ago)

So it worked out for you it seems, that's nice to hear and I bet youre happy. I used my syringes on grains and BRF and its working out fine. About to start agar myself. Good for you dude.


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OfflineKyngwhatt
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Re: Spore age working with agar. [Re: Gorguss]
    #26525794 - 03/09/20 04:43 PM (3 years, 10 months ago)

Quote:

Gorguss said:
Upon more and more transfers, especially to different medias, it seemed to stimulate the mycelium to do different things.

Here are some plates with growth on the charcoal agar:




what was your ratio of the carbon and what did you mean by different things, good things?


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OfflineGorguss
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Registered: 02/03/10
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Re: Spore age working with agar. [Re: Kyngwhatt]
    #26526181 - 03/09/20 09:05 PM (3 years, 10 months ago)

Quote:

Kyngwhatt said:
Quote:

Gorguss said:
Upon more and more transfers, especially to different medias, it seemed to stimulate the mycelium to do different things.

Here are some plates with growth on the charcoal agar:




what was your ratio of the carbon and what did you mean by different things, good things?




Yes I meant good different things. I found the mycelium turning rhizomorphic and ropey growing out on this medium. I only added 1g Activated Charcoal per, 20g agar, 20g ME. This recipe is basically my germination mix.
I also add a small amount of yeast every now and then 1-2g.  Its an old wine yeast, pastor champagne I think. I don't know if this is an acceptable yeast, especially cause it is older and wasn't refrigerated.

Then I do a pink agar with food coloring, 20g Agar 20g ME and some peroxide, this is my H2O2 recipe.

And finally I do an antibiotic agar that is 20g agar 20g ME and 4 bags of black tea cold soaked in 1L of water for a few hours or so. I've boiled the tea to, I didn't make any notes or controls to experiment with the results.

I am still in the process of identifying clean culture on a dish. I am now dropping wedges onto grain with the intentions of fruiting them out in shoeboxes with plastic liners in a SGFC or a MARTHA set up.

The shoebox method seems to suit my goals of weeding out sub par genetics and searching for my own master culures of each variety I now possess.


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Offlinevinnie boombotz
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Registered: 04/29/19
Posts: 492
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Re: Spore age working with agar. [Re: Gorguss]
    #26528311 - 03/10/20 10:53 PM (3 years, 10 months ago)

I recently read that tryptophan is the most important amino acid for Spore germination. It also makes the spores grown from those mushrooms more viable


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