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OfflineZenn
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Little questions. Spore print scraping to agar
    #26355963 - 12/01/19 05:34 PM (4 years, 1 month ago)

Hi guys.

So I have put some P.Cyanescens spore on agar a couple weeks ago. I'm just wondering what's the best temps and other conditions?

Roughly how long does it take for spore on agar to hydrate and germinate?

I don't see sign of contamination yet, so that's good I think. Shine light through the agar and small black specks are visible, my guess where the spores settled. But no other visual signs as of yet. Had read P.Cyanescens can take a long time to germinate


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OfflineGrimsweeper
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356033 - 12/01/19 06:28 PM (4 years, 1 month ago)

Usually a couple days but up to a few weeks. 3 weeks it's probably not going to happen.


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: Grimsweeper]
    #26356147 - 12/01/19 07:37 PM (4 years, 1 month ago)

Quote:

Grimsweeper said:
Usually a couple days but up to a few weeks. 3 weeks it's probably not going to happen.



Ok, thank you.
Is that from a dry or fresh spore print? (If that makes difference....noob and this is my 1st print to agar) I have seen spore from syringe show signs pretty quick but thought that was due to them being hydrated?

I tried to find the info I found before:

"Spores will pull moisture from the agar plate but if they are to dry, it can take 4 weeks to germinate."

So I guess was wondering on others personal experience with how long spores off foil take to germinate on agar? Does it take different times depending on agar firmness, dryness of spores? Other factors?

All info is very much appreciated!


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OfflineGrimsweeper
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356188 - 12/01/19 08:10 PM (4 years, 1 month ago)

Personally I've never had spores not germinate and it's always been inside of a week. That being said I've probably never worked with dried out spores that were years old.


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: Grimsweeper]
    #26356337 - 12/01/19 09:51 PM (4 years, 1 month ago)

Quote:

Grimsweeper said:
Personally I've never had spores not germinate and it's always been inside of a week. That being said I've probably never worked with dried out spores that were years old.





Ok that's fair. This print is wild P.Cyanescens from this season, but it would have dried before I got it. Dry but not years old dry


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OfflineJohnRainy
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356345 - 12/01/19 09:54 PM (4 years, 1 month ago)

Have you tried incubating them?


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: JohnRainy]
    #26356582 - 12/02/19 02:14 AM (4 years, 1 month ago)

Yes, it's in the incubator :smile:


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InvisibleAyePlusS
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356589 - 12/02/19 02:31 AM (4 years, 1 month ago)

What kind of agar? For some reason I’ve had trouble germinating ps. cyans on malt extract agar.

Dark streak of spores just sitting there for like 3 months before I finally tossed it,

I’d say at three weeks its definitely time to swab some more spores, try using a softer PDA. They seem to like that a little better IME.

And the incubator is unnecessary. Cyans germinate outdoors in rainy cold weather just fine


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: AyePlus]
    #26356592 - 12/02/19 02:44 AM (4 years, 1 month ago)

Quote:

AyePlus said:
What kind of agar? For some reason I’ve had trouble germinating ps. cyans on malt extract agar.

Dark streak of spores just sitting there for like 3 months before I finally tossed it,

I’d say at three weeks its definitely time to swab some more spores, try using a softer PDA. They seem to like that a little better IME.

And the incubator is unnecessary. Cyans germinate outdoors in rainy cold weather just fine





Aaaah ok, yeah the agar is light malt extract and dextrose.  I have some potato flakes I could use but I'm guessing you need to strain it after as last time I tried using potato flake for agar the potato particulate settled at the bottom after being in the PC .... when I experimented with mini lc the potatoed agar fermented and gave off little bubbles lol. That's why I switched to LME agar.

As for incubation I have one already set up for my cubensis (it's fucking cold here this time of year... infact just passed wild cyanescens fruiting season I think) it was recommended to me to try them on agar and as I already had incubator I used it to see if it help them along.

....do you think perhaps boiling some oak chips, straining then using that water with agar would work better for the P.Cyanescens? Or is that a dumb idea


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InvisibleMice-Helium
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356595 - 12/02/19 02:50 AM (4 years, 1 month ago)

I used a LME agar and a loop to try to streak the plates but ended up scraping them onto the agar.

I tried plates with the agar kept on the bottom when stored and some flipped upside down.
I found from my experience that the ones that were flipped upside down germinated first and showed the fastest growth. Possibly to do with the condensation, i don't know? This was only a small sample though so take with a pinch of salt.

After one transfer, i have tried a few wedges in some WBS and that's taking really well, also a Honey/Water LC seems to be going well too.

So far these Cyans seem to be far more aggressive than the Cubensis i've tried in my opinion.


Side note;
I have once successfully hydrated an agar plate with some 2-3yr old spores.
In a SAB, using a sterilised syringe with filtered water inside that had been boiling for 10m, sucked up and evacuated 3x and then left to cool with a cap on.
1 plate germinated and stayed clean for further transfers, the other two germinated but became contaminated.
Both a success and failure, so take what you want from it.


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: Mice-Helium]
    #26356608 - 12/02/19 03:21 AM (4 years, 1 month ago)

Quote:

Mice-Helium said:
I found from my experience that the ones that were flipped upside down germinated first and showed the fastest growth. Possibly to do with the condensation, i don't know? This was only a small sample though so take with a pinch of salt




I flip some things upside down especially if in an incubator. Even my brf jars (plastic with micropore taped holes) and it seems to go well. On occasion I also flip them periodically but mostly kept bottom side up and notice better growth (agar and cakes). But like you say pinch of salt :smile:

Considering trying to hydrate a little of the spores in a syringe, then try on different agar

Thank you for your info :smile:


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InvisibleMice-Helium
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26356613 - 12/02/19 03:27 AM (4 years, 1 month ago)

Quote:

Zenn said:
Thank you for your info :smile:




No worries, I'm still a beginner with mixed success but I find as long as I have a clean "back up" plate or more spores, I quite enjoy the experimentation.

Best of luck to you, update us on any success or fails :thumbup:


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OfflinemeaculpaUIO
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Re: Little questions. Spore print scraping to agar [Re: AyePlus]
    #26356655 - 12/02/19 04:51 AM (4 years, 1 month ago)

Quote:

AyePlus said:
What kind of agar? For some reason I’ve had trouble germinating ps. cyans on malt extract agar.

Dark streak of spores just sitting there for like 3 months before I finally tossed it,

I’d say at three weeks its definitely time to swab some more spores, try using a softer PDA. They seem to like that a little better IME.

And the incubator is unnecessary. Cyans germinate outdoors in rainy cold weather just fine




What recipe for agar did you end up using, Aye?
I have the same problem with semperviva, azurens, cyanens like you.
They simply arent germinating. I have a low number of attempts
since I'm being told to stick to cubes, but I find it
strange that I get cube myc to germinate but not azur or cyans


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: Mice-Helium]
    #26360265 - 12/04/19 12:38 AM (4 years, 1 month ago)

Well, I've done a couple different things and will do another experiment to see if it has better results.

I sterilized a pasty plate with about 10ml water in It, into the SAB and scraped maybe 1/5 of the print into the water and made a syringe to see if the spores would hydrate in there. Good news they have! :laugh:

I also made some potato agar by boiling some potato flakes and draining off the water (no potato bits this time) with a little dextrose  and dripped a little of the P.Cyanescens syringe in there to see if it likes the potato better.

The 3rd thing I am going to do is make some agar but instead of boiling the potato I am going to boil oak shavings and do the same as I did above.

Here's hoping they like the different agar. I am very happy that the spores germinated in sterile water, I take that as a great sign!


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InvisibleMice-Helium
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Re: Little questions. Spore print scraping to agar [Re: Zenn]
    #26360312 - 12/04/19 01:43 AM (4 years, 1 month ago)

Nice!

Now they have germinated, which is a great sign, the mycelium should grow fairly rapidly on future transfers when you clean up the samples.

Interested to hear about the oak shaving agar. I've not heard of that before. I'd think that as a saprotrophic species that it'd probably love that.
Have you read about this or just doing it on a whim?


Also, are you planning on starting an outdoor patch for yourself?


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Offlinefeldman114
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Re: Little questions. Spore print scraping to agar [Re: Mice-Helium]
    #26360691 - 12/04/19 09:21 AM (4 years, 1 month ago)

Wait what? You put spores on water (no nutes) and they germinated? Never seen myc in a MS syringe🤷🏻‍♂️


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Re: Little questions. Spore print scraping to agar [Re: AyePlus]
    #26360799 - 12/04/19 10:30 AM (4 years, 1 month ago)

Quote:

AyePlus said:
What kind of agar? For some reason I’ve had trouble germinating ps. cyans on malt extract agar.

Dark streak of spores just sitting there for like 3 months before I finally tossed it,

I’d say at three weeks its definitely time to swab some more spores, try using a softer PDA. They seem to like that a little better IME.

And the incubator is unnecessary. Cyans germinate outdoors in rainy cold weather just fine



Here's the thing though...spores really only need spores to germinate. Most times they will germinate in plain old water they need food to live afterward but store enough energy before hand .
Just a stoner thought lol but back to the o.p
How many plates did you Try? Swiping an s pattern ?


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Offlinecurious.psychonaut
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Re: Little questions. Spore print scraping to agar [Re: feldman114]
    #26360818 - 12/04/19 10:40 AM (4 years, 1 month ago)

Quote:

feldman114 said:
Wait what? You put spores on water (no nutes) and they germinated? Never seen myc in a MS syringe🤷🏻‍♂️




Also makes me wonder what the exact trigger is. I've read that spores can germinate and grow a bit in nutrient-less agar. When I transfered spores from print to agar some time ago, I found some lying around in the SAB. I put a few drops of water on them and after a couple of hours there were tiny arms of myc around the clump. (I checked how they look before & after with a handheld microscope.) I assume there might have been some nutrients from air/dirt in the water though. Now we have germination in sterile water. What is it in spore syringes that preserves them through international shipping w/o refrigeration? (At least I assume they are delivered without myc growing in them; I've only ever gotten prints.)


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Re: Little questions. Spore print scraping to agar [Re: curious.psychonaut]
    #26360846 - 12/04/19 10:53 AM (4 years, 1 month ago)

How would a spore eat nutrients?


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InvisibleAyePlusS
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Re: Little questions. Spore print scraping to agar [Re: cronicr]
    #26360911 - 12/04/19 11:32 AM (4 years, 1 month ago)

Quote:

cronicr said:
Here's the thing though...spores really only need spores to germinate. Most times they will germinate in plain old water they need food to live afterward but store enough energy before hand .
Just a stoner thought lol but back to the o.p
How many plates did you Try? Swiping an s pattern ?



I totally agree and  I can’t explain it tbh, dark streaks in an S pattern from a print I made maybe a month prior, 3+ plates with nothing for months. Se la vie, have a dank clone running on pda now see POTD


Sounds like OP finally got germ, yay


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Offlinecurious.psychonaut
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Re: Little questions. Spore print scraping to agar [Re: cronicr]
    #26360950 - 12/04/19 11:53 AM (4 years, 1 month ago)

Quote:

cronicr said:
How would a spore eat nutrients?




Not eat but sense. Apparently (at least) some fungal spores have nutrient receptors. Maybe there are others.

Quote:


From this page quoting this book

Spores of Penicillium digitatum, the green mold fungus which attacks only citrus fruit, germinate to a minor extent in pure water, whereas the addition of fruit juice greatly accelerates germination (Pelser and Eckert, 1977). Testing the effect of the juice components on spore germination revealed that of the sugars within the juice (glucose, sucrose and fructose), glucose is the best stimulant. A greatly enhanced germination is stimulated by the ascorbic acid, whereas the citric acid has no stimulating effect. The combination of glucose and ascorbic acid results in a germination rate quite close to that stimulated by the whole juice.




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Re: Little questions. Spore print scraping to agar [Re: curious.psychonaut]
    #26360970 - 12/04/19 12:08 PM (4 years, 1 month ago)

:cheers:


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OfflineZenn
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Re: Little questions. Spore print scraping to agar [Re: AyePlus]
    #26362011 - 12/04/19 11:29 PM (4 years, 1 month ago)

Quote:

Mice-Helium said:
Nice!

Now they have germinated, which is a great sign, the mycelium should grow fairly rapidly on future transfers when you clean up the samples.

Interested to hear about the oak shaving agar. I've not heard of that before. I'd think that as a saprotrophic species that it'd probably love that.
Have you read about this or just doing it on a whim?


Also, are you planning on starting an outdoor patch for yourself?





Well the wood water agar I did wonder about, I think some people have tried it before but it was more I had a curiousity about it and ha e looked into it a little. Will do more research before I try to, but I think it could work. Some say the woodlovers are picky so I thought why not try make agar with what they like.
I may well do outdoor patch but I am going to try indoor in my pantry (this time of year that room is cold enough) I have read people have had success with them indoors started in the fridge (a little warmer than they would usually have the fridge) I want to do PF type cakes but would add some fine oak chips/shavings to the brf/verm mix. Then spawn to bulk with wood chips :smile:

Quote:

feldman114 said:
Wait what? You put spores on water (no nutes) and they germinated? Never seen myc in a MS syringe🤷🏻‍♂️




Yeah they hydrated. Not quite myc but you can see visible signs. I didn't know this could happen either till I witnessed it in a previous syringe I bought. It basically just looked like water when I got It, a while later I went back to use it and I could see the germinated spores. I asked guys here if it was myc. Since they hydrated in the water I've kept it cool untill I get to use it soon (waiting on supplies).


Quote:

cronicr said:
Quote:

AyePlus said:
What kind of agar? For some reason I’ve had trouble germinating ps. cyans on malt extract agar.

Dark streak of spores just sitting there for like 3 months before I finally tossed it,

I’d say at three weeks its definitely time to swab some more spores, try using a softer PDA. They seem to like that a little better IME.

And the incubator is unnecessary. Cyans germinate outdoors in rainy cold weather just fine



Here's the thing though...spores really only need spores to germinate. Most times they will germinate in plain old water they need food to live afterward but store enough energy before hand .
Just a stoner thought lol but back to the o.p
How many plates did you Try? Swiping an s pattern ?





I only tried 1 LME plate, but I have a couple  potato and dextrose plates I want to try a little of the spore on.
Also want to try the wood water agar idea to see what it prefers out of agar types since it doesn't seem to like LME. I just need to make more plates. I still have like 4/5 of the print left to work with.

The way I see it, is like you said, the fact it germinated with water is like when a seed germinates in a moist environment, but it will only "grow" to a certain point, after which it needs the right nutrients to thrive and survive. Some aren't so picky like GT seem to love PDA and LME agar. The p.cyan I do think "germinated" on the LME due to the moisture but hasn't grown since it's not the right nute for it. And maybe that agar was also too firm *shrug * on to the next plates!




Quote:

AyePlus said:
Se la vie, have a dank clone running on pda now see POTD


Sounds like OP finally got germ, yay




Yeah alot of trial and error in this haha. And nice one on the clone!

Yay indeed, just need to make more plates to try out! :laugh:


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