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Offlineh3aling
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Spore solution on agar
    #22183823 - 09/03/15 04:57 PM (8 years, 4 months ago)

Hello shroomers,
I have decided to give another go to agar after having failed a couple of times with cloning and spore print inoculation.

What I did was inoculate two agar plates with spores suspended in water solution (all done as sterile as possible).

Now after 5~ days I see some growth, but one plate was contaminated with green mold, so it's gone. The other one has some fuzzy fluff growing all over but it does not seem like any mycelium I've seen around (it is not stringy / rhizomorphic), yet it's completely white.

I'm wondering, which contaminants are white and fluffy? Does anyone have a picture of spores germinating on agar?
In the "how it should / shouldn't look like" thread I can't find anything similar to mine.

To end, I know pictures would help you understand better, I'll post one later.

Thank you  :regularshroom:


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Re: Spore solution on agar [Re: h3aling]
    #22183846 - 09/03/15 05:04 PM (8 years, 4 months ago)



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Re: Spore solution on agar [Re: blindingleaf]
    #22184135 - 09/03/15 06:13 PM (8 years, 4 months ago)

None of those are even cubes BL. :lol:


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Re: Spore solution on agar [Re: Inocuole]
    #22184140 - 09/03/15 06:14 PM (8 years, 4 months ago)

i know, i couldn't find any, was hoping no one would see :facepalm:

but they look similar i think.  i have a pic of PE spore swipe, but its bacterial


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Re: Spore solution on agar [Re: blindingleaf]
    #22186484 - 09/04/15 05:43 AM (8 years, 4 months ago)

Hello again,
Thanks for your initial guesses.

Here are some (hopefully helpful)  pictures:


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Re: Spore solution on agar [Re: h3aling]
    #22186494 - 09/04/15 05:49 AM (8 years, 4 months ago)

Those dont look quite right to me, I would start new plates if it were me. But to be fair I cant see the pics too well right now

Next time, squirt the spore solution on a sterile cotton swab or loop, and do a quick swipe. It should give you a good idea of where spore germination should occur vs the solution sliding all over the plate. Then take a transfer as soon as you see healthy growth


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Edited by mushpunx (09/04/15 05:50 AM)


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Re: Spore solution on agar [Re: mushpunx]
    #22186903 - 09/04/15 08:45 AM (8 years, 4 months ago)

yea, looks like mold to me too, but maybe some spore germination on top of first pic, the more white stuff.

mark the date of ur plates when u noc em up, or write it down somewhere,
mold grows very fast, and many times, the date i write down will be more of an indication to me about what it is (compared to the day I'm looking at it) then the actual growth pattern, especially for MS swipes.


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Re: Spore solution on agar [Re: blindingleaf]
    #22187663 - 09/04/15 11:47 AM (8 years, 4 months ago)

Jesus Christ I will never have this shit work for me then.
I know the date, it was the 31st of July.
Might it be that the agar is too dry?
I bought it from an european supplier and never had any luck with spores germinating on it, and not even with putting living fruit pieces on it.

The spores solution was made by myself from a spore print I got from a previous growth, and I know they are good because the WBS I inoculated got some mycelium starting to show up.


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Re: Spore solution on agar [Re: h3aling]
    #22187875 - 09/04/15 12:35 PM (8 years, 4 months ago)

Quote:

h3aling said:
Jesus Christ I will never have this shit work for me then.




Why's everybody freaking out today?


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Re: Spore solution on agar [Re: h3aling]
    #22188614 - 09/04/15 03:29 PM (8 years, 4 months ago)

Quote:

h3aling said:
Jesus Christ I will never have this shit work for me then.
I know the date, it was the 31st of July.
Might it be that the agar is too dry?
I bought it from an european supplier and never had any luck with spores germinating on it, and not even with putting living fruit pieces on it.

The spores solution was made by myself from a spore print I got from a previous growth, and I know they are good because the WBS I inoculated got some mycelium starting to show up.





All the mycelium means is the spores are viable. Usually you wont see mold in grain spawn , it will show up after your substrate is fully colonized and in fruiting conditions. It is the frusyerating part of this hobby, and why agar is such a usefull tool to clean up cultures.

Next time you might be better to swipe the spores directly from the print onto the agar with a loop, as spore syringes are notoriously difficult to get tottally clean.

But if you try like I said and drip it onto a sterile swab, and swipe onto your plate you unless your syringe is beyond redemption you should be able to isolate some clean growth away dude!

Youre only at agar stage, its too early to get mad haha


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Offlineh3aling
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Re: Spore solution on agar [Re: mushpunx]
    #22191598 - 09/05/15 07:14 AM (8 years, 4 months ago)

Thank you.

I also tried direct spores from a print to agar but they never even germinated.
With a chill mind I would say that dry spores on dry agar don't have much hope though. Also, I thought spore prints were notoriously more prone to contamination, is that wrong?

Another thing, in which way would a cotton swab help me achieve germination more than directly dripping spore solution on the agar? It seems like an unnecessary step which might introduce complexity (more chances to contam).

Keep in mind it's a genuine question and I am not trying to discredit your advice/experience.


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Re: Spore solution on agar [Re: h3aling]
    #22192973 - 09/05/15 02:27 PM (8 years, 4 months ago)

Quote:

h3aling said:
Thank you.

Also, I thought spore prints were notoriously more prone to contamination, is that wrong?




In my experience that is not the case. Syringes are made from prints so if it's a print with a contam then no matter how you use it it's going to have contamination.


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OfflineLogicaL ChaosM
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Re: Spore solution on agar [Re: h3aling]
    #22193291 - 09/05/15 03:52 PM (8 years, 4 months ago)

Those pics looks like mycelium.

Theres basically two types of mycelium. The fluffy, "cotton ball" type and the thick, ropey rhizomorphic type.

You want rhizomorphic type. They are typically higher-potent strains of mycelium.

So dont worry, make more plates and try another squirt of your sporoe solution :thumbup:


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Re: Spore solution on agar [Re: LogicaL Chaos]
    #22193431 - 09/05/15 04:25 PM (8 years, 4 months ago)

Quote:

LogicaL Chaos said:
You want rhizomorphic type. They are typically higher-potent strains of mycelium.




no no no, dude, NO! :nojustno:

its a reaction from the myc stretching out looking for nutrients.
rhizomorphic mycelium can go tomentose if you transfer it to a more nutrient rich agar plate.

the beauty of agar is that you're able to see whats growing, and transfer small pieces of healthy myc
to new plates, and leave the contam behind. once you have clean growth on a plate
you cut a wedge and put it to grains.

if you just did spore syringe -> grain you dont get the chance to remove the contaminants.


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Re: Spore solution on agar [Re: h3aling]
    #22193448 - 09/05/15 04:28 PM (8 years, 4 months ago)

Quote:

h3aling said:
1) I also tried direct spores from a print to agar but they never even germinated.
With a chill mind I would say that dry spores on dry agar don't have much hope though.

2) Another thing, in which way would a cotton swab help me achieve germination more than directly dripping spore solution on the agar? It seems like an unnecessary step which might introduce complexity (more chances to contam).




1) dry spores to agar is what most of us do, you can use less agar powder in the recipe for a more watery agar which helps spore germination.
but the agar itself has more than enough water to let spores germinate.
how long did you wait before you concluded nothing grew?

2) you could do it, PC a q-tip in a jar or wrapped in foil, like this


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Re: Spore solution on agar [Re: spacechildo]
    #22193890 - 09/05/15 06:15 PM (8 years, 4 months ago)

If you cant get a swab you can flame sterilize  loop instead.

The reason it helps is, spores in solution have excess water.. one drop will slosh around on the plate and you will have groeth all over the place, potentially making it hard to pick out clean germination to isolate.

If you swipe the spores on it will hopefully germinate alomg the general line it was swiped on, should be easy to see whats clean myc to transfer vs a fluffy mess thst might be growing over mold colonies or tricky bacteria.

I use a sterile swab out of a single use wrapper or a looop, but you cn sterilize q tips in PC cycle wrapped in foil or in jar with GE lid


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Re: Spore solution on agar [Re: mushpunx]
    #22194421 - 09/05/15 08:31 PM (8 years, 4 months ago)

Lol higher potency strains from rhizomorphic growth. :rofl:

How are you such a noob if you've been here since 07 LC?


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Re: Spore solution on agar [Re: Inocuole]
    #22195929 - 09/06/15 07:08 AM (8 years, 4 months ago)

Wow so many answers, thank all of you.

The fact that you recognize it as mycelium just brightened my day :smile:

I have more plates sitting in the fridge so I could give it another go with the Q-tip method, sounds smart after reading your arguments.

@spacechildo
I just scraped spores on the agar in as sterile as possible conditions, and that was back in May. At the end of June there was nothing showing yet, neither good nor bad, so I assumed it was just screwed.

Again, I also tried putting a live pin on agar at the same time, and all that happened is the pin dried out and died, and no growth/contam whatsoever ever showed up. I think I still have that plate lying around somewhere.

So you would advise keeping this (in OP) plate and trying to isolate good mycelial growth from it?


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Re: Spore solution on agar [Re: h3aling]
    #22212585 - 09/09/15 02:47 PM (8 years, 4 months ago)

shameless
self
bump


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Re: Spore solution on agar [Re: h3aling]
    #22212643 - 09/09/15 03:01 PM (8 years, 4 months ago)

Do both! Why not?

Make up a new batch of plates, put some more spore solution (one drop no more) on some, scrape spores with a loop on a couple, and take a flamed scalpel scrape a bit of the healthy myc onto it and transfer it to a few more.

The transferred myc should show growth more quickly than the others, but if it ends up completely contammed you'll have the others to fall back on.


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Re: Spore solution on agar [Re: Inocuole]
    #22212802 - 09/09/15 03:32 PM (8 years, 4 months ago)

Quote:

Inocuole said:
Lol higher potency strains from rhizomorphic growth. :rofl:

How are you such a noob if you've been here since 07 LC?




Ok, prove me wrong.

Show me a proven potent strain of mycelium that has fluffy, wispy, cotton-ish mycelium.


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Re: Spore solution on agar [Re: LogicaL Chaos] * 1
    #22212816 - 09/09/15 03:35 PM (8 years, 4 months ago)

Quote:

LogicaL Chaos said:
Quote:

Inocuole said:
Lol higher potency strains from rhizomorphic growth. :rofl:

How are you such a noob if you've been here since 07 LC?




Ok, prove me wrong.

Show me a proven potent strain of mycelium that has fluffy, wispy, cotton-ish mycelium.




pans
the growth has fuck all to do with potency it has to do with nutrition, it can go from tomentose to rhizzo with a simple recipe change


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Re: Spore solution on agar [Re: cronicr]
    #22212862 - 09/09/15 03:46 PM (8 years, 4 months ago)

Interesting...

So the food is what determines the type of mycelium growth, not the aggressive/fitness of the strain.

i was under the impression that aggressive or fit strains growth thick rhizzo mycelium which are the most potent (because they are most aggressive at colonizing a substrate).


Thanks for the explaination cronicr.


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Re: Spore solution on agar [Re: LogicaL Chaos]
    #22212868 - 09/09/15 03:47 PM (8 years, 4 months ago)

yep more nutes will make it go tomentose while less will make it go rhizzo:super:


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Re: Spore solution on agar [Re: cronicr]
    #22213228 - 09/09/15 05:02 PM (8 years, 4 months ago)



See this tub? I lost the photo of the culture plate but it was veryy aggressive, awesome looking culture. Looked freaking amazing. And the tub grew out pretty strong too.

But not potent. Pretty dissapointing actually haha.

JUst goes to show you that you need to test each and every culture out, regardless of how it looks. You might have a super fast growing isolate that puts out a crazy yeild but if it isnt potent its pretty worthless IMO


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Re: Spore solution on agar [Re: mushpunx]
    #22216523 - 09/10/15 11:37 AM (8 years, 4 months ago)

Heyo again,
I'm glad my thread helped you guys clear doubts, the more you know.


So I'm back with some (hopefully decent but of smartphone-shitty quality) of the incriminated plate.
Still, I don't see any of the radial patterns of mycelium seen in the other threads/pictures, but as it seems this one's totmentose rather than rhizomorphic so that's the reason, is that right?

Here are the pictures:


[I realize the camera picks up the reflection/glare of the plate and the focus on the agar is gone.]

I also come with another question.
Is it possible tha mycelium appears as a film inside a jar of liquid culture?
I suspect that is just a film of honey which solidified on the walls of the jar while I was PCing, as it slid off as I was gently swirling the jar and is now floating around happily. But if it was honey, would it not be dissolved?
I'd appreciate any input.


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Edited by h3aling (09/10/15 11:39 AM)


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Re: Spore solution on agar [Re: h3aling]
    #22216554 - 09/10/15 11:45 AM (8 years, 4 months ago)

That looks very contaminated to me..


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Re: Spore solution on agar [Re: Mad Season] * 1
    #22216620 - 09/10/15 12:05 PM (8 years, 4 months ago)

Quote:

Mad Season said:
That looks very contaminated to me..



Fuck.
You mean the white fluff is not mycelium or that the growth pattern is weird?

I came back to post this that I found googling around

The top picture looks similar to my plate.
Still, I have no real experience with agar so I might be very wrong.


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Re: Spore solution on agar [Re: h3aling]
    #22216631 - 09/10/15 12:08 PM (8 years, 4 months ago)

Just all of it lol. The white growths might actually be the mycelium. Just the reaction to the (what looks like) grey shit all around it.


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Re: Spore solution on agar [Re: Mad Season]
    #22216678 - 09/10/15 12:23 PM (8 years, 4 months ago)

Quote:

Mad Season said:
Just all of it lol. The white growths might actually be the mycelium. Just the reaction to the (what looks like) grey shit all around it.



The grey shit is yellow agar but the pics came out shitty as always. You can see for reference the OP.


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Re: Spore solution on agar [Re: h3aling]
    #22216707 - 09/10/15 12:30 PM (8 years, 4 months ago)

You need to read through this: http://www.shroomery.org/forums/showflat.php/Number/22020260

the moral is that if it isn't organized, don't trust it. Honestly I wasn't there the whole time that grew, so I can't say for sure. But if it was with my other dishes right now, it wouldn't be for long.

For initial transfers, don't let it grow out too much.

like at this point. If not even sooner.


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Edited by Mad Season (09/10/15 12:30 PM)


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Re: Spore solution on agar [Re: Mad Season]
    #22216932 - 09/10/15 01:33 PM (8 years, 4 months ago)

That a good thread, thank you.
So your suggestion is I transfer the mycelium to a new plate?
It does not look like contaminated anymore?


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Re: Spore solution on agar [Re: h3aling]
    #22217138 - 09/10/15 02:28 PM (8 years, 4 months ago)

Try it out. If you can get it organized, then :thumbup:. I've seen crazier things done with agar.

Besides it almost looks like there's knotting. If you get a pin you most definitely should cut it out and transfer the pin


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