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Offlinejbogart
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Registered: 10/01/22
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Last seen: 2 years, 22 days
Spore germination at 100X magnification * 2
    #27978568 - 10/02/22 07:24 PM (2 years, 3 months ago)

Hey all, my first post on the Shroomery!

I'm new to growing - one flush from a PF tek cake 2 years ago. But I do have a background in labwork and still have access to the facilities.

I am trying to ressurect these cube spores from this grow 2 years ago by plating on MEA. Let me be clear, these spores have been stored POORLY. Although they were only opened in sanitary conditions, they have remained folded in a foil square, unsealed, taped to a box in my garage this whole time.

EDIT: I should have added, the plates were inoculated in a flow hood onto autclaved substrate, so any contamination is 99% likely from the spore print.

So I have three plates at 5 days old. They are all covered in teeny brown specks that look like colonies. I put them under the microscope and found what appears to be two different fungi germinating.

First, what appears to be a clean spec of mycelium (~15 per plate):



Secondly, and more abundantly, the brown spots (~200 per plate):


These brown spots, I'm not sure what to make of them. They could just be mold. I also wonder if they could be the clumps of spores that were visible in the inoculum, and now I'm just looking at a ball of hyphal growth. That would explain why there are so many of these. But then again, I don't see why they would all be the same size and perfectly round! That makes them seem like a contaminant.

Anyways, I thought this might be interesting to share since I couldn't find too many images of germinating spores. I will let them grow, do some transfers and isolate whatever these things are. Then I'll come back here and let you all know what's up :smile:

And of course if anyone can chip in and put me out of my misery now that would be great!

JB

Edited by jbogart (10/03/22 09:48 PM)

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OfflineKROM
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Re: Spore germination at 100X magnification [Re: jbogart] * 1
    #27978577 - 10/02/22 07:29 PM (2 years, 3 months ago)



--------------------
πŸ…ƒ πŸ„΄ πŸ„° πŸ„Ό    πŸ„² πŸ„» πŸ„Έ πŸ„½ πŸ„Ά πŸ…† πŸ… πŸ„° πŸ„Ώ

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Invisiblesandman420
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Registered: 06/17/04
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Re: Spore germination at 100X magnification [Re: KROM]
    #27978630 - 10/02/22 08:21 PM (2 years, 3 months ago)



is it perhaps germinated chlamydospore of Hypomyces pernicious or similar?


--------------------
- Sandbag Tek - How To Sterilize Spawn Bags - All About Static Pressure / Pressure Drop for DIY Flow Hoods - Sandman's LC Tek-

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Offlinejbogart
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Registered: 10/01/22
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Re: Spore germination at 100X magnification [Re: jbogart]
    #27978649 - 10/02/22 08:31 PM (2 years, 3 months ago)

Hmm... I don't think so. Based on the images I just looked at those spores are not much larger than the hyphae, but if you look at the third image you can see the hyphae growing out of one of these clumps for scale. The clumps are 50-100x the width of the hyphae and are visible with the naked eye!

If the chlamydospores are larger than I thought then it could be the case.

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OfflineSymPlayTon
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Re: Spore germination at 100X magnification [Re: jbogart]
    #27978738 - 10/02/22 09:06 PM (2 years, 3 months ago)

Interesting.  I need a scope.

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OfflineRusty2096
rah rah raw in Lady gaga
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Registered: 08/23/22
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Re: Spore germination at 100X magnification [Re: SymPlayTon]
    #27978744 - 10/02/22 09:09 PM (2 years, 3 months ago)

Quote:

SymPlayTon said:
Interesting.  I need a scope.




Why am I not surprised you said that? If you can afford it, I say go for it : you seem the be digging the hobby a lot and it would open a whole new world to you.


--------------------
Currently looking for nothing. You guys who sent me stuff are straight up awesome!. :mushroom2:

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Offlineschpat
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Re: Spore germination at 100X magnification [Re: Rusty2096]
    #27978928 - 10/03/22 01:00 AM (2 years, 3 months ago)

@jbogart, what magnification are these taken at?

Edit: Never mind, I just read the title, what a dumbass I am.  :smile:


--------------------

Edited by schpat (10/03/22 01:08 AM)

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OfflineSymPlayTon
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Re: Spore germination at 100X magnification [Re: Rusty2096]
    #27979065 - 10/03/22 06:52 AM (2 years, 3 months ago)

Quote:

Rusty2096 said:
Quote:

SymPlayTon said:
Interesting.  I need a scope.




Why am I not surprised you said that? If you can afford it, I say go for it : you seem the be digging the hobby a lot and it would open a whole new world to you.





I am very much enjoying the hobby.  I have been looking for information on mycology, and have found more articles on how there are almost no professional mycologists, and universities won't fund mycology research than information on mycology.

I have been looking at microscopes thinking about getting one, but the picture on this thread locked in that want to get one lol.

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Offlinejbogart
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Registered: 10/01/22
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Re: Spore germination at 100X magnification [Re: schpat]
    #27979602 - 10/03/22 01:48 PM (2 years, 3 months ago)

Quote:

schpat said:
@jbogart, what magnification are these taken at?

Edit: Never mind, I just read the title, what a dumbass I am.  :smile:




Not so much, I should have been clearer! The second picture is actually 40X to show how consistent the brown spots are. I will have to figure out how to add a scale in future, like in sandman420's image. That would have been helpful here :grin:

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Offlinejbogart
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Re: Spore germination at 100X magnification [Re: SymPlayTon]
    #27979615 - 10/03/22 01:56 PM (2 years, 3 months ago)

Quote:

SymPlayTon said:

I have been looking at microscopes thinking about getting one, but the picture on this thread locked in that want to get one lol.




Go for it! If you're lucky you might find a second hand one but they're cheaper than I thought they'd be. Handy for contaminant issues, at least :smile:

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OfflineTheUsualSuspect
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Re: Spore germination at 100X magnification [Re: jbogart]
    #27979771 - 10/03/22 04:25 PM (2 years, 3 months ago)

This may be a dumb question but given how many people are on the board its a maybe...

anyone ever gone from glass microscope slide sample, transfer to agar to grain bag?

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Offlinejbogart
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Registered: 10/01/22
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Last seen: 2 years, 22 days
Re: Spore germination at 100X magnification [Re: TheUsualSuspect]
    #27980443 - 10/03/22 11:08 PM (2 years, 3 months ago)

It occurred to me while transferring today, that the mycelial colonies probably originate from single spores and are therefore monokaryotic. So when I transfer, I actually need to make two transfers of these teeny colonies and hope that I get a +/- pair that can mate!

Weird fungi...

So... I transferred 4 pairs onto new plates, assuming this gives me a 15/16 chance that I'd get a successful mate (and also assuming these are even cubensis). I guess this is not something that growers are usually concerned with as we tend to make transfers from dikaryotic mycelium, after the 'mate' has occurred (please correct me if wrong, I'm still learning the science!).

Anyway... the green circles here show (probable) mycelial colonies and the red circle shows the vastly more abundant mystery blobs. The left plate is the original and the right is the transfer:



I also collected one plate with a single brown blob so we can find out what that eventually turns into. Probably something gross.

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InvisibleGuerrilla
Original Asshole


Registered: 01/30/21
Posts: 3,179
Loc: United Kingdom Flag
Re: Spore germination at 100X magnification [Re: jbogart]
    #27980555 - 10/04/22 02:21 AM (2 years, 3 months ago)

Highly unlikely you have monokaryons unless serial dilution was involved


--------------------
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Offlinejbogart
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Registered: 10/01/22
Posts: 18
Last seen: 2 years, 22 days
Re: Spore germination at 100X magnification [Re: Guerrilla]
    #27980612 - 10/04/22 04:43 AM (2 years, 3 months ago)

@Guerrilla No serial dilution, there are hundreds of thousands of spores visible on the plate for sure (a single drop of inoculum, evenly spread). But 99.9% of them haven't germinated yet as far as I can see.

Is a monokaryon physically distinctive in any way, other than the single nucleus? Might I find one if I zoom in to look at the spores more closely?

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OfflineSymPlayTon
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Registered: 09/03/22
Posts: 420 420 Posts!
Loc: In a state of confusion
Last seen: 1 year, 3 months
Re: Spore germination at 100X magnification [Re: jbogart]
    #27981167 - 10/04/22 01:02 PM (2 years, 3 months ago)

Quote:

jbogart said:
It occurred to me while transferring today, that the mycelial colonies probably originate from single spores and are therefore monokaryotic. So when I transfer, I actually need to make two transfers of these teeny colonies and hope that I get a +/- pair that can mate!

Weird fungi...

So... I transferred 4 pairs onto new plates, assuming this gives me a 15/16 chance that I'd get a successful mate (and also assuming these are even cubensis). I guess this is not something that growers are usually concerned with as we tend to make transfers from dikaryotic mycelium, after the 'mate' has occurred (please correct me if wrong, I'm still learning the science!).

Anyway... the green circles here show (probable) mycelial colonies and the red circle shows the vastly more abundant mystery blobs. The left plate is the original and the right is the transfer:



I also collected one plate with a single brown blob so we can find out what that eventually turns into. Probably something gross.





This is exactly where my current research is. 

I am also questioning if senescence is related to the heavily isolated nature of our "lab" cultures, and not a problem if we regularly "mate" high functioning samples to refresh the DNA pool. 

I also haven't seen any examples of senescence, but lots of conversations about the evils of it. 

At what extent does that impact cubes and how do we track the prevalence of that phenomenon.

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Offlinejbogart
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Registered: 10/01/22
Posts: 18
Last seen: 2 years, 22 days
Re: Spore germination at 100X magnification [Re: SymPlayTon]
    #27981394 - 10/04/22 03:14 PM (2 years, 3 months ago)

Quote:

SymPlayTon said:
This is exactly where my current research is. 

I am also questioning if senescence is related to the heavily isolated nature of our "lab" cultures, and not a problem if we regularly "mate" high functioning samples to refresh the DNA pool. 

I also haven't seen any examples of senescence, but lots of conversations about the evils of it. 

At what extent does that impact cubes and how do we track the prevalence of that phenomenon.




How funny, this seems like a response to a different post I made last night.

I was wondering about senescence in relation to the biggest-organism-on-earth super mycelium in Oregon. I think it's an issue in the lab/grow because we clone an individual over and over again, without the sporulation process (whatever that is) to re-set the DNA.

The post I linked above shares a process to select your desired traits by switching between clone/spore grows while selecting the mushroom of choice. Quite neat, the genetics aspect is very interesting.

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OfflineSymPlayTon
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Registered: 09/03/22
Posts: 420 420 Posts!
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Last seen: 1 year, 3 months
Re: Spore germination at 100X magnification [Re: jbogart]
    #27981441 - 10/04/22 03:34 PM (2 years, 3 months ago)

Quote:

jbogart said:
Quote:

SymPlayTon said:
This is exactly where my current research is. 

I am also questioning if senescence is related to the heavily isolated nature of our "lab" cultures, and not a problem if we regularly "mate" high functioning samples to refresh the DNA pool. 

I also haven't seen any examples of senescence, but lots of conversations about the evils of it. 

At what extent does that impact cubes and how do we track the prevalence of that phenomenon.




How funny, this seems like a response to a different post I made last night.

I was wondering about senescence in relation to the biggest-organism-on-earth super mycelium in Oregon. I think it's an issue in the lab/grow because we clone an individual over and over again, without the sporulation process (whatever that is) to re-set the DNA.

The post I linked above shares a process to select your desired traits by switching between clone/spore grows while selecting the mushroom of choice. Quite neat, the genetics aspect is very interesting.





I think that is why mycology is so iimportant. 

I will have to find the articles, but mycelium naturally overcome senescence in the wild with the spore cycle and the "mating" process.  When a mycelium network "mates" that DNA is shared through the network in certain groups of fungi. 

So a cell with old DNA can get new DNA through a mating cycle because it will replicate it through the entire organism. 

In the wild it maintains the fruiting genetics because only the fruiting genetics make spores to replicate future generations. 

We attempt to force that and forget to add new genetics over time.

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OfflineSymPlayTon
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Registered: 09/03/22
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Re: Spore germination at 100X magnification [Re: SymPlayTon]
    #27981451 - 10/04/22 03:37 PM (2 years, 3 months ago)

I had another thought right after posting that last one.

I think that is why PF tek cakes are so successful, and why longer "consolodation" cycles on them produce better fruiting conditions. 

You get more genetic diversity, and allow the kycelium to form a single organism from numerous spores.  The consolidation time allows the DNA replication to pass through the entire organism, and maximize nutrition sharing, and maximize distribution of fruiting DNA.

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Offlinejbogart
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Registered: 10/01/22
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Re: Spore germination at 100X magnification [Re: SymPlayTon]
    #27982195 - 10/05/22 01:05 AM (2 years, 3 months ago)

Quote:

SymPlayTon said:
I think that is why mycology is so iimportant. 

I will have to find the articles, but mycelium naturally overcome senescence in the wild with the spore cycle and the "mating" process.  When a mycelium network "mates" that DNA is shared through the network in certain groups of fungi. 

So a cell with old DNA can get new DNA through a mating cycle because it will replicate it through the entire organism. 

In the wild it maintains the fruiting genetics because only the fruiting genetics make spores to replicate future generations. 





That's crazy, I can't believe that wasn't featured in "Fantastic fungi" :laugh: I found an old Nature paper that describes it pretty well (free PDF download).

Crazy. Can you imagine if humans just merged with their partners to form a mega-human? :laugh: Thanks for the info @SymPlayTon!

EDIT: This is still confusing my brain :wink: In the lab we go through the spore/clone cycle but to introduce new genetics we would want our monokaryon to mate with a new monokaryon (rather than one from the same spore print). Which should happen regularly in the wild, cause the spores get mixed up in the wind. I think I'm starting to make sense of it?

Edited by jbogart (10/05/22 01:14 AM)

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OfflineRusty2096
rah rah raw in Lady gaga
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Registered: 08/23/22
Posts: 4,946
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Re: Spore germination at 100X magnification [Re: jbogart]
    #27982299 - 10/05/22 05:54 AM (2 years, 3 months ago)

Quote:

jbogart said:
Quote:

SymPlayTon said:
I think that is why mycology is so iimportant. 

I will have to find the articles, but mycelium naturally overcome senescence in the wild with the spore cycle and the "mating" process.  When a mycelium network "mates" that DNA is shared through the network in certain groups of fungi. 

So a cell with old DNA can get new DNA through a mating cycle because it will replicate it through the entire organism. 

In the wild it maintains the fruiting genetics because only the fruiting genetics make spores to replicate future generations. 





That's crazy, I can't believe that wasn't featured in "Fantastic fungi" :laugh: I found an old Nature paper that describes it pretty well (free PDF download).

Crazy. Can you imagine if humans just merged with their partners to form a mega-human? :laugh: Thanks for the info @SymPlayTon!

EDIT: This is still confusing my brain :wink: In the lab we go through the spore/clone cycle but to introduce new genetics we would want our monokaryon to mate with a new monokaryon (rather than one from the same spore print). Which should happen regularly in the wild, cause the spores get mixed up in the wind. I think I'm starting to make sense of it?




I don't think you can call that to overcome senescence. I see it more like humans having babies. The parent don't get any younger and still die to senescence (or McHeartAttacks)


--------------------
Currently looking for nothing. You guys who sent me stuff are straight up awesome!. :mushroom2:

We don't own things - things own us.

Semi-solid liquid culture (SSLC)

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