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InvisibleMandalaMind
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Where should i transfer from? - Enoki
    #26559251 - 03/26/20 02:08 PM (3 years, 9 months ago)

I have a hard time to choose where to transfer from as the mycelium seems to grow  in 2 different ways, one fluffy and more aerial and other more dotted and compact to say, you'll see what i mean. Both looked the same in early stage.

I already made 2 transfers from the orginal petri.
Here is the original petri 7 days after the first transfer:

From here i made 2 transfers, T1 and T2.
Here are T1 and T2 7 days after the transfer:


They grew differently, on T2 i think it spread like that from the condensation/water on agar.
T1 grew more like T2 after transfer but it kept the aerial fluffynes on top.
T2 grew dotted and spotted but it kept some parts of aerial mycelium tho.
Both of them became yellow-brownish after some time which i've read that is normal for enoki mycelium as it ages.

Here are the transfers today, 13 days after the transfer:

T1:

T1 in light:

T2:

T2 in light:


I want to isolate further these days but i am not sure where to transfer from. It's my first enoki grow and my first grow ever too so i am not familiar with how enoki mycelium should look like.
There seems to be 2 kind of mycelium growing. The aerial one is white maybe yellowish, it doesn't look gray or dark so i don't know if it is cobweb or not. They may be the same mycelium just growing a bit different because of different conditions on plates and is confusing me.

So, where should i make the transfer from?


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OfflineForresterM
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Re: Where should i transfer from? - Enoki [Re: MandalaMind]
    #26559708 - 03/26/20 05:48 PM (3 years, 9 months ago)

Wait, you're starting enoki from spores? 

Just need to clarify.  It's not all that common folks would have a reason to do that...


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InvisibleJawn876
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Re: Where should i transfer from? - Enoki [Re: Forrester] * 1
    #26560185 - 03/26/20 10:33 PM (3 years, 9 months ago)

In my experience its easier to get a wild culture of Flammulina velutipes through spores than tissue culture but that might just have been me. The mycelium does look a little different than an oyster or something but I think you may have some contamination there.
heres a cool video to compare your mycelium with:

If you have happen to have a microscope, you can take a piece of the mycelium to a slide and look for clamp connections. Clamp connections are unique to basidiomycetes so it can help distinguish target species


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OfflineForresterM
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Re: Where should i transfer from? - Enoki [Re: Jawn876]
    #26560655 - 03/27/20 07:26 AM (3 years, 9 months ago)

Quote:

Jawn876 said:
In my experience its easier to get a wild culture of Flammulina velutipes through spores than tissue culture but that might just have been me.




No that makes sense, I wasn't saying it wasn't a good idea - just confirming that the OP was in fact starting from spore. 

A lot of times folks come in here from the cube world and ask about isolating all this stuff from, in the end, what turns out to be an already isolated culture.  So I always ask :lol:


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Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleMandalaMind
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Re: Where should i transfer from? - Enoki [Re: Forrester]
    #26560838 - 03/27/20 09:52 AM (3 years, 9 months ago)

Started from spores because i found them in the wild and i spore printed them. I tried to clone one specimen once but it didn't work out well, fast forward some weeks now i have petri dishes and only spores.

Well i do have a microscope i just found my old one from my childhood.
Here are some photos of the aerial fluffy mycelium i'm talking about(i think it's the fluffy one in this pics)(also the mycelium is some days old between the slides in these photos, it looked better fresh):
-x100 magnification:

-x450 magnification:

-x900 magnification(i know it's blurry i couldn't make it any better i think it's the lens):


I watched that video but i am still confused because in early stage on original plate it began exactly like that, then at T1 it began to grow aerial and fluffy, much like in the video and on T2(from original) half grew fluffy and half of it began to grow dotted like is seen in T2 plate.
That's why i isolated both kind of mycelium to see if they are indeed different.
Again, at the beginning they looked similar, on T1 after 13 days is seen that in the middle it grew more like T2( with that dusted compact white appearance) but on the edge it is fluffy and cloudy.
T2 grew exactly opposite, majority dusty white compact and some spots of fluffy cloudy.

I am a noob in this and i don't know which one is the one i am looking for.
Another observation i made is that in T1 in light it's seen that the middle is becoming yellow/brown. Now in the middle is the growth of the dusty type. I've read that enoki mycelium browns as it ages(as also found in the wild where the stem goes into the wood and is yellowish). On T2 some parts are becoming yellow(slowly than T1 tho) and the yellowing seems to follow the dusty type of mycelium.
The fluffy type doesn't seem to yellow.

Right now as i type this i realize that if i put light over T1 the fluffy type looks very much like spider web.
My assumption is that T2 is the right one and on T1 the fluffy one is some sort of contamination, maybe cobweb mold, that is growing on top of the enoki mycelium.
I may have just answered my own question.
I'll look for clamp connections tho but i think my x900 is too blurry to see that clearly. That's a neat trick i can learn if you say it's specific to basidiomycetes, thx for the tip!

I'll transfer further from the zones that are yellowing on T2 and see where that goes.


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InvisibleMandalaMind
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Re: Where should i transfer from? - Enoki [Re: MandalaMind]
    #26560979 - 03/27/20 11:22 AM (3 years, 9 months ago)

I found some useful info that mostly back-up my assumption, i'll write it here so that anyone looking for the same problem as i have will have more info compacted:

Someone here on shroomery was concerned that his enoki mycelium(inoculated from an LC syringe) had a yeast contam. He asked if the enoki mycelium have almost powdery apperance several times and someone said that it looks like a frosty appearance and not so defined.
The mycelium on my T2 looks like it has that porperties.

On another thread someone said that Enokitake mycelium is always a bit cottony looking. T2 still have some cottony mycelium in some places, but it seems different than the one on T1. On T1 it spread faster, T2 almost stalled, on T2 it's only seen over some areas where the "powder" is more dense and is yellowing. Also on T1 it looks more stringish while on T2 it looks more powdery but actually they looks pretty much the same.

Now on "How it should look" thread it say that enoki mycelium looks very cobweb-like, that's normal for this species. In the attached photos there is one where it looks more like the fluffy type i have and nothing like the powder type.

I am again as confused as before.

If they are the same mycelium, why does it have different apperances? I don't understand. Right now i'm going to look for clump connections on both of them.

Still waiting for someone with enoki experience to help me here.


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InvisibleMandalaMind
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Re: Where should i transfer from? - Enoki [Re: MandalaMind]
    #26561355 - 03/27/20 03:04 PM (3 years, 9 months ago)

I'm back with the results.

I made 2 slides from the original petri dish(don't want to risk and contaminate the transfered plates for now), both with 1-3 samples from both types of mycelium.
The results were confusing at first but i think i figured it out.

Here is the first slide; all photos are made at x900 magnification:
First i checked the aerial/cotton type and it seems that it have clamp connections and the hyphae is septate:

Then i checked some dotted mycelium from the first slide. Here it gets weird, it seems to have a lot of little cells and very few to no hypahe:

Then i found some mycelium with clamp connections tho:

Here are more weirdness from second sample of dotted mycelium on the first slide:


Then i made the second slide, where i took one big sample of aerial mycelium and several samples of powder/dots/spots as isolated as possible.
Here is the aerial type with septate hyphae:

Here is supposed dotted mycelium:

I haven't seen any sign of hyphae on dotted mycelium samples on this slide, only little orbs forming those weird clumps.

At first i thought that those orbs were something from agar, and it is very possible but the fact that i barely find any hyphae makes me believe it is not mycelium at all.
I think the mycelium i found on the dotted samples are just aerial type mixed or that have been growing over the dotted type.

I conclude that the T2 transfer is not enoki mycelium but some type of contamination, maybe bacterial i really don't know enough to say. The real enoki mycelium is the one from T1 i believed is cobweb.

One more observation i made now is that the mycelium from the original petri dish seems to begin to "gather" and i think it's forming primordia(but i may be wrong). I put it in the fridge for 2 days to see if they begin to fruit on the petri dish and check if i have enoki mycelium at all.

I hope this helps future problems regarding enoki mycelium as i've seen that it isn't much documented here on shroomery(or i couldn't find it).


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InvisibleJawn876
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Re: Where should i transfer from? - Enoki [Re: MandalaMind] * 1
    #26563062 - 03/28/20 12:06 PM (3 years, 9 months ago)

@ Forrester haha so true!! I hope I didn't come off as tryin to be argumentative or anything, just sharing my experience. I agree it is important to clarify

@ MandalaMind, Cool! I always love microscopy photos. The T1 plate after 13 days looks a little more colorful than something I might look for. And where did you put the transfer wedge on the T2 plate? I would usually expect to see radial growth from the point of inoculation with enoki.
As for the microscopy, might be best to try and separate each species/culture onto its own slide.  All the dots in the microscopy images might be mold spores, but its hard to tell from the photos. I think it is possible that you still have more than one species on each plate. The clamp connections look like you might have your target species in there somewhere.
I personally don't think I would describe enoki mycelium as "powdery" or "aerial" but other folks might have different opinions on descriptions. For example, Enoki is certainly not as aerial and powdery as Rhizopus sp. or something similar. If your plates do pin that will help distinguish things a lot.
I dug out my wild enoki plates to compare with yours. They've seen better days for sure. It is turning a little brown but this plate is from January.


Here are some microscopy images. since the plate was old, the mycelium was rather dense/consolidated and difficult to get good samples from.
This is 400x

1000x


I transferred some wedges to a new plate and I can take photos as it grows if you want but I don't I will be able to get better documentation than that video. Cool project by the way. Hope this helps


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InvisibleMandalaMind
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Re: Where should i transfer from? - Enoki [Re: Jawn876]
    #26564046 - 03/28/20 07:40 PM (3 years, 9 months ago)

The wedge fell off(i intended to put in the middle) at 9:30 on the T2 after 7 days photo. It is the big white square in the fluffy mycelium near the center of the plate.

Yeah i used "aerial" because at first it grew in 3d/aerial for like half a centimeter or more and i found that weird. Now all the mycelium seems to be mostly leveled to the agar.

After i posted last night i looked again directly on the original dish with x450 magnification. I was able to see mycelium and clamp connection better than on x900 because anything was clearer and i didn't know that.

On the dots i was talking about there was indeed the same mycelium but there were clumps of a few hyphae with a lot of little cells i was talking about. Something is off about that so i transfered today from the T1 plate(5-6:00 on T1 after 13 days). I found a zone with mycelium looking very much like yours and the video posted above. I'll see how it grows on the new dish. I am pretty sure i isolated enoki mycelium this time.

Thank you very much, you really helped me.


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