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Myco-pathic
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Ms agar question
#27431950 - 08/17/21 04:34 PM (2 years, 8 months ago) |
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Hello and long time no see. I’m returning experienced cultivator but I don’t have a lot of experience on agar. I’ve been able to find most of all the questions I could ask with the search tool but getting frustrated at this point when I have a small minute question that is so specific I cannot find the answer.
I have a clean room (lab) flow hood with a Hepa filter also the room is positively pressured with a secondary Hepa
I just thought I would mention that so we can get a lot of the sterile technique blah blah blah out of the way.
I’m not going to mention the vendors name but I was getting syringes always leading to Trichoderma. I used the same vendor because I understand the syringe might be the issue and now I’m using spore print.
Trichoderma often fools me, it’s not green until it sporulates. But I have noticed that it might form very tiny knots.
On my multi sport plates I noticed growth on day three. Everywhere I’ve noticed the spores had landed I have a very fine my cellular growth very soft almost looks like a gel. On day four close to the site where the spores landed I’m seeing those knots. I figure I have a 50-50 chance it’s healthy mycelium or Trichoderma and I will attempt to isolate away from it.
I would like to know if anybody who does this regularly can let me know if MS plates often knot so soon or if it’s a tale sign of trich. (Pic 1 Below)
Edited by Myco-pathic (08/17/21 05:02 PM)
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Myco-pathic
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Here they are!

Picture above has the knots in question. Hard to see I understand but Hold CNTRL and mouse wheel up to zoom in more.

Above is what appears to be normal.

Underside of plate.
Edited by Myco-pathic (08/17/21 05:05 PM)
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Myco-pathic
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24 hour update. I’m just not thinking that’s the mycelium I was looking for. Might pull a transfer of one of the softer colored sites.
I 
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Myco-pathic
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So I decided to open one up under the hood and take a closer look. To me it appears that clumps of multiple spores landed and slightly submerged in the agar growing slightly beneath the top layer and the explosive white growth I might just be finally exiting.
Would love some input
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The Mycologist
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I dont like much if any of the growth on those plates
-------------------- "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

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Myco-pathic
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Keep in mind this is a multispore plate not a isolate.
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c10h12n2o
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None of that looks good
I would recommend streaking a plate using the technique in my sig and agar guide using as little liquid as possible, then youll probably be able to differentiate the colonies well enough to get some clean cultures
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  C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide "Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing." "Convictions are more dangerous enemies of truth than lies" ― Friedrich Nietzsche
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27434154 - 08/19/21 08:07 AM (2 years, 8 months ago) |
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I just checked him this morning everything is bright fluffy white my concern about Trichoderma is behind me.
I did not do the streaking technique but I am familiar with it I will update some pictures today its looking much better.
I did use as a little liquid as possible I poor it very slowly until there is still gaps on the plate and then drop by drop until the liquid Connects.
I’m still not sure how the spores submerged in the agar but that is what happened and that’s why the growth was throwing me off it was literally growing beneath the agar top layer and then bursting out resembling Trichoderma before sporulation.
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Myco-pathic
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Not all colonies have Breached the surface yet but these were done on the 12th if it was Trichoderma I would’ve seen its ugly face by now.
On a sidenote a lot of the texture in the agar is light malt extract. From what I have read it’s normal to have that texture, I did my best to avoid it I dissolved the entire solution on a heated stir plate for at least half an hour before I sterilized.

Also I have no intention on differentiating colonies at this moment I just want clean culture for Grain jar I will begin isolations once I have a fruit body to sample from.
Edited by Myco-pathic (08/19/21 08:24 AM)
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PBJ710
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Thats a bacterial mess from using too much MSS. c10h12n2o is giving you good advice - streak the plate to isolate the mycelium from everything else. Then you won't have to make 6 transfers to *hopefully* get rid of the crap that's riding with it.
How is this advanced mycology?
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Myco-pathic
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Re: Ms agar question [Re: PBJ710]
#27434187 - 08/19/21 08:30 AM (2 years, 8 months ago) |
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I’m going to respectfully disagree with you there. With my microscope I can clearly see what growth is underneath the agar.
As far as advanced mycology goes no this is probably not advanced but I did not see a section labeled mycology.
This doesn’t quite fit mushroom cultivation either because I am not cultivating mushrooms at this point in time and I have no questions on how many holes I need an a Mano tub.
I did however assume there were mycologist here that could answer a mycology question. 🤷🏻
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The Mycologist
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There probably is mycelium in there, but its all about the ease of cleaning.
Streaking and taking from a big leading edge of growth is going to be way easier to get cleaned up then trying to perform microscopy with what is usable on the plate.
Check out these links
https://www.shroomery.org/forums/showflat.php/Number/22721954
https://www.shroomery.org/forums/showflat.php/Number/24806569
-------------------- "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

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Myco-pathic
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As you can see more clearly as time goes by this is not a bacterial infection I’m not able to post pictures with my scope under 40x but what I was able to see is this was just my Mycelium growing underneath agar and what look like Trichoderma was just the mycelium making a breach.
This is day seven and no signs of bacterial colors or a Trichoderma sporulation just nice white culture.
I totally agree with you using that technique would be beneficial but I’m only hoping to be dealing with a multispore just this once below I will post pictures of the original plate and the transfer.
Less than 24 hours after transfer the mycelium has already climbs off of the transfer wedge onto the new plate, very happy with this growth.


Hope this was helpful to anybody who shared my concerns with your own agar plates just be patient and see how it goes.
As far as cleanup and isolation I stated above that was not some thing I was looking to do with these transfers I actually would like to fruit this as a multispore and begin isolation after I’ve identified a strong fruit body. I will take a biopsy and begin isolations from that point. I don’t see the use in isolating from multispore Because that would require so many plates and so much time for something that might not being isolate I was looking for. I would hate to end up isolating the weakest shortest runt of the bunch and that is very possible.
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Myco-pathic
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c10h12n2o
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Ehhh, believe what you want man but those colonies are not axenic, there is definitely more than 1 organism present. There are very obviously multiple things growing in those colonies, intermingled, and one is outpacing the other. Clean cube myc is not slimy like that
Quote:
Myco-pathic said: I’m going to respectfully disagree with you there. With my microscope I can clearly see what growth is underneath the agar.
40x is nowhere near enough magnification to see bacteria. Clean cube myc will not grow underneath agar, it will grow across the surface
This type of comingling of organisms is very common in spore syringes, since spores are never sterile, since mushrooms are typically not fruited in sterile environments
We are not talking about isolating single strains, we are talking about isolating clean axenic cube myc from bacteria and mold mycelium by doing a serial streak
Bacterial spawn usually still fruits but clean cultures do much better. Even cultures with some trich intermingled can still get a flush
Run those if you want, but i highly recommend starting fresh w a streak and working towards a clean axenic culture while you are playing with those
Btw this is the kind of question you should post in the Mushroom Cultivation forum not Advanced Mycology
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  C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide "Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing." "Convictions are more dangerous enemies of truth than lies" ― Friedrich Nietzsche
Edited by c10h12n2o (08/20/21 09:07 PM)
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27437028 - 08/21/21 08:50 AM (2 years, 8 months ago) |
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Day 8 still nothing like what you are saying. Even more and more white. I’m an expert Trichoderma farmer, trust me if it was there I’d see it by now. I’m keeping the original MS plate and I will keep this post updated on how it looks as it goes but I’ve already moved onto my T1. I posted a picture above but I will post it here again.
This is 24 hours old I haven’t been to the lab yet.

40 is more than enough to see mycelium. I don’t need to see the bacteria because it would’ve outpaced all growth on this plate And from my experience Trichoderma would have too. Day 8 most all the slimy growth has breached pure white.
I made four more plates. T1 All from my best performer.
Will update pictures today so we can grow together. Also this was a print. Not syringe.
Edited by Myco-pathic (08/21/21 08:51 AM)
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Myco-pathic
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T1’s looking good!



LME LC made with agar wedge today, 1.5G LME/1500ml


Unrelated Lc contamination test (clean)

Coworker busted the lids learning how to use parafilm 😂 It’s ok 👍🏻
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Myco-pathic
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Here is the MS-GT plate #1 All t1’s cut from this plate. Still not a bacterial mess… Patch on the bottom right what the entire plate looked like At the start until the mycelium started to breach. That patch should breach soon. Just keeping it for study but it’s spent as far as my needs go.

The granular texture seen is LME that didn’t dissolve. I think the 10g/10g LME/MEA recipe was a bit heavy for 600 ml.
Adding Nutritional yeast for future plates. 10-MEA 2.5-YE 7.5LME @20 plates
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The Mycologist
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The T1s that you think look good are bacterial. The cube mycelium is thick because its stressed and the slime on the edge of it isnt supposed to be there.
-------------------- "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

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PBJ710
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That hazy growth around your cultures es no bueno.
Are you actaully using 10g MEA (along with 7.5 LME and 2.5 YE) or was that a typo and MEA was supposed to be agar?
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