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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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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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c10h12n2o
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Quote:
Myco-pathic said: 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
That plate definitely IS a bacterial mess. This is very basic stuff.
Im not gonna keep repeating if you arent gonna hear it but clean myc is not slimy, those are not axenic cultures, there is definitely more than 1 organism present
I have no idea what you are talking about when you say "breach" but that plate is 100% definitely bacterial (at least)
This us what a clean culyure looks like 
Notice the leading edge.
This culture is not super rhiz , and a culture doesnt need to be rhizo to be clean, but even a tomentose culture should be fairly well organized, have a clean leading edge, and not be slimy
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27439996 - 08/23/21 06:48 PM (2 years, 8 months ago) |
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Malt Extract Agar is used for the cultivation of fungi and is not intended for use in the diagnosis of disease or other conditions in humans. An acidic medium which will support the growth of most yeasts and molds whilst inhibiting most bacteria.
(MEA)
Mycologists: are you referring to the light grey around the transfer wedge or the texture in the media?
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27440004 - 08/23/21 06:57 PM (2 years, 8 months ago) |
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Comment on the t1 .. you Quoted the ms plate and I already said it’s spent as I’m done with it.
Something is defiantly strange with the agar mix. It’s very very soft like it almost didn’t completely solidify.
That’s what I mean when I say the mycelium is growing though the top layer and not fully on top.
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Myco-pathic
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As close as I could get without the camera freaking out.
Again ignore the undissolved LME.


That soft ring will follow the growth out. Has a stranded texture. I have plates with bacteria and it’s way different. I really think this agar is too soft.
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shthd
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Quote:
Myco-pathic said: 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
OMG that lil guy running is so CUTE
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c10h12n2o
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lol... dude we know what MEA is. You said:
Quote:
Myco-pathic said: 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
MEA = malt extract + agar + water (15g + 20g + 1L being pretty standard)
LME= light malt extract
MEA is the final product, agar powder and LME and water are the ingredients. this is why PBJ asked you if that was a typo. this could be what is "strange about the agar mix"
and you said:
Quote:
Myco-pathic said: 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.

obviously i understood that you are done with that plate, the part i was taking issue with is where you said that it is "not a bacterial mess." it absolutely is a bacterial mess
the t1s do not look good, because you transferred them from bacterial colonies. might still fruit because bacteria =/= guaranteed failure, they usually fruit anyway. but the bacteria makes it much more susceptible to other contams (which might already be present as well)
as i said before, roll the dice on these if you want, but you should serial streak a fresh plate and start working toward an actually clean culture which you can use with 100% success whether these work out or notQuote:
Myco-pathic said: As close as I could get without the camera freaking out.
Again ignore the undissolved LME.


That soft ring will follow the growth out. Has a stranded texture. I have plates with bacteria and it’s way different. I really think this agar is too soft.
like we keep telling you, that does not look good. clean myc (tomentose or rhizo) is not slimy like that, there are multiple organisms present there
bacteria by itself does not look the same as bacteria mixed with cube myc. same with trich: trich myc by itself does not look the same as trich myc mixed with cube myc.
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frikiriki
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No offense, but i agree with most comments saying this is not good growth. You might be wasting your time on something that is not healthy/is stressed.
The spore syringes in general are a no from me. The spores cannot set and grow well in liquid.
I use sterile spore swabs and transfer part of the cotton swab to the plate. The cotton is clean and boosts growth time. You can tell a lot more quickly if a dish is good or not.
(you can use sterile spore swabs in conjunction with plates, just swipe and go)
(Also good for swabbing gills for spores and starting a library of your own. That way you don't have to purchase from vendors. )
Nice set up though...quite envious!
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Myco-pathic
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Re: Ms agar question [Re: frikiriki]
#27440062 - 08/23/21 07:42 PM (2 years, 8 months ago) |
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It was a print. Had syringes from way back. All of them went funky on me.
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27440078 - 08/23/21 07:51 PM (2 years, 8 months ago) |
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Your saying the agar mix is jacked… I was thinking something was up with it because before I bought the LME it was much more of what I expected.
I will scrape some more into some new mixes.
I really do appreciate everyone’s advice. Hang with me until I get it.
I have tons of material to keep trying with. It’s been 10 years since I fruited anything. So really looking forward to it!
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Myco-pathic
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Round 2 FIGHT!
Just using MEA solo this time.
Ordered some agar agar. Really disliking the naming scheme they used for agars
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c10h12n2o
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If you are using a premixed MEA, just follow the instructions on the packaging
Are you heating it in a media bottle on a hotplate? That might be part of your problem with dissolving, those arent made to be heated that way
Check out the guide in my sig
For the most consistent, clear, even agar, i like to first mix it into cold water, then boil it in a beaker in a microwave on low power and mix well
If you really want to use a hot plate, use a flask
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Myco-pathic
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Re: Ms agar question [Re: c10h12n2o]
#27440805 - 08/24/21 10:40 AM (2 years, 8 months ago) |
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No heat just stir bar being used.
I will check it out. New plates look much better. Very firm.
Last ones were like Jell-O shots. That’s what I meant by the mycelium submerged and breached. 😂
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The Mycologist
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Your agar mix is not to blame
-------------------- "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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Quote:
The Mycologist said: Your agar mix is not to blame
That comment was not very constructive or helpful.
Yes the agar was part of the problem as I stated above I remade the agar and I’m starting over and doing some things differently.
At the beginning this post I respectfully disagreed because not every comment on the Internet is the nugget of gold.
I haven’t been here in 10 years and I don’t know who knows what they’re actually talking about. Thankfully guys like C 10 are here to be credible and helpful. 👍🏻
The Shroomery has always been a friendly and engaging environment for a very nerdy hobby. Please don’t treat this like Reddit.👎🏻
Edited by Myco-pathic (08/24/21 12:10 PM)
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The Mycologist
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Bro, soft or hard agar. That growth isnt right.
You are blaming the rain for leaks in your roof.
-------------------- "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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frikiriki
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They could just be shit genetics. (excuse my french)
Do you store them at room temp? Or fridge?
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PBJ710
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Quote:
The Mycologist said: Bro, soft or hard agar. That growth isnt right.
You are blaming the rain for leaks in your roof.

The agar firmness/nutrition content is something that needs to be addressed, but it's not the real issue on those plates.
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c10h12n2o
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Re: Ms agar question [Re: PBJ710]
#27441801 - 08/24/21 11:49 PM (2 years, 8 months ago) |
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Are you boiling your agar at all? You should really check out my agar guide in my sig
Like they said the agar isnt really the issue here. Thats more of a cosmetic thing.
Check this thread as well, it shows some examples of hidden contams
https://www.shroomery.org/forums/showflat.php/Number/22020260
Also, i just started a new project i will post in a few weeks (this thread and a few others gave me the idea). Basically i combined spores and clean myc with various contams to show what cultures look like when they are intermingled with various contams, should be pretty helpful for people.
Contams look different intermingled w cube myc vs on their own
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karri0n
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Quote:
Myco-pathic said:
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.
  
This is legit nightmare fuel.
As I pictured your plate, pooling with bacterial slurry, I winced and shouted "NO!" with each and every drop, methodically and meticulously dripping away all hope from your gorgeous plates in front of your epic hood and immaculate lab environment.
You absolutely are cultivating bacteria and mold like this.
As little liquid as possible would be one drop. That would still be too much, and is likely not going to be axenic.
You should put a single drop onto an inoculation loop or sterile swab and streak several plates. This is the first thing you learn about cultivating mushrooms from spore on agar. Better yet, if you have a print, just streak the print itself using the swab or loop and skip the syringe altogether.
If you did this with the vendor syringes, that's also why you didn't have any success. I'd wager that you had better luck because you made this syringe in front of a hood, from a print,which had time to dry and wouldn't have motile bacteria suspended in water.
Water is basically the enemy on an agar plate, because it give bacteria the ability to be motile, whereas the 2d surface *should* give mycelium a motility advantage. If there is no water and 1 bacterial cell, the mycelium can outcompete it or destroy it with antibacterial metabolites. If there is water, that one bacterial cell can rapidly multiply and move anywhere else water is present, quickly out competing the mycelium.
If there are no bacteria at all, water isn't a problem(hence people do transfers of clean growth to plates with condensation/pooling and are ok sometimes), but there can't be truly axenic prints because mushrooms fruit in open air and necessarily will be exposed to bacteria and mold spores.
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Panaeolus Bisporus
Edited by karri0n (08/26/21 02:11 AM)
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shthd
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Re: Ms agar question [Re: karri0n]
#27443306 - 08/26/21 03:44 AM (2 years, 8 months ago) |
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I took their statement as referring to pouring their agar mix, not the spore solution. (They do mention they used a print after failed attempts with syringes.) That much spore solution would certainly lead to primordial soup lol
You say you haven't grown in 10 years? Where was the lab equipment stored in that time, specifically the flow hood? Is the HEPA filter new?
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karri0n
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Re: Ms agar question [Re: shthd]
#27443667 - 08/26/21 10:37 AM (2 years, 8 months ago) |
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That does sound like it could be explaining how they pour plates now that you mention it.
They mentioned that they used the print to make a mss syringe too though. And the germination is across the whole plate, so that really could be what's happening.
In any case, you want to put as little genetic material on the plate as possible to have the least chance of something that isn't the target organism to get in there.
If using a syringe, stretching one drop to several plates by streaking with a loop or sterile swab is a good way.
If using a print, lightly dabbing the print on one very small spot then streaking several plates is a good way.
Remember that any visible spores are actually hundreds to thousands, and that there are millions suspended in the water that you cannot see. You only need two.
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Panaeolus Bisporus
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c10h12n2o
serial dilutor



Registered: 01/21/15
Posts: 3,200
Loc: the abyss
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Re: Ms agar question [Re: karri0n]
#27444064 - 08/26/21 04:04 PM (2 years, 8 months ago) |
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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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TheBoJim
Strangest


Registered: 07/23/20
Posts: 449
Last seen: 1 year, 2 months
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Re: Ms agar question [Re: c10h12n2o]
#27468664 - 09/14/21 08:56 PM (2 years, 7 months ago) |
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Quote:
c10h12n2o said: Are you boiling your agar at all? You should really check out my agar guide in my sig
Like they said the agar isnt really the issue here. Thats more of a cosmetic thing.
Check this thread as well, it shows some examples of hidden contams
https://www.shroomery.org/forums/showflat.php/Number/22020260
Also, i just started a new project i will post in a few weeks (this thread and a few others gave me the idea). Basically i combined spores and clean myc with various contams to show what cultures look like when they are intermingled with various contams, should be pretty helpful for people.
Contams look different intermingled w cube myc vs on their own
Great thread. I found his last sentence very fitting to op.
Quote:
Mad Season said:
This right here is why I never say that I don't need to improve my techniques, or that I'm 100% clean. I'm not, and I'm constantly working on my sterile procedures.
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