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Believe to have bacterial plates and not sure what happened please help! Old gene library revival
    #29595856 -

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The user is experiencing persistent bacterial contamination in their agar plates despite following a detailed sterilization and transfer process. They describe their agar preparation, including the use of water agar and low-nutrient agar, and their strict protocols for maintaining sterility, such as using a SAB, cleaning with alcohol, and sterilizing equipment. Despite these efforts, recent transfers have resulted in bacterial growth on both water and nutrient agar, even from seemingly healthy parent cultures. The user is unsure if the contamination is due to their agar, procedure, or something else. They plan to test new agar types, avoid food dye, and use sterile water, while also checking plates under a microscope. They seek advice on whether transferring sterilized 500ml jars into a larger 1000ml jar would maintain sterility and are eager for feedback to resolve the issue.

This is going to be long and I apologize but I have to explain the whole procedure otherwise we may not be able to figure out what I'm doing wrong.

I'll start by saying how I make my plates. I have both water agar and low nutrients agar. The set this started with was 500ml water with 9 grams agar agar from the good ol telephone brand. I used 16 grams of malt from a pretty old malt tub I have. The second set of plates I made that's been bad was only different with the nutrients which was 10 grams instead. I will mention I am seeing weird growth on my water agar plates too though so I'm not sure it has anything to do with the ingredients or this part of the procedure.

I PC two 500ml jars of my agar usually one just water agar and the other low nutrients. I PC them at 20psi for 20 minutes. I wait for them to cool down to about 130 degrees then pour some in condiment cups and some in regular Petri dishes. Almost always both new bags that have been sealed. If not I try to clean them all with alcohol and keep them sealed in a zip lock bag.

I use a SAB so I spray everything I use with a spray bottle filled with water and a cap of bleach. I let it sit for a while till it dries. I re-go over everything with 70% ISO diluted to about 1/3 iso water ratio.

I get everything clean and put it all in my SAB and close it up. Whatever I'm transferring from I go ahead and get in there with the plates I'm transferring to with the dates and names already written on them.

When I do my transfers I always try to get the strongest genes unless I'm trying to get away from contam then I transfer from the farthest points from contam. I always wear gloves and clean my gloves off with iso and my surgical blade handle every single time I do pretty much anything.I change the blade between every few transfers. I use a torch between transfers on the same blade.

Now down to the issues. So I just got back into things using genes that have sat for about 3 years maybe more. Starting off I used GT and P. Orchas. I made a post recently about it all. Things have been going well. I have a lot of good looking plates! On my second to last set of transfers things went side ways at some point. Everything I transfer now comes up bacterial. No matter what.

I had a good set of P. Orchas and Gts. The night before I started trying to get some other spore and LC syringes to agar. I can't remember all I did other than Enigma.

I made some Enigma transfers, P Orcha transfers, and Gt transfers. I transferred 4 of each plate and 2 to water agar and 2 to nutrients agar of each plate. Everything turned up bacterial.

I did some transfers from other plates of the same genes and those all also came up bacterial. Here's what I believe to be bacterial,

These are the water agar ones all put to agar from 5/26-6/1








These are the nutrient ones










You get the point with the contam. These are the parents plates in order of oldest to newest. Starting with the first plates
Enigma




P Orchas from LC Syringe







First transfer




Second Transfer


The plates I transferred from all looked good and still look good. So I have no idea what's going on and maybe they're not bacterial but they definitely look like it. I have no idea what to do from this point.

I have some good plates still which I'm saving so I don't screw them up too. Any help would be greatly appreciated. I have no idea what to do and have thought about just doing trenches for them all. The problem is they're bacterial even on the water agar so I'm worried nothing will work. These are my good plates







The trench plate is my best plate and is just now touching the other side so I'll make a transfer from there soon. I have this one plate from around the time I did my first transfers which looks a little weird




That's all I got for now! Little update on my old gene library I'm trying to revive as well as the issues I'm having. I need some help and ideas please. Thanks everyone! Took me awhile to write this 😅

Edit: so it seems a few people have mentioned food dye and the agar I'm using. So I'm going to do a few different things. I'm going to make some plates with no dye, make some plates with different agar, and I'm going to start back with T1s and drop some drops of the LC's I got of the same genes. That my plan at least.

Thank you everyone for helping me try to figure out what's going on! I got a few ideas thanks to all of you and I think we are going to get to the bottom of this. You all rock! Mush love everyone. I'll update when I get some new plates going.

Edit 2: I forgot to mention that I will be using a microscope to check my plates. If there's a lot of bacteria in them I'll know. I'll take pics and update it here later to show everyone. I'm super curious about the spiky myc but the microscope pics could help us all out here :smile: I will also finally be making my new plates today with the new recipe. Extremely curious to see how this changes the way my current crazy P. Orchas grow as well as my slow to grow GT's. I hope we get some growth on the slower ones like the TAT's as well. Oh and I'll finally be making a nice sterile water container that I'll PC and use a syringe to get it out. I'll post a picture of that too.

On that note, does anyone know if I PC two 500ml jars of water and pour them into an extremely clean 1000ml jar that it will stay sterile? I'll leave the 1000ml jar in a bucket of alcohol water till I'm ready for it then I'll do the pouring in an SAB. Thanks everyone :smile:


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Knowledge above all none sense.

Edited by No-Pilot-555 (06/07/26 12:16 PM)

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: No-Pilot-555]
    #29595908 -

Bacteria typically form visible colonies on agar and I'm not seeing any visible colonies in your pics. I do see what looks like it could be a fast growing mould contam in the blue dyed plates in the first set of pics though. Is that what you're talking about? The ring of thin wispy mycelium around your transfers? If so how fast is it growing?


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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dave Bennett]
    #29596126 -

Yes! That is what I'm talking about. That and the yellowish plates below the blue ones. The second yellowish plate if you click it and zoom in, you can see the same type of growth. It's almost clear or in the plate and super thin or something.

Since it's clear or thin, I assumed bacteria. I just wasn't sure. I also believed they formed colonies that you could see buttt I definitely haven't seen it all so I just thought, you never know.

The first set of blue plates the myc is almost growing spiky and intertwined with the contam. I've seen this before years and years  ago. I might have a picture too hold on.. I do! Prepare for some weirdness,






You can see the clear contam piggy backing on these old plates. The mycelium would get weirdly wispy and spiky too. This was from back in like '22 I just wanted to show you.

Anyways back to the current contam! The blue plates are water agar so it should already be slow moving. If you look at the first plate all the growth on it has been growing since 5/31. Day one and two wasn't much. Since Tuesday night it's grown about that much. Suggestions? What do you think I should do? Could I trench them? I'm at a loss but, this is why I love mycology. I love learning :smile: please halp though, thank you in advance! 🤙

Oh also on a side note, I'll be busting out the microscope to take a peak at the contam if I get time today. I'll upload those pics as well.


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Knowledge above all none sense.

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: No-Pilot-555]
    #29596174 -

I think the wet looking ring around the mycelium colonies is caused by the digestive enzymes from either the mycelium or the fungal contam or both diffusing out and breaking down the agar and liquifying it which gives it a gel like appearance rather than being caused by bacteria.

And looking at the pics in your last post it looks like something is attacking and killing the mycelium (especially the first pic with the blue agar) and maybe causing the spiky growth on the other plates too, which combined with the appearance and fast growth of the contam mycelium indicates that it could be Trichoderma mycelium you're dealing with.

Unfortunately there's no way to clean up Trich once it's growing intermingled with another species mycelium as it always outpaces it on agar so there's nowhere to take a clean transfer. If it is Trich it will sporulate and go green in the presence of oxygen so could I suggest you sacrifice the first plate to fully colonise with the contam mycelium and leave it open to the air and observe it to see what happens. There are other fast growing contams that have fine wispy mycelium like that that don't sporulate green but it would help prove or rule out a Trich infection.

So sorry to be the bearer of bad news but you may have to go back to spores and start again, I'm afraid.


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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: No-Pilot-555]
    #29596233 -

No-Pilot-555 said:





I've had spiky myc like that before. I'm my case, it didn't like the agar recipe I used (i used molasses in place of honey in PDA, it did not like the molasses). When I transferred the myc to different agar it behaved like normal. Could be what's happening with your spiky myc.


Edit: welcome back to SHROOMS and good luck reviving your library :mushroomgrow:

Edited by Dala (06/05/26 03:05 PM)

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dala] * 1
    #29596309 -

That's super interesting information and points towards a theory I'm having with a stubborn woodlover.

It spiked like hell, but started growing super sluggish by T3.

It was on a 1% LME that I used for pans, so it's not surprising that it didn't like it.

@OP: It's worth noting that some people have had issues with Telephone brand agar, and others have not. If you're noticing weird unexplainable behavior, it might be good to rule the brand out.


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I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: PuffM4gicDr4gon] * 2
    #29596316 -

The spiky stuff is funny lookin. It's like the myc is playing "the floor is lava".


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MUSHROOMS RULE

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dala] * 1
    #29596342 -

I am having very similar issues right now, and in my case, pretty sure it's two things happening at once:


1. They don't like this particular plate style, which contains both a blue/purple color with the lightly fungicidal red3, as well as a higher than usual dose of peptone + yeast. Transferring them to regular uncolored 1% plates, they do fine. Some of them also adapt to the blue after 10-15mm of growth.


2. There actually IS an outbreak of some other mycelium that's rampant in my basement rn. Didn't have these issues in winter, but now that spring/summer is around, I guess it's in the air. This thing grows low, light, and fast like your pics before fluffing up. But so does real myc on the blue plates.


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.~`  𝔅 𝔞 𝔤 𝔢 𝔩  🥯  𝔅 𝔦 𝔱 𝔢 𝔰  `~.
:salute: CAKE WARS III HAPPENING NOW :salute:

🧪 Castellani Preservation | 🔬 The Slidewich | ☠️ Touchless skull sterilizer | 📸 Agar plate scanner


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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Rasmodius]
    #29596486 -

I'm a chronic serial plate abandoner.

Currently in the process of reviving cultures that sat at room temperature for a YEAR. Unwrapped. Dried-out little crusts of agar that I have to rip out with flamed tweezers and drop onto fresh agar. Haven't kept track of my odds so far, but luckily I'm also heavily into making multiple backup plates, so at least a couple from each culture line are reviving just fine. Some others turn the color of the rainbow, and a few just don't do anything at all.

Spiky myc, scalloping myc, weirdly fuzzy myc with little moats of raised material, and a few other things I'm not recalling at the moment have all proven to be signs of bacterial cultures. Sometimes. And then sometimes not. The only surefire way I've found to test them is to grow them out and see what happens. I have yet to run into a problem with my agar or SAB or whatever that manifested itself across ALL of my cultures. It's always the same ones that end up being problematic, but some are just too cool to let go, yah know? Or they do OK enough, often squeezing out a few flushes before crapping out. But my tolerance for all that definitely diminishes with each passing year. Especially when it's occurring alongside some rockstar culture that revived from the dead looking no worse for the wear.

There's so many techniques people have written up here for trying to outrun bacteria- Josex poke, cabin sequestration, etc. I still haven't gotten around to any of that. I've just relied on taking huge numbers of transfers, and for the first few rounds trying to always catch the growing edge of mycelium before the plate is fully colonized. That's worked out reasonably well for me. Though doing pre-pour on a set pool of reusable plates is also crucial for me. Doing extra transfers doesn't really cost much of anything, and I also have a limit which forces me to make tough choices.

You know what's funny, though. I think my success rate has been better with reviving dried-out room temperature plates than it has been with refrigerated slants and plates. Could just be that improvements in my culture game have coincided with me being more cavalier about storage. But I swear, the usual cycling and condensation of the typical fridge sometimes feel worse for the growth of random shit than just letting everything dry out.


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ACAB  🖕🧊


Edited by B Traven (06/05/26 06:17 PM)

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: B Traven] * 2
    #29596496 -

B Traven said:
I'm a chronic serial plate abandoner.

Currently in the process of reviving cultures that sat at room temperature for a YEAR. Unwrapped. Dried-out little crusts of agar that I have to rip out with flamed tweezers and drop onto fresh agar. Haven't kept track of my odds so far, but luckily I'm also heavily into making multiple backup plates, so at least a couple from each culture line are reviving just fine. Some others turn the color of the rainbow, and a few nust don't do anything at all.....

.....Though doing pre-pour on a set pool of reusable plates is also crucfor me. Doing extra transfers doesn't really cost much of anything, and I also have a limit which forces me to make tough choices.


I've had good luck just doing a hot pour over dried up plates.

+1 for prepours and just being prolific with plates. It lets you be picky and helps with learning to read agar


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I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: PuffM4gicDr4gon]
    #29596508 -

PuffM4gicDr4gon said:
I've had good luck just doing a hot pour over dried up plates.



:mindblown:


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ACAB  🖕🧊


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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dave Bennett] * 1
    #29596573 -

Dave Bennett said:
I think the wet looking ring around the mycelium colonies is caused by the digestive enzymes from either the mycelium or the fungal contam or both diffusing out and breaking down the agar and liquifying it which gives it a gel like appearance rather than being caused by bacteria.

And looking at the pics in your last post it looks like something is attacking and killing the mycelium (especially the first pic with the blue agar) and maybe causing the spiky growth on the other plates too, which combined with the appearance and fast growth of the contam mycelium indicates that it could be Trichoderma mycelium you're dealing with.

Unfortunately there's no way to clean up Trich once it's growing intermingled with another species mycelium as it always outpaces it on agar so there's nowhere to take a clean transfer. If it is Trich it will sporulate and go green in the presence of oxygen so could I suggest you sacrifice the first plate to fully colonise with the contam mycelium and leave it open to the air and observe it to see what happens. There are other fast growing contams that have fine wispy mycelium like that that don't sporulate green but it would help prove or rule out a Trich infection.

So sorry to be the bearer of bad news but you may have to go back to spores and start again, I'm afraid.



Every one of your posts gives me a headache and makes me drink more. In fact, I'll crack another. Time for a dart...:smoker:

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Stipe-n Cap] * 1
    #29596591 -

Stipe-n Cap said:
Every one of your posts gives me a headache and makes me drink more. In fact, I'll crack another.


:chugbeer:

Serious note, when I get that wet look on my plates I get worried, then I stick my scalpel in that shit and I realize the new agar is weak and my plate is fucking jello :lol:


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“All Fungi are edible.

Some fungi are only edible once.”
― Terry Pratchett


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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dave Bennett]
    #29597007 -

Ahhh that's good to know. I didn't know that. It could be both here like you said. It's possible but those pics and plates are from years ago. If you think any of the ones in my original post look like that, I have a few so I could sacrifice one to see what happens. That would be unfortunate but definitely worth knowing.

The myc growing on these plates were all like T3-T4 from old LC syringes. I could go ahead and drop a few drops on some plates anyway and restart from T1. I saw someone else mentioning it could be the agar as well. I may attempt a different brand of agar and no coloring to see if that helps. I'm wondering if it's my brand of agar or food dye.


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Knowledge above all none sense.

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dala] * 1
    #29597012 -

Thank you for the welcome back!! It's so exciting to be back. I love mycology. I think you're on to something here. I'm going to change up my recipe and change the brand of agar I use as well as try no food coloring.


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Knowledge above all none sense.

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: No-Pilot-555]
    #29597197 -

It's just the blue agar plate/plates with the fast growing ring of thin, translucent disorganised mycelium in the first set of pics in your OP that looks questionable and perhaps beyond saving. If that's not present in any of the other plates you should be able to continue to work with them. I also agree with the poster who suggested your agar recipe might be too rich in carbs which could be leading to the abnormal growth on your other plates.

Don't worry about the clear halos that appear on the agar just in front of the leading edge of an expanding colony that go all the way round the circumference. There was an "embedded bacteria" scare a few years ago where people thought the halo was caused by bacteria. It's actually caused by digestive enzymes secreted by the hyphal tips diffusing out into the agar and as the enzymes start degrading the nutrients and altering the pH of the agar it can give it a slightly wet look and change it's optical properties which results in the clearer halo.

Halo assays are regularly used in mycological research to assess and screen strains for specific enzyme production. The wider the halo the more digestive enzymes the culture is producing. So if a halo appears on plates from an isolated culture in home cultivation it can be regarded as a sign that it's pumping out a lot of digestive enzymes and will perform really well on grain.


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Edited by Dave Bennett (06/07/26 05:47 PM)

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dave Bennett]
    #29597218 -

That's honestly so awesome. Good news too but also bad news.

First could I test the agar out with the enzymes by making the agar thicker? Would that change anything?

Second, you believe it's possible that my blue plates are then probably contaminated with trich. Those plates are the water agar plates which blows my mind in the conditions that trich can trive then. I can test to see if it's trich by sacrificing one to basically let it sit in open air and see what happens?

Sorry for all the questions but I love this stuff and the more I can learn about it the better off I'll be in the future. You know, teach a man to fish and all.  It to mention I can share the information and pass it a long as well. I really appreciate all the help. Thank you again.

Blown away by the way that mycelium can break down the agar like that. It makes a lot of sense with how fungi break things down in nature in general.


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Knowledge above all none sense.

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: No-Pilot-555]
    #29597233 -

Apologies! I completely missed that the blue plates were water agar. You can scratch what I said about a potential fungal contam unless it's growing way faster than cube mycelium normally does (like filling the plate in a matter of three or four days). Cube mycelium grows thin and wispy like that on water agar, especially if the transfer is small which limits the available nutrients for any growth. So it looks like it's good news all round.

Some agar brands seem to be more prone to showing halos but if you wanted to enhance their visibility on agar add some soluble starch, like potato or corn starch, to your agar recipe. Making the agar slightly softer helps the enzymes diffuse out more efficiently too. Exactly the same process is happening when mycelium consumes the dye or colourant in agar plates too.


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Edited by Dave Bennett (06/06/26 05:08 AM)

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Re: Believe to have bacterial plates and not sure what happened please help! Old gene library revival [Re: Dave Bennett]
    #29597490 -

Oh really?! Oh few! That's such good news. Youve had me worried the whole time! 😂 either way it's still spiky. You don't think the weird spikiness is sus?

I have noticed on water agar the myc grows more rhizo then the other way. I can't remember the terms at all right now and don't have them written down. It's bothering me 😑 all I can remember is scalloping because I had that with my enigma plates recently. I'll look into it then I'll make it my signature so I don't forget 😂

When I first started studying mycology, I learned everything you could about it. I put it all on flash cards and did flash cards with my wife. This being years ago now and since I'm just getting back into the hobby, I'm a bit rrrrustyyyy. No not crusty like crusty the clown but rusty like the type of rusty that you get when you don't do something for awhile and instead of doing your normal chemical mix while cleaning, you accidentally mix alcohol and bleach and chlorine gas yourself in the garage. I definitely didn't do that.

Anyway, I'm going to experiment with it. Now that I know what's going on at least. I got a really good gene library and hope to revive what I can. I tell people this when I talk about the hobby, I just love mushrooms in their entirety. I love the way they grow, I love the way they look, some of them smell good, some taste good, isolated genes is fun, the microscopy behind it is awesome, doing things with agar is my favorite, then there liquid culture, fighting contam, honing your skills, actually growing the mushroom, I could go on and on. I tell them it's not about growing actives and meeting god. It's about learning and learning more. Its way better and way more then something like gardening (even though I garden and garden requires significant effort as well just in other ways,) just in its own way.

I realized I loved it when I stopped doing it for getting money or the main reason people do it. I do it now because it just makes me happy. I bought my first set of gourmet mushrooms and I'm so excited about it! Anyway sorry about the yap. Another awesome thing is the community. I've never seen such a kind community willing to work together.

On that note I thank you again man. You're awesome and I appreciate all the help. So blue plates, which are water agar, you believe are good as well as the others then? Even with the spikiness? I'm going to make my new plates tonight or tomorrow without any coloring and different agar. I'll look into some safe food coloring as well. I've had a lot of people say to just use activated charcoal.

Also last thing before I forget, checkout MillyWyco. Not WillyMyco but MillyWyco. Great guy, has an awesome discord for stuff like this (which now that I think about it, maybe the shroomery does too 🤔) and check out Wizard of Barge. Awesome artist to look at while tripping but he's an awesome artist no matter what and a really good guy too :smile: thanks again my friend! Will update soon.


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Knowledge above all none sense.

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ignore, was a mess up. Phone wasn't working people. [Re: No-Pilot-555]
    #29597497 -

ignore this, phone was acting up and idk how to delete it :frown: sorry everyone!


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Knowledge above all none sense.

Edited by No-Pilot-555 (06/06/26 01:11 PM)

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