GenesisCorrupted said:

That still has value even in the trashcan.
You won’t make that mistake again. Next time you’re agar plates are going to come out clean.

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Thanks for the encouragement. There are 10 LME plates and 10 water agar plates waiting to prove you right. We'll know soon

Phaethon said:

Looks suspicious to me too. Could be pouring/sterile technique, but I think 20 minutes may be too short to properly sterilize the amount of media you used, especially with more particulate than you may usually have. See e.g. https://www.shroomery.org/forums/showflat.php/Number/28166056

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Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

Smellyhobbit said:

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

This solidified, right? Pour plates with it. It won’t look like that when it’s thinner

---

Nichrome said:

SWBZA said:

Hi all.

First time making agar plates with yeast: 2% agar, 1.6% LME, 0.2% nutritional yeast.  Same process followed for all my other agar - slowly vent PC, sterilise for 20 minutes, let cool down and pour at 50 degrees celsius.  Besides the plates looking butt ugly, today (day 4), my plates look some variation of this:



That looks very suspicious to me   Is this what yeast plates are supposed to look like?

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That is bacteria. Toss all plates that look like that right in the trash. Some kind of flaw in your method.

---

This is a long post exploring "some kind of flaw in your method", so <TL; DR>:

I reviewed my agar process carefully.  There were two changes from when I had mostly success, to where I now have high failure rates.

1 - I changed my Petri dish supplier
2 - I changed from cling wrap to Parafilm to grafting Parafilm tape

I have 10 Petri dishes left from the current supplier.  I'll make them, wrap them with cling wrap, and see what happens.  All new plates to be purchased from the old, more expensive dealer.  Joining Team Cling Wrap again. I'll up my PC time from 20 minutes to 45 minutes, although 20 minutes worked fine in the beginning.

I still have a lot of condensation around the edges of the dishes, even while pouring at 50 C and letting the plates sit in my SAB overnight.  The water sits around the edges of the plates when I pick up a clean, new one, and that is where the recent bacterial growth started.  My only thought around this is that the Petri dish is not sealed properly, and bacteria enter through the Parafilm grafting tape into this water ring.  That feels like reaching, though.



============  The long post =======================

When I started this hobby, I was scared of agar, just like all noobs, I supposed.  When I started using it, it actually became one of my favourite parts.  My wife called me a "mad scientist", always in my SAB busy with a scalpel or something.  I also had rather good results.  Recently I've had a spate of problems, and I've spammed The Shroomery with a bunch of questions and complaints.  This last week reached a low point for me when you guys helped me identify most of a recent batch of plates as bacterial.  I've thrown out half of them, and I suspect the remaining ones are dodge, too - the ones I've just used!  In addition, I almost gave up on LAGM this year as my process ended in all-green mould.  This week I decided to give it another go, and yesterday did four new spore plates, but all of them on my new batch of plates - and now I'm pretty sure all four of those plates are probably doomed   Much effort, with nothing to show for it.  Except maybe learning and improvement of my process. Still sux.  My agar fun fell on its nose, there is blood all over the floor, and teary eyes.

Working with you guys and identifying PC time as a potential problem, yesterday I poured 10 LME plates and 10 water agar plates with the change being PCing for 45 minutes, not 20, after venting for 10.  When I got up today, I thought about those 4 LAGM plates that are probably worthless and hit a downer again.  Having my first coffee, I thought fuck this (sorry for my language), I'm not a quitter!! I have plates that are hopefully now clean.  I also made spore prints yesterday - the caps are still on foil, and I hope when I lift them up this morning I have clear, crips black prints.  So why not put some of these brand-new spores on a brand-new plate?  That was my plan.

Picking up my new LME plates I looked for the purest, bestest, prettiest one to use for the new spore streak, I noticed that all of them have little pools of water right around the edges of the plates.  When I pick one up to look at it, I can see the water running over the plate making little arms:



I'd say the condensation I have around the plates after pouring has succumbed to gravity, hence the water moat.  Here you can see the drops on the edges of the plates after a pour:



(Colour is all wrong - I played around with editing to make the condensation clear - photo was taken through the SAB, so a little blurry.)

After a major hissy fit, I calmed down, and thought of the bacterial plates I threw out yesterday.  Here is one:



Those bacteria started growing around the edge, right where I'm seeing this pooled water ring on my new plates.  Not guaranteed connected, but suspicious coincidence, no?  After another temper tantrum, I made a second cup of coffee, played with my cat a bit, and thought about things.  I reviewed what could have changed from when I was a mad scientist having fun and having success with agar, until where I'm now making too many posts in the agar forum whining about flops and feeling like taking up scuba diving again. 

1 - My workflow process has not changed.  Same LME and agar brands.  Same way of prepping the agar, same recipes. (Granted, for the recent plates I added yeast, but the plates before those were also bad and were stock standard for me.)  Same PC.  Same water bath method to cool agar broth to 50 C before pouring in a SAB.  Still putting a hot water container on the top of a stack of plates to reduce condensation on the top plate, and I'm still leaving the plates overnight before wrapping. Used to do 20 minutes then, too.  I will, however up my PC time to 45 minutes.  Costs me nothing and eliminates one variable.  Can't do harm as far as I can tell.

2 - I used to use blue colouring.  I've asked enough questions here to know that is not a good idea as it makes it hard to read plates.  Unless that blue powder somehow kills bacteria, leaving it out is not a cause of failure.  And some of the problem plates that I started posting here were still blue, so I'm confident this change is not a contaminated vector.

3 - I've changed my vendor for Petri dishes.  I found a shop that sells to schools and their plates are reasonably priced. When I did my first pour with them, I made a mess, pouring "between plates" - I stack pour, not sure there is another way.  I realised that when I picked up the plates by grabbing the top part, sometimes the bottom half of the plate lifts up as well - almost as if there is a little "lug" that keeps the two plates together.  It just takes being mindful of this and making sure the bottom part of the plate does not come with when I lift the top during my stack-pour, and that solves the problem.  Reading the label on the petri dish plastic sleeve carefully this morning after digging it out the trash (yuck!!!!!), I see this sentence: "vented for better gas exchange".  Only thing I can think this means is that the lid fits horribly by design.  This is what the plate looks like from the side:



I don't have one of my old plates to compare with, so no idea whether this is a big step or not.

So, this is the first major change in process that I will fix: my old supplier was more expensive, but the plates were easy to pour, and for a few bucks more it will give me peace of mind.

4 - I started off using cling wrap.  It worked, but in the SAB there was always a bit of a wrestling match getting the stuff off of plates and into my "garbage area" in the SAB (I try to put all used stuff in one corner to keep the space uncluttered.  My workflow is from far right corner to garbage space front left, transfering in the middle of the SAB from closest to me to the back middle of the SAB.).  That cling wrap sticks to anything and everything.  I bought myself a small strip of Parafilm when I just started out to use "one day" for a "very important plate" like "real growers do", thinking Parafilm is the more professional option.  I recently realised that the short strip of Parafilm is still sitting in my supplies box waiting for who-knows-what.  I used it and liked it - mainly because it comes off a plate clean and manageable.  The waxy stuff does not stick to my gloves.  But where I live it is expensive.  I found out about "Parafilm grafting tape", read up here and elsewhere, and it sounded like the stuff works.  It certainly is cheaper.  I bought a roll and have been using it since.  It is really easy to put on a plate and comes off clean and non-sticky when I open a plate.  But it is a change, and thus a potential contamination vector that entered my process.  I need to go do shopping just now, and I'll be returning with a roll of cling wrap and go back to the swearing, shaking of gloved hands, jerking arms out of the SAB and jumping around until I can get the stuff off of me, and then waiting until my SAB is still again before continuing my transfers

So those are the two changes I'm going to make - essentially going back to what I did initially:  I'm going back to my original supplier of Petri dishes, and I'm going back to cling wrap. 


I have no idea what to do about the ring of water and the condensation around the edges of the Petri dishes after I pour.  I can't pour colder than 50 C.  I already have to work fast.  I'm not rushing. I'm working steadily and calmly, but if there is a hiccup, the agar sets by the time I get to the top plate.  It's as cold as I can go.

Anyway, there is my long story.  When I return from the shops, I'll make that spore streak from my brand-new prints and wrap the plate in cling wrap. There will still be water tears all over that plate, but I can't change that now, so I'll do what I can with what I have.  Hopefully that condensed water is at least uncontaminated.

Cheers, peeps, and thanks for listening.  Hope you all have fantastically boringly successful grows.

SWBZA said:

when I was a mad scientist having fun and having success with agar

---

Just do that part again. All the rest is where you went wrong.

That level of legs (drips) at the edge is not out of the ordinary. The liquid is often where you will see bacteria flourish first. Most bacteria need liquid. Very few move fast across solid surfaces without it. The liquid doesn't cause the bacteria, it just gives it a good foothold.

Those dirty plates are exactly what I'd expect to see made with poorly manufactured/cheapo plates and borderline sterile media. PC the media for 45 and switching to new plates like you plan to is a good move.

Nichrome said:

SWBZA said:

when I was a mad scientist having fun and having success with agar

---

Just do that part again. All the rest is where you went wrong.

That level of legs (drips) at the edge is not out of the ordinary. The liquid is often where you will see bacteria flourish first. Most bacteria need liquid. Very few move fast across solid surfaces without it. The liquid doesn't cause the bacteria, it just gives it a good foothold.

Those dirty plates are exactly what I'd expect to see made with poorly manufactured/cheapo plates and borderline sterile media. PC the media for 45 and switching to new plates like you plan to is a good move.

---

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

SwabMarley said:

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

2 hours is massive overkill also for agar in an insta at 12psi. As a general rule I tend to almost double my times compared to 15-18 in a presto. 40 mins is more than fine for agar in an insta in my experience. As I would usually give LC’s and agar 20 mins or so in the presto.

---

SWBZA said:

Nichrome said:

SWBZA said:

when I was a mad scientist having fun and having success with agar

---

Just do that part again. All the rest is where you went wrong.

That level of legs (drips) at the edge is not out of the ordinary. The liquid is often where you will see bacteria flourish first. Most bacteria need liquid. Very few move fast across solid surfaces without it. The liquid doesn't cause the bacteria, it just gives it a good foothold.

Those dirty plates are exactly what I'd expect to see made with poorly manufactured/cheapo plates and borderline sterile media. PC the media for 45 and switching to new plates like you plan to is a good move.

---

---

Most everyone goes through issues like this from time to time. Either with agar or grains or something. Don't let it get you down, all part of the fun.
If those changes don't work out, you can always try pre-pour/no-pour to remove variables. And while it's not as cool, I "wrap" my plates with individual ziplock baggies.

chris77 said:

SwabMarley said:

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---

2 hours is massive overkill also for agar in an insta at 12psi. As a general rule I tend to almost double my times compared to 15-18 in a presto. 40 mins is more than fine for agar in an insta in my experience. As I would usually give LC’s and agar 20 mins or so in the presto.

---

---

Soloist said:

Why is my LMEA cloudy?
I used D3’s recipe but cut in half
-so 10g agar (telephone)
8g LME
500mg distilled

2 hours in an “instant pot type” pc
Manufacturer specs @12psi, 116 F.

Every LMEA I see it’sclear, did I caramelize it?
Should still be viable for germ plates no?

---