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n0mad
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Adventures with agar in PE - cleaning up contamination, lots of questions
#26167939 - 09/03/19 11:37 PM (4 years, 4 months ago) |
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I'm super new to this, and I'm learning as things go along. A friend of mine gave me a grain jar of PE that was ... rather bacterial from what I gather. I would love for someone to [1] Confirm that it is indeed bacterial, and not something else. [2] Am I on the right track? I could use some encouragement here. I tried to give it a go with a shoebox, but clearly it was contaminated and it didn't pin. So I started digging into the forum here and learning AGAR in attempt to clean this shit up.
Before I did the shoebox, I made a g2g transfer, which unsurprisingly was also contaminated. Side view:

Top view1:
 Top view2:

I have a bag over it with holes to perhaps see if I get anywhere, but the black wet spots sadly have been growing.
I got started on agar and poured some of my first plates around Aug 13:
 As you can see the black wet spots, which I understand are bacterial traveled with it. [3] What bacteria is it?
Since then I also made a few transfers, most looked "ok to me" first few days and a few additional transfers were made. Some of T2 weren't so hot after a few more days. Plate 1 Aug18:
 Plate 2 Aug18:

Plate 3 & 4 Aug 23... look at those black spots again.


Here are some of the better plates. I believe all of these were done Aug 23. [4] Should I transfer again? If so, which parts [5] Does the mycelium look ok to you? - PE variety [6] Is this futile? Plate 5:
 Plate 6, [7] does this one looks a little bacterial to you?
 Plate 7:

Interestingly enough, this plate #8 was transferred at the same time as 5,6, and 7, yet took over plate completely. This is BRF agar, and is with more sediment, (perhaps better nutrition???) , but is not transparent, and is actually really hard to photo.
 [8] is this now clean? [9] Should I try to continue to clean all of these up?
I've actually run many more plates, but didn't post them mostly due to spectacular failures.
So here is a full list of the questions in that wall of text:
[1] Confirm that it is indeed bacterial, and not something else. [2] Am I on the right track? I could use some encouragement here. [3] What bacteria is it? [4] Should I transfer again? If so, which parts [5] Does the mycelium look ok to you? - PE variety [6] Is this futile? [7] does plate 6 looks a little bacterial to you? [8] Is this now clean? [9] Should I try to continue to clean all of these up?
And a few extra: [10] How do you take better pictures? [11] Should I actually be posting in this subforum, or AGAR one?
Edited by n0mad (09/03/19 11:39 PM)
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Geinstein
Shroomery addict



Registered: 01/25/18
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Re: Adventures with agar in PE, lots of questions [Re: n0mad]
#26167941 - 09/03/19 11:40 PM (4 years, 4 months ago) |
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I'll PM you
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Nothing breads nothing
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MLPismyOPSEC
That One Ponyfucker


Registered: 11/13/18
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Re: Adventures with agar in PE, lots of questions [Re: Geinstein]
#26172142 - 09/06/19 09:20 AM (4 years, 4 months ago) |
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1) Definitely bacterial, the black is likely mold. 2) I would say no. It seems you have some agar experience down for sure, but i believe you're just transferring mold. 4/9) I wouldn't. Grab some spores/swabs and start fresh, you'll save a lot of time and headache. Again, i don't believe you're transferring any mycelium. 5) No. Yes. Mold, not bacteria. No. No. 6) Yes. 7) Mold, not bacteria. 8) No. 9) No. 10) Play with lighting; directly overhead or at an angle. Different camera angles too. One good way is to have a light source behind the plate, with the picture taken through the plate. Light -> plate -> camera. 11) Either, or the Mush Cult forum, i think that's one of the busier ones.
Now my own!  1) What is your agar recipe? 2) How did you transfer to those plates with the blue tape around them? 3) You are using a SAB right? 4) Have you tested any blank plates to ensure your sterile tek is up to snuff?
https://www.shroomery.org/forums/showflat.php/Number/17231150
Your post in the Agar Envy thread; those plates do look a lot better, but with the condensation and quality of pictures, it's hard to tell.
Edited by MLPismyOPSEC (09/06/19 09:23 AM)
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n0mad
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Re: Adventures with agar in PE, lots of questions [Re: Geinstein]
#26172947 - 09/06/19 04:57 PM (4 years, 4 months ago) |
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Amazing thank you so much! Really appreciate this.
I don't have access to the source anymore of the original culture for the plates in this thread.
Thank you for the words of support! This helps enormously to have someone look over the shoulder and tell me whether I'm shooting in the dark. 1. 20g/L for either BRF or Dry Malt Extract 2. Blue tape were the very first plates I poured, and the transfer was from the contaminated grain. I believe I dug at the middle of the cake and pulled what looked like "clean mycelium". 3. I am using SAB 4. I have certainly tested blank plates, and I probably get about 10% contam rate when I'm interrupted doing the lab work.
As for the most recent post in Agar Envy - those are liquid culture plates. I was able to source a syringe and start with those.
1. What do you suggest best way to deal with condensation? If I wait longer temperature of agar drops too low and it comes out lumpy. 2. Would preheating the plate sleeve help with condensation?
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MLPismyOPSEC
That One Ponyfucker


Registered: 11/13/18
Posts: 884
Loc: Equestria? Mordor? Wester...
Last seen: 10 days, 13 minutes
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Re: Adventures with agar in PE, lots of questions [Re: n0mad] 1
#26175013 - 09/07/19 11:11 PM (4 years, 4 months ago) |
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Anytimw man! We all started somewhere, i'm glad i can start to call myself experienced enough to help out.
1) a hot cup of coffee, set on top of the plate! Has to be freshly made, the mug will need a lot of heat to work. when you set the mug down, it'll take a few minutes. I also have major condensation problems, it's such a PITA. 2) it would but there's probably not a good or perfectly sterile way to do it.
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