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Doctor Mario
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Mistakes Were Made **And Corrected**
#26910941 - 08/31/20 10:33 PM (3 years, 4 months ago) |
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My feet are officially wet. I made a bunch of mistakes the biggest one being that I didn't let the agar cool enough before pouring. I realized it when a lot of the plates had so much condensation. A few of the ones I nocced up were still pretty liquefied.
Hopefully something good comes out of it and even though I screwed up, it wasnt a failure because I learned what to not do and what to do better.
I intentionally contammed one plate and maybe unintentionally contammed even more π
***UPDATE***

I went for round 2 tonight and I feel like I nailed it. My sterile tek was on point, I poured properly and over all, I feel pretty good about it.

I didnt start pouring until the agar was right around 120Β°F which made little to no condensation in the lid of the plates. I didnt let anything go over the plates and I poured precisely and swift yet careful and cautious as well. All of the pours are pretty even and I still had enough to pour one more plate.

I'll wrap these soon and let them sit for a few days and see if they contam. If the ones from yesterday contam (they probably will) then I'll inoculate some of these and go from there. If they don't contam then these will be ready for transfers. If these don't grow some weird shit, should I store them in a fridge or are they fine just going on a shelf somewhere?
Thanks for reading.
Edited by Doctor Mario (09/01/20 11:23 PM)
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PsiloPsychIn
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To help reduce condensation... Pour a little cooler yes, but also stack your plates. The bottom plates will be prevented from condensation by the heat and slow cooling of the plate on top. For plates that already have condensation you can store them upside down (after securing the lid with prafilm or syran wrap) and after a few days the condensation often evaporates with extra moisture being absorbed by the agar above.
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meowjinx
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Congrats on getting started! Good luck, hope it goes well
What did you use to inoculate your plates? A spore syringe? A print?
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Doctor Mario
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Re: Mistakes Were Made [Re: meowjinx]
#26910987 - 08/31/20 11:14 PM (3 years, 4 months ago) |
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Thanks for the tip. Turning them all upside down right now. I poured a little heavy on them too.
I have 40 more blank plates and another 500mL media bottle of agar ready to go. I'll wait a week or so and see what happens with these plates and if they dont grow anything at all, I know I fked up making and it and will start over. The agar clumped up while I was making it which is weird cause the water was boiling pretty good. No matter how much I stirred it it wouldn't liquefy.
I knocked them up from print.
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A.k.a
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With the stacking thing I was getting really irritated with condensation so I started heating water after I was done pouring and put it into old empty plates and added those to the stack so all my agar ones would be clear.
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WunFunGai
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Quote:
Doctor Mario said: Thanks for the tip. Turning them all upside down right now. I poured a little heavy on them too.
I have 40 more blank plates and another 500mL media bottle of agar ready to go. I'll wait a week or so and see what happens with these plates and if they dont grow anything at all, I know I fked up making and it and will start over. The agar clumped up while I was making it which is weird cause the water was boiling pretty good. No matter how much I stirred it it wouldn't liquefy.
I knocked them up from print.
Congrats on trying! My first Agar runs were with PastyPlates and 4oz Glad cups - they worked, and are pretty stealthy, but petri's are clearer and easier to visually see what's going on.
What kind of agar are you using? I've only used the Thai "telephone" agar-agar shown in Bod's and others TEKs. It comes in a powder and works and dissolves great. They're pretty cheap, and you can usually find them at asian food stores. Or you can get 3x25g packs on Amazon for about $8.
I use honey and potato flakes (that's the cloudiness you see in my plate pics) and a few drops of food coloring for contrast.
WFG
-------------------- The Official TNF Reccomended Teks & Methods Bod's Simplified Cultivation Methods <-- Awesome!!! D3monics Perfect Transfers and Agar Tek No matter where you go, there you are... - Buckaroo Banzai You gotta be here, if you're not all there...
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Doctor Mario
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Re: Mistakes Were Made [Re: WunFunGai]
#26911495 - 09/01/20 09:12 AM (3 years, 4 months ago) |
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I think I'm gonna try again tonight and I think I'll do a lot better now that I've identified my errors from the first run.
1. Pouring while the agar was too warm 2. Innoculating while the agar was too warm 3. Innoculating too deeply. (I'm not sure if this matters) I pushed the loop inside of the agar rather than using it to lightly swab the top. I assume the myc won't be able to breathe and likely won't grow at all. 4. When I sterilized the agar I brought the pressure to 17psi and maintained it but I didn't bring the heat down enough to minimize water loss. This was okay for a 20 min agar run but If it were a longer run for grain I could have screwed up the cycle or even warped my pc. I found the sweet spot for the heat about 15 minutes into the run. I have 2 quarters tapes to the weight but it was still really noisy. I'll probably add another quarter and see if I can make it quieter.
Quote:
WunFunGai said:
What kind of agar are you using?
Im using this agar agar powder and following this agar tek.
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WunFunGai
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Quote:
Doctor Mario said: I think I'm gonna try again tonight and I think I'll do a lot better now that I've identified my errors from the first run.
1. Pouring while the agar was too warm 2. Innoculating while the agar was too warm 3. Innoculating too deeply. (I'm not sure if this matters) I pushed the loop inside of the agar rather than using it to lightly swab the top. I assume the myc won't be able to breathe and likely won't grow at all. 4. When I sterilized the agar I brought the pressure to 17psi and maintained it but I didn't bring the heat down enough to minimize water loss. This was okay for a 20 min agar run but If it were a longer run for grain I could have screwed up the cycle or even warped my pc. I found the sweet spot for the heat about 15 minutes into the run. I have 2 quarters tapes to the weight but it was still really noisy. I'll probably add another quarter and see if I can make it quieter.
Quote:
WunFunGai said:
What kind of agar are you using?
Im using this agar agar powder and following this agar tek.
Ahhh..showing my age here, but I've seen this around since I was a kid in Chinatown and the quaint graphics are comforting: Telephone Agar and they're the perfect size for what I use so I don't have a bag open forever.
Not that I'm doing it any better, but things I've done differently than you've mentioned: 1) I poured too hot as well. Picked up a IR temp sensor and will try it on a new batch I'm making tonight. I'm pretty sketch on a water bath - hurt like hell the last time but I was able to pour 20 plates fairly quickly just need to let temp down a bit more. I only feel comfortable stacking 10 in my SAB and will have two cups of hot water to put on top of last plate. (I'll put pics in my isolation log)
2) After pouring agar, I let my plates sit for 48hrs to observe for contams. They also were cool and were firmed up pretty well. Between Jello and Gummy Bear consistency, a bit closer to Gummy.
3) I did two different styles of innoculating my two plates, zig zag, and the method were you streak overlapping and turning clockwise working toward center - both worked fine and are pictured in my isolation post. I used a light amount of pressure to "swab" it vs. drag/scrape the loop over the agar.
4) I have a 12psi PC and I'm worried about playing with pressure so I just increased my PC time from 20 to 30 minutes and came out just fine. I didn't have time when I finished so after it cooled, I put it in the fridge with foil on the cap still and let it sit there before "thawing" out with microwave on the weekend to do my pours. I'll probably do two bottles of media at a time using this method from now on to save myself trouble.
I need to also thank you. I found the tek you mentioned in your sig, and although I do stuff similarly for agar, I did like the idea of the "punch" D3monic uses for isolation and came up with my own $2 variant that works great. I'll post up some new pics on the log later with my 3rd xfer using a punch to get myc.
WFG
-------------------- The Official TNF Reccomended Teks & Methods Bod's Simplified Cultivation Methods <-- Awesome!!! D3monics Perfect Transfers and Agar Tek No matter where you go, there you are... - Buckaroo Banzai You gotta be here, if you're not all there...
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Doctor Mario
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Re: Mistakes Were Made [Re: WunFunGai]
#26912342 - 09/01/20 04:44 PM (3 years, 4 months ago) |
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Do contams usually show after at around 48 hours? Im curious to see what monstrosity may be on some of my plates. I made so many errors that I'm sure there's got to be some on at least a few of the plates. Im gonna make some more plates tonight and really try to get some sterile tek experience under my belt. Im a lot more confident now.
I've enjoyed reading your log this far and look forward to more.
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WunFunGai
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Quote:
Doctor Mario said: Do contams usually show after at around 48 hours? Im curious to see what monstrosity may be on some of my plates. I made so many errors that I'm sure there's got to be some on at least a few of the plates. Im gonna make some more plates tonight and really try to get some sterile tek experience under my belt. Im a lot more confident now.
I've enjoyed reading your log this far and look forward to more.
YMMV but in my environment, warm/humid tropical they show up pretty quick - as evidenced by that possible contam on my agar wedge that I fumbled into my jar.
I nocc'd the jar on 08/24 evening, saw a dot on 08/25, and by 08/26 this:

Agar is meant to be grown on, so based on my conditions, I'll see something within a day. Most of my spore and agar2agar transfers show signs within 24hrs.
WFG 
* updates on the isolation thread later today, just gotta clean up pics.
-------------------- The Official TNF Reccomended Teks & Methods Bod's Simplified Cultivation Methods <-- Awesome!!! D3monics Perfect Transfers and Agar Tek No matter where you go, there you are... - Buckaroo Banzai You gotta be here, if you're not all there...
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Doctor Mario
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Re: Mistakes Were Made [Re: WunFunGai]
#26912416 - 09/01/20 05:18 PM (3 years, 4 months ago) |
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I live in a warm humid area as well. Mold grows outside and inside everywhere. When I first moved into this house there was a mushroom growing in the corner on the bathroom floor... I also have a dog so I've been super worried about contam.
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A.k.a
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Itβs super dry where Iβm at, idk if it matters, but I almost always see bacteria before mold. Bacteria will show up in the first day or two but with mold Iβll see it around a week usually. It used to trick me and Iβd think it was a myc colony at first before it went green but I can tell the slight differences apart now.
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Doctor Mario
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Re: Mistakes Were Made [Re: A.k.a]
#26912979 - 09/01/20 11:25 PM (3 years, 4 months ago) |
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Quote:
A.k.a said: Itβs super dry where Iβm at, idk if it matters, but I almost always see bacteria before mold. Bacteria will show up in the first day or two but with mold Iβll see it around a week usually. It used to trick me and Iβd think it was a myc colony at first before it went green but I can tell the slight differences apart now.
That's good to know. Hopefully I see something soon. Im getting anxious now.
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