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 AspectOfTheCreator
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 What can be my problem? 
#28235296 - 03/18/23 03:31 PM (8 days, 9 hours ago) |
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I've been at this for ~6 months with 100% failure rate. For the life of me I can not attain a single fully colonized grain jar. I've used a combination of oats, WBS, unmodded lids, MP tape (3 layers), soak, no soak, boil, 24 H soak with a boil. Jars are at 70 F with indirect sunlight. Inoculated with a small wedge of agar (agar always seems to go well, seldom do I experience contam on agar).
The myc will jump off from the agar onto a few surrounding grains at which point it will just stall out. I'll shake and sometimes that will bring them to colonize a tiny bit more before again stalling. After a month I open the jars, grains still smell fresh. No slime or green. Cut grains in half they're soft and mushy inside as I believe they're supposed to be. There's no condensation inside the jars. Don't seem either too dry or too wet.
The tiny bit of mycelium in there will smell of fresh mushrooms if I examine a single colonized grain. I have read extensively trying to find an answer or some type of solution but I come up with nothing. To me it is completely inexplicable. Any thoughts/ideas? I have microposse SFDs now but I seriously doubt their use will suddenly solve this issue.
Jars, WBS ones are over a month old.
Typical stalled jar (inoculated 2-20)
1.5 month old stalled jar that got further along (lid is very loose now incase they were being suffocated but it hasn't helped)
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 Way
The Way
Registered: 01/14/23
Posts: 615
Loc: A long way away
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Why is there foil still on your lids? What type of filter on the lids are you using to provide gas exchange?
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 That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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AspectOfTheCreator
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Re: What can be my problem? [Re: Way]
#28235317 - 03/18/23 03:37 PM (8 days, 9 hours ago) |
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The ones with foil are unmodded lids, the ones without foil have 3 layers of MP tape.
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dowodenum
Noob Pope
Registered: 08/05/19
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Loc: 🇨🇦
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Use 4 layers with a 1/4" hole. Or get some good SFDs (amazon/ebay/aliexpress generally suck).
Can you detail your grain prep?
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Way
The Way
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Unmodded metal lids? I didn't know you could provide GE with those. I'm camp not getting enough GE, especially if you've tried many different grain prep teks and followed them as you should.
Are all the agar wedges from the same colony?
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That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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dowodenum
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Re: What can be my problem? [Re: Way]
#28235324 - 03/18/23 03:42 PM (8 days, 9 hours ago) |
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Quote:
Way said:
Unmodded metal lids? I didn't know you could provide GE with those. I'm camp not getting enough GE, especially if you've tried many different grain prep teks and followed them as you should.
Flipped lids and back a quarter turn. I don't trust em anymore, but plenty get away with it.
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Way
The Way
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Posts: 615
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Re: What can be my problem? [Re: dowodenum]
#28235326 - 03/18/23 03:43 PM (8 days, 9 hours ago) |
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Quote:
dowodenum said:
Quote:
Way said:
Unmodded metal lids? I didn't know you could provide GE with those. I'm camp not getting enough GE, especially if you've tried many different grain prep teks and followed them as you should.
Flipped lids and back a quarter turn. I don't trust em anymore, but plenty get away with it.
Yeah that sounds sus. 
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That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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AspectOfTheCreator
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Re: What can be my problem? [Re: dowodenum]
#28235330 - 03/18/23 03:46 PM (8 days, 9 hours ago) |
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Using a 1/4' hole already as that seems to be the standard size. Grain prep starts with 5x washes regardless of grain. For WBS I do 24 H soak, 5 minute simmer, strain, dry. Oats I've done 24 H soak plus boil strain dry but normally I'll boil for ~40 minutes before removing at the first sight of burst grains. I throw a little gypsum in soak/boil water. PC for 2 hours at 15 psi. The oats are producer's pride oats. WBS I'm unsure of the brand as the bag is long gone.
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AspectOfTheCreator
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Re: What can be my problem? [Re: Way]
#28235334 - 03/18/23 03:48 PM (8 days, 9 hours ago) |
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I've used many different cultures and varieties. Tidal wave, natalensis, APE, pan cambo. My agar work seems on point to me.
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 dyel
growing mushrooms in the desert
Registered: 10/15/21
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Re: What can be my problem? [Re: dowodenum]
#28235336 - 03/18/23 03:50 PM (8 days, 9 hours ago) |
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what are the tempretures?
or maybe it's the lack of gas exchange as the guys above mentioned
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AspectOfTheCreator
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Re: What can be my problem? [Re: dyel]
#28235341 - 03/18/23 03:54 PM (8 days, 9 hours ago) |
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Temp is 70-72 F, might go down to ~65 F at night. I have microposse SFDs and pennington WBS for the next round of attempts. I figured GE was the most likely suspect but I've seen plenty others using the same lid teks with nowhere near this many issues.
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dowodenum
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1/4" plus 3 layers MPT should be plenty of GE, but maybe letting in too much bacteria? Mold would definitely show...
Maybe a second rinse after soak would help?
But I've used SPG's oat prep recently with better results than what you're seeing, and that process doesn't have either a rinse or a soak. They're bacterial, but they still hit full colonization at least.
What do your source cultures look like? You're noccing in a SAB with no airflow in the room?
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Way
The Way
Registered: 01/14/23
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Well your grain prep seems okay, and if it wasn't one of the many methods you tried should have panned out.
You have multiple different colonies you've tried, if one or two of them was bad and contamination stopped the spread of myc, at least one should have worked, or turned severely bacterial.
You have two different types of grain, so we can rule that out.
Your temperature is slightly low, but that would just result in slower colonization.
All that's left is not enough air, right? Unless I'm missing something extremely obvious here, I don't know what else could cause that. You could also be using the wrong type of micropore tape...
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That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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AspectOfTheCreator
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Re: What can be my problem? [Re: dowodenum]
#28235357 - 03/18/23 04:03 PM (8 days, 8 hours ago) |
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Still air box in the morning before the heat goes on, so all fans would have been off for several hours. Butane torch for sterilizing tools. Using Kerr 1/2 pint jars for no pours. Been using 1% agar recently (2 g agar, 1 g LME, 100 dH2O). Unfortunately I have no pics. Don't usually keep the culture after using it for inoculation. From what I can tell they always look and grow out healthily, no slime or wrinkly surface on the agar.
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dowodenum
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Re: What can be my problem? [Re: Way]
#28235360 - 03/18/23 04:05 PM (8 days, 8 hours ago) |
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Maybe a fresh start with new spore syringes to pftek is warranted. That would work around: genetics, A2G sterile technique, WBS/oat grain prep, and filter choice. If you succeed there, it'll be easier to point the finger at one of those.
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sonson
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Re: What can be my problem? [Re: Way] 2
#28235361 - 03/18/23 04:06 PM (8 days, 8 hours ago) |
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(I realized my GE mistakes after reading your comments. Hopefully it is not too late to fix my bottles Thank you!)
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AspectOfTheCreator
Stranger
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Re: What can be my problem? [Re: Way]
#28235362 - 03/18/23 04:09 PM (8 days, 8 hours ago) |
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I'm using 3M paper micropore tape.
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dyel
growing mushrooms in the desert
Registered: 10/15/21
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do you have pics of agar?
i use almost the same gas exchange without issue, 3M 3 layers almost same size holes
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AspectOfTheCreator
Stranger
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Re: What can be my problem? [Re: dowodenum] 1
#28235370 - 03/18/23 04:16 PM (8 days, 8 hours ago) |
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I have PF tek cakes successfully colonized. I keep them in the fridge as an easy way to start from MS on agar and skip the germination time. Haven't used them exclusively, some A2G jars were started from streaked plates. Usually go to A2G at T3. PF Tek is no problem. Want to do bigger things though just don't understand where I'm going wrong.
Should be in a smaller jar, I know. This was unmodified lid with foil kept on top.
Unfortunately I really don't have any pics of agar. I use no pours so good pics are kind of impossible to do.
Edited by AspectOfTheCreator (03/18/23 04:23 PM)
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phenyl
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Bummer. My stalled jars have always been the result of an inoculant or substrate that wasn't as clean as I thought it was. Those pics are very familiar.
How are you running the pressure cooker? Vented and ran for at least 90 minutes at the pressure appropriate to your altitude (≥15PSI), then cooled overnight?
Do you have any agar pics from a T3/T4 transfer? Contamination on agar isn't always obvious.
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