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stroopwafelman


Registered: 03/21/23
Posts: 386
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Agar not colonizing grain, help.
#28388628 - 07/07/23 05:19 PM (1 year, 6 months ago) |
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Transferred an agar slice to a prepped corn jar, 5 days later there's no visible growth of any sort. Popcorn was first rinsed, then soaked for about 12 hours, then cooked on a low simmer for 40-45min until just the very center of the kernel was a tiny bit dry, left to steam on a dring rack for about 30-40min, then loaded up into jars with a tablespoon of dry verm on the bottom and PCed at 15-16PSI for 2hours. The agar slice itself looks completely empty as well (looked that way right after the transfer). Are the grains too dry? Could the mycelium have slipped off the agar slice during transfer or something like that?
   Here are the pics of the plate the slice was sourced from. It's a pin from a sealed BRF jar (with filter, no dry verm layer - BRF puck tek) transferred over to water agar.
 
What am I doing wrong?
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Screwup
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Registered: 01/27/22
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Nothing about those plates looks good IMO
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stroopwafelman


Registered: 03/21/23
Posts: 386
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Re: Agar not colonizing grain, help. [Re: Screwup]
#28388658 - 07/07/23 05:52 PM (1 year, 6 months ago) |
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Quote:
Screwup said: Nothing about those plates looks good IMO
I was lead to believe that that's the way water agar plates looked. Like this pic from the water agar tek post

What exactly is wrong with my plate? None of the plates I have have rhizo growth, if that's the problem, and out of 10 BRF jars I made only 1 had visible rhizomorphic growth, all others didn't.
Here are 2 more water agar plates, do these also look bad? Is it bacterial? Mold? I can't honestly tell what to even look for, how are contams even surviving on frigging water?
 
Edited by stroopwafelman (07/07/23 06:08 PM)
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Screwup
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Registered: 01/27/22
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Well Iβd wait for someone else. Water agar ainβt my forte then.
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PBJ710
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Re: Agar not colonizing grain, help. [Re: Screwup] 1
#28388679 - 07/07/23 06:21 PM (1 year, 6 months ago) |
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That donor plate doesn't look good to me either. Is that an abort that you are trying to clone from? If so, you need to do at least 1 transfer away from sketchy material before you go to grain.
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stroopwafelman


Registered: 03/21/23
Posts: 386
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Re: Agar not colonizing grain, help. [Re: PBJ710]
#28388681 - 07/07/23 06:23 PM (1 year, 6 months ago) |
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Those are all small mushrooms from inside a BRF puck jar (with filter no dry verm layer) that was opened inside a SAB. The donor plate was from a pin that was growing on the glass side of the jar iirc.
These are the teks I was following. I also made LC from another plate, which currently has a few mycelium "balls" floating around (no doubt it's also contaminated then if these plates are shit).
Honestly I'm starting to feel agar was a waste of time and money at this point. I have no idea where the contam is coming from (did spores on LME agar before plates were also called "bad").
Edited by stroopwafelman (07/07/23 06:35 PM)
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PBJ710
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If it was mine, I would take a transfer of the best growth from that plate and put it on some 'normal' strength agar and see how it grows out. Depriving a culture of nutrients makes it hard to tell if the mycelium is healthy/contaminated or not.
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stroopwafelman


Registered: 03/21/23
Posts: 386
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Re: Agar not colonizing grain, help. [Re: PBJ710]
#28388696 - 07/07/23 06:37 PM (1 year, 6 months ago) |
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In this case there's no way to tell if the contam is coming from my no-pour tek, my SAB sterility tek, the material I'm working with, or god knows what else. So if I put it on nutrient agar and get contams, trying to guess where those contams may have originated from would be the same as in this case. My whole bet was on using water agar to prevent/halt/stop/whatever the contams and get some clean growth for transfers/LC, seems like it's not happening and I am none the wise where I went wrong.
I'm tired, I give up. Thanks for help.
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CocaineBuffet
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Quote:
stroopwafelman said:
I'm tired, I give up. Thanks for help.
Thats bitch made coward talk.
Transfer that plate pin to a few new plates and share how they look in a couple weeks. Put LC out of your mind for a bit so you can get used to A2G first. Make regular MAE agar to start with too.
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stroopwafelman


Registered: 03/21/23
Posts: 386
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Quote:
CocaineBuffet said: Transfer that plate pin to a few new plates and share how they look in a couple weeks. Put LC out of your mind for a bit so you can get used to A2G first. Make regular MAE agar to start with too.
Made 4 plates (2x water agar and 2x LME agar) from one of the more "evenly growing" water agar plates. I think 1 started slowly growing out 2 days later (small ring of what looks like mycelium strands just about touching the LME agar from the donor slice). Don't have particularly high hopes, all those "fresh" plates are pretty old at this point and were stored at quite high temps (28-30C at times) and are probably quite dry already if not also contaminated in case my no-pour tek is also the problem.
Good news on the popcorn jars however - out of 4 quart jars that I made 3 are showing at least some level of growth on the agar slice and in very close proximity to it (touching the grains 1-2mm from the slice), the slices in those 3 jars are also significantly smaller than the big slice in the earlier post that failed to produce any mycelium growth so I expect that to be a very slow colonization if it doesen't stall out or contaminate first.
The 6 LC jars that I made all have a fair bit of mycelium in them as well as sediment that was present there post-PCing, liquid looks more or less as clear as it was before shaking and about the same clarity as it was post shaking as it used to be after a shake before inoculation. It took longer than a week (about 10 days or so to get to roughly 60-70% LC colonization with the mycelium) compared to what LC teks say, however I believe my variety or whatnot is just that slow, since it shown similarly tardy growth on every other media so far without exception.
Edited by stroopwafelman (07/10/23 02:50 PM)
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stroopwafelman


Registered: 03/21/23
Posts: 386
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3 grain jars
  
2 LC jars (darker one is one out of three from a batch with 0.5% LME while the lighter one of three from a batch is 0.3%, the lighter ones were also inoculated 3-4 days after the darker ones, all were inoculated using the Josex' poke)
 
Fresh LME petri with a water agar slice transferred to it showing some initial growth
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SplendidSwimmer
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I hope you take this the right way, but you're setting yourself up for failure.
Why are you making 6 LCs? LC is more prone to contamination than agar and you can't see the contamination. If you can't get the agar right why would you be able to get LC right.
Stick to the basics and let's get some solid wins under our belt before we move to the next step - regardless of what your long term goals are.
Make loads of agar plates. Make lots of transfers. Learn what contaminated plates look like. You can even test your LCs on those plates.
Right now you're skipping steps and adding complication (water agar with minimal growth to grain?!). Just follow a basic agar recipe and try to make plates that look like the agar envy thread.
I feel like you're really frustrated right now, and when you blast some of that questionable LC onto grain have three days of great growth followed by stalling or complete contam meltdown you're going to be even more frustrated.
Take it slow, follow the steps in order, have some success, and then progress.
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starvinghooker
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Registered: 04/16/23
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Good to see something happening with your grain. Don't be discouraged if the growth is slow. Be happy if it's continuous π
Definitely do lots more transfers. Xfer from the water plate to multiple nutritious agar plates and your grains will be happy af!
Had some helpful folks here lead me toward agar when I first noobed the fuck out of an AI1 bag and I've not looked back.
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MadSeasonStudent
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What is the temperature where you have your jars at?
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stroopwafelman


Registered: 03/21/23
Posts: 386
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Quote:
MadSeasonStudent said: What is the temperature where you have your jars at?
Everything (LC/Plates/Grain jars) is stored at 23-24C
Quote:
starvinghooker said: Definitely do lots more transfers. Xfer from the water plate to multiple nutritious agar plates and your grains will be happy af!
Did a few transfers 2x to water agar and 2x to LME agar. Here's the most grown-out plate so far (posted in agar envy however received 0 commentary on it). It's an 80mm glass no-pour with visible smudging on the lid on the inside that I can't seem to get rid of no matter how I make the no-pours (especially visible with a flash on).
The other LME plate has a smaller piece that broke during transfer so it grew out only about half the size of this one, the growth also looks even just like the pate in the pic above. So do the water agar plates, but it's a lot less "fluffy" probably due to nutrition as the previous water agar plates were also pretty "flat".
Edited by stroopwafelman (07/15/23 08:03 AM)
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