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SMmack
Stranger
Registered: 10/01/23
Posts: 39
Last seen: 1 day, 38 minutes
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Contam update, I think its coming from the grain.
#28630746 - 01/22/24 07:58 AM (5 days, 23 hours ago) |
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In my last thread I was ranting about a fungicola contam that kept following me around like the plague for months, across Josex pucks and water plates. Then I had some replies saying my latest plates looked clean, so I tried those plates on grain. To be fair I haven't seen any of the same mould that used to haunt me recently, but that didn't mean they were clean. https://www.shroomery.org/forums/showflat.php/Number/28614849
I have some results, and although inconsistent I've got a hunch the bad is all coming from the grain prep. If your impatient skip to TLDR below.
First I sent GT and APE to grain, I had control jars for 5 weeks which I then used to test the samples (more on these later): GT Agar Day 3 Day 7 Day 11 + Back side
    
APE (The spot at 12 o'clock is just where I took a transfer previously) Day 3 Day 7 Day 11 + Back side
     Initially GT looks ok, but APE is yeasty as fuck. Later on the myc stalls out, that sour rot kicks back in as per usual and it turns into my normal grain jar of trich fighting wet rot. I don't know if opening the lid to inoculate triggers this, or what, because things don't generally start to show up until the myc grows 3 grains out.
I also dumped one wedge of that GT in some honey LC [LC-A] Control on the left, 10 Days on the right. Both so clear you can still see writing through them. Probably should have put a marble in there because I can't break those clumps up now.
 
I had *some* issues with sour rot every previous batch, so I ran some controls: Three modified jars, one with micropore tape, two with micropose filters. After 5 weeks I ended up using the two filtered grain jars in my agar-grain above, leaving the micropore tape jar for later. One interesting note. I noticed some growth at 6 weeks on that last grain jar in the top right prior to inoculation. I plan to spawn to bulk by 6 weeks normally, but sus, what usually happens if I inoculate straight away is the myc stalls and sour rot, trich, (or fungicola back when I was growing fungicola) take over, I haven't touched it so did it survive the pressure cooking? I'll post my grain prep below if you are interested.
 
A couple days later I grew my best looking rhizo sample yet.
 And also some of my standard cross style growth, more tomentose than rhizo though so lets call it tomentose.

Sent GT Rhizo [LC-B] (Left) and Tomentose [LC-C] (Right) to LC. After 1 day LC-C is crap. After 6 days LC-B is also looking weird, LC-A, B, C.
  
TLDR_________________________________
So to conclude:
I've got one GT plate that went half to LC and stayed looking clean but the other half went baccy in grain. The other GT & APE plates that looked clean got baccy in LC and in grain. My GT consistently seems cleaner than my APE. The agar plates do not grow without inoculation. The LC's did not get baccy without inoculation. I've had my SAB technique reviewed prior, no-ones is perfect but it's good enough to not deserve 100% grain contam rates. The control grains did show trich after 6 weeks but became baccy a couple days after a week of inoculation, regardless of how soon or late inoculation occurs.
So the results just contradict eachother. 
I don't think the agar samples are the problem though. I can't imagine them looking so clean and then being rank with 3-4 different contams each. I think if I use a smaller inoculation wedge and more tries I'll be able to get some decent looking LC's. LC-B & C could be from using a whole agar plate whereas in LC-A I only used one wedge, therefore less points of failure. Granted LC-A has not been fully tested yet.
I think the problem might be coming from the grain, because that has been consistently terrible ever since I stopped using uncle bens in 2022. I get the same results from Brown rice, so I use WBS because it's cheaper. They've all looked ok until inoculated so I initially thought it was the agar, but now I've seen some growth after 6 weeks, granted the wet rot doesn't show up until inoculated still. There is also the possibility that I have contam that is undetectable on agar, but then that kind of defeats the point of using agar in the first place.
I follow Frank's WBS TEK, my WBS process is below have a look if there's anything that sticks out to you:
Pour WBS in a pot with cold water, drain off any floaters and until the water runs clear. Fill with cold water and soak for 12-24hrs, afterwards put on high heat and once simmering continue to simmer for 8 minutes. Strain and let some of the moisture evaporate off. I'm unfortunate enough to live in a cold climate, so I use a blow-dryer or spread in the oven on low for 10minutes in order to get them to not stick to the walls of the glass. Into modified jars, covered with tinfoil to prevent wetness and PC for 100 minutes @ 15psi. Let cool for 2 hours and shake while still warm. I leave the foil on for extra protection and as a label.
Do you think the wet rot is coming from the grain. If so or if not what should I try on the next batch this week?
My best canopy so far of 2023-2024.
Edited by SMmack (01/22/24 08:01 AM)
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 1,655
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Re: Contam update, I think its coming from the grain. [Re: SMmack]
#28630753 - 01/22/24 08:12 AM (5 days, 23 hours ago) |
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Considering your issues, I would try to continue agar xfers until the middle isn't fuzzy. They still don't look right to me
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zmicroinquisitor
donkey


Registered: 07/27/22
Posts: 129
Loc: Land of Awes
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Re: Contam update, I think its coming from the grain. [Re: SMmack]
#28630766 - 01/22/24 08:27 AM (5 days, 23 hours ago) |
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First focus would be the grain. Sounds like your plates need work aswell, looking under a plate can help identify contam spots that aren't as visable on the surface With lc less is more and contam is pretty much impossible to detect by eye.
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SMmack
Stranger
Registered: 10/01/23
Posts: 39
Last seen: 1 day, 38 minutes
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Re: Contam update, I think its coming from the grain. [Re: LadysKnight]
#28630782 - 01/22/24 08:40 AM (5 days, 23 hours ago) |
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Quote:
LadysKnight said: Considering your issues, I would try to continue agar xfers until the middle isn't fuzzy. They still don't look right to me
So I didn't like it initially, but the middle is always fuzzy, I got enough comments on here saying that's the norm for some genetics to convince me otherwise. T2

T7

Some different spores T6 T1 T3 T6 
That fuzz is always there regardless of if they're new spores or isolated leading edge transfers.
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SMmack
Stranger
Registered: 10/01/23
Posts: 39
Last seen: 1 day, 38 minutes
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Re: Contam update, I think its coming from the grain. [Re: zmicroinquisitor]
#28630788 - 01/22/24 08:44 AM (5 days, 23 hours ago) |
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Quote:
zmicroinquisitor said: First focus would be the grain. Sounds like your plates need work aswell, looking under a plate can help identify contam spots that aren't as visable on the surface With lc less is more and contam is pretty much impossible to detect by eye.
I check under every plate already they appear clean but I seem to get more consistent results when using smaller wedges. Any ideas on how to better the grain?
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 1,655
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Re: Contam update, I think its coming from the grain. [Re: SMmack]
#28630827 - 01/22/24 09:20 AM (5 days, 22 hours ago) |
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As you can see i transferred this one so many times I lost count. I never escaped the fuzz. Tried to pretend it was normal. When this went to grain it took way too long to colonize. Then when spawned to shoe boxes, same. Took 3x as long to colonize. Half the tubs ended in aborts. Clones and spore prints had it. I eventually surrendered and called it fusarium. Had to get new spores and moved houses.
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OldManRiver
Fisherman at large


Registered: 11/12/17
Posts: 416
Loc: Pacific NW USA
Last seen: 7 hours, 39 minutes
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Re: Contam update, I think its coming from the grain. [Re: LadysKnight] 1
#28630838 - 01/22/24 09:40 AM (5 days, 22 hours ago) |
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The hardest problems to solve are those with multiple components. I was struck by your jars going bad before inoculation, but only after some weeks went by. If your sterilization were bad, I would expect them to go south fairly quickly, like within a week or so. If they go bad after sitting for several weeks, I'd be suspicious of my filters. I would investigate this area first.
I'd make a batch of jars, and put two or three of them on the shelf and watch. I expect my grain bags to be able to sit on a shelf indefinitely without contamination.
I had a period of time when the culture I was using was sneakily bad. It would show white on agar, but then would go bad only after a couple weeks on the agar, which I refused to believe was the culture itself for a while.
Good luck in your analysis.
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