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InvisibleJHOVAS
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Re: PC'd Grain - Life Span? [Re: Mateah]
    #25846167 - 03/01/19 06:10 PM (21 days, 21 hours ago)

Bacteria gives no fucks


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OfflineBobbit
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Re: PC'd Grain - Life Span? [Re: JHOVA]
    #25870970 - 03/13/19 03:38 AM (10 days, 12 hours ago)

So I got bored at page 2 of cultivation guru 1 says X and I reckon Y? And what would you know. . .

Can anyone fill me in on whether anyone used science here? Did we get to the bottom of this?

I PC'd 12 jars of rye 2 months ago.
PC'd for 2hr.
These have remained refrigerated with foil hats on.
Stored lying down
Food including dairy and meat Stored in same fridge.
Some grain water from the soak was left in the same fridge with holes in the lid and went green and fluffy (removed at 6 weeks, yep I pay heaps of attention)

I am now watching 3 x G2G jars colonise with p.allenii. . . Yet that ain't sciency enough right.

What say I inoculate some agar plates with individual grains from a few of the jars?

Wouldn't that (hides under chair) prove if anything was alive in there?


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Invisiblevan hatton
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Re: PC'd Grain - Life Span? [Re: Bobbit]
    #25870990 - 03/13/19 03:55 AM (10 days, 12 hours ago)

You would in theory use the same grains soak water for agar. The reason I say same (same 50 lb bag) is because grain varies so fucking much.

Some things don't grow on lme and pda that grow on our grains.

That would probably be your best route imo


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OfflineBobbit
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Re: PC'd Grain - Life Span? [Re: van hatton]
    #25871052 - 03/13/19 04:41 AM (10 days, 11 hours ago)

Well, I have some MEA poured, and not sure I'll soak any rye before making more agar as much of this thread tells me I'm wasting my time anyways...

However if there is a need to put speculation to sleep, then I may be convinced.


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Invisiblestareatclouds
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Re: PC'd Grain - Life Span? [Re: Bobbit]
    #25871102 - 03/13/19 06:16 AM (10 days, 10 hours ago)

No idea why you stored them in the fridge or what you think this proves either way? If you'd bother to read the thread, I think you'd have a better understanding of the issues and be able to answer your own questions.

Here are random thoughts:

  • There's a huge difference between "is this jar viable?" and "is every single living thing within this jar, including currently dormant endospores, rendered completely sterile?" And people keep interchanging them, making the debate a PITA.

  • The currently accepted idea of our "sterilization" is just killing enough vectors for a "window" where mycelium can get a head start. Thus, we believe that even WITH proper "sterilization" for mycology, we're still dealing with SOME bad guys (and obviously there are people debating that concept now).

  • If the above is correct, fruiting a jar successfully, no matter how long it sits after a PC cycle, doesn't prove everything in the jar was sterile. You don't need true sterility to spawn something and get fruits. People fruit bacterial jars all the time.

  • An endospore is specifically designed LIKE THE TERMINATOR and can withstand extreme conditions and lie in stasis until favorable germination conditions are met. Those conditions, to my knowledge, are primarily influenced by moisture (not dry), and temperature (not cold, not super hot).

  • If endospores survive your PC cycle, it's not likely they'll immediately germinate once it cools down to room temperature. As I said to Mateah, you don't open a bag of dry grains and puke from nasty bacteria stench. But if you soak your shit for 24 hours and leave it out a day or so, it reeks. Because those are conditions that catalyze the vegetative state (active bacteria), as well as endospore formation. So pointing to a dried out jar afterwards like, "See? Nothing!" is not indicative of everything being fine.

  • If the above is true, you'd be more likely to eyeball something's wrong when you introduce moisture and warmer conditions, like after inoculating. This is just my guess and could be wrong. And not all endospores are the same species and behave the exact same.

  • Dropping a single grain on agar doesn't speak for the entire jar. Why would it? Why can't another grain be harboring bacteria that wasn't killed like others? No idea if this is optimal, but I suggested repeatedly to mix in water to cover the grains and leave it within activation/germination temperatures. I think this would be a better test than a grain on agar. Google for endospore activation/germination conditions.

  • No matter how many times someone repeats any success with this, we aren't closer to knowing at which point in any given jar is truly sterile (or even if they WERE sterile). Original endospore count, grain type, prep method, sterilization time, PSI used, steam vented, and 3753 other things all make my situation different than yours. Maybe your grains are sterile and your inoculant isn't? Maybe my sterile technique sucks and yours is good? Perhaps I'm using shitty SFDs? Both things muddy our results, but aren't the fault of the grain. And again, even if it works, viability is different from sterility.

  • Personally, I care most about viability, and that's why I engage in these discussions. That's why I've just told people to try and use their old jars, not point at them. And while it's not concrete, I would feel better about using older grains if I knew that 20 other people had replicated this success over 100 jars each, at least establishing that the old idea of "a few days" was not as concrete with a certain baseline of time.

  • Related to above + people interchanging viability and sterility: while viability is most important, the science aspect of it is dope, and I wish I knew more. It would be amazing to know how many endospores were present AND the minimum time to truly sterilize. But we can't, and likely won't. And it's unnecessary for our purposes. So let's focus on those.

  • For what it's worth, I've used old grains successfully before. And they'd possibly been inoculated previously. I inoculated 5 WBS quarts with APE BLI. It showed no growth and sat on my shelf for 2 months (plastic lids IIRC). Curious if I even inoculated them in the first place, I smelled each of them and found no foul smell. So I resterilized them and inoculated them via AA+ G2G. They wound up sitting at 100% for 3-weeks on accident. I broke them up to spawn and somehow forgot them again so the grain starting recovering. I eventually spawned, cased, and fruited it on 5/26/18. The first flush was 26 days later, but average was 20 for that clone. Yield average for that clone/tub was 1407g. Our old, resterilized and neglected WBS was 1897g, a record for that variety for that spawn volume. No green or anything and smelled fine, but looked shot after harvest so I tossed it.

  • I've also used a bunch of older jars that looked fine with very poor results. No mold or obvious bacteria, but unreal low yields on proven cultures, which could be related to grain. But I'm not 100% either.

That was longer than I intended. And I could be wrong about any of it, so feel free to correct me if so! Also, anybody interested in documenting this? Then check this.


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InvisibleMad Season
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Re: PC'd Grain - Life Span? [Re: stareatclouds]
    #25871342 - 03/13/19 10:57 AM (10 days, 5 hours ago)

Another thought to consider is if grains could get truly sterile, why aren't there any grains freshly harvested, then canned? It would be nice af to get fresh, hydrated grains. It would then mean we wouldn't need to hydrate, and potentially if people did things right, wouldn't need to PC. However, it's clear that it won't work. Companies know grains won't last like that. They know that for long term storage, like in the span of years, drying them, or canning liquid form like oat meal is the way to do it.


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Re: PC'd Grain - Life Span? [Re: Mad Season]
    #25871374 - 03/13/19 11:32 AM (10 days, 4 hours ago)

I had one jar leftover that I couldn't fit into the PC.. I just had to throw it out a few days ago because of green mold. The others seem and look fine.. also look hydrated still.

My agar will be finished by this weekend. I was going to drop my wedges to these grains... after all of this reading I'm not sure if that's a good idea or not lol. But they're already all prepped and do look fine. So I don't fucking know. Maybe I'm just better off prepping all new jars because I've read it could mean lower yields... though that's not completely backed up.

It will be about 15 days since PC'd. I don't think thats extremely long.. but two weeks vs 1 or 2 days...


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Re: PC'd Grain - Life Span? [Re: Mad Season] * 1
    #25871375 - 03/13/19 11:32 AM (10 days, 4 hours ago)

They make canned corn. Canned oats or barley might not be a hot seller


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InvisibleMad Season
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Re: PC'd Grain - Life Span? [Re: bodhisatta]
    #25871383 - 03/13/19 11:38 AM (10 days, 4 hours ago)

True that. It's hard to say for sure, just a thought I was having the other day.


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Re: PC'd Grain - Life Span? [Re: Mad Season] * 1
    #25871463 - 03/13/19 12:22 PM (10 days, 3 hours ago)

Canned grains are in liquid tho. Never loose hydrated grain like ours. No places to trap air if you've got it all in liquid


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Re: PC'd Grain - Life Span? *DELETED* [Re: stareatclouds]
    #25871592 - 03/13/19 01:40 PM (10 days, 2 hours ago)

Post deleted by bodhisatta

Reason for deletion: If you're going to call people ignorant you can have your entire rebuttal deleted.


Edited by bodhisatta (03/13/19 02:44 PM)


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Invisiblestareatclouds
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Re: PC'd Grain - Life Span? [Re: Bobbit]
    #25871669 - 03/13/19 02:27 PM (10 days, 1 hour ago)

I spent quite a bit of time trying to recap relevant points to my understanding to catch you specifically up to speed. And you're being an ass. What are any of us going to have that is beyond circumstantial evidence?

We put plates and slants in fridges to slow down growth, yes, but that's for storing clean cultures. The point of these experiments is to figure out if you have bacteria/living things in your jars, not mask it by slowing life down. That's why it's recommended to let stuff grow out a bit before using from fridge, because in a few days, all the bacteria and pr mold spores and shit that wasn't showing will bloom.


Quote:

"what was I trying to prove?" Was more a verification process that being busy (read: absent minded and lazy) can still yield results, and at least some learning for myself, if not the community.




Great. I was asking for this exact clarification because people confuse sterility with viability and your goals would change my answer to you.

You're the only one being smug. You taking a single grain to an agar plate doesn't prove anything and isn't good science. How does taking 3 change anything? Did you skip over the previous discussion AND my bullet points?

If you're trying to test viability, why would sampling a few grains to agar do more than just using the jars? I suggested what I would do if not using them normally: dump sterile water in the grains and see what happens. Even if a single grain contaminated, that doesn't mean the whole jar would've, or even that the single grain would've had it been in the jar noc'd with mycelium and not your agar plate.


Quote:

I hope this encourages staresatignorance to get over their 'we'll never be able to tell so lets make it into a religion' stance.




Yeah, not like I'm the main dude campaigning for a large-scale documented test where we all record results with old grain.

The main point of my post is summed up in the first bullet point. And yet again, you completely miss it. go be lazy and absent minded when you're not involving others in it.


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Re: PC'd Grain - Life Span? [Re: stareatclouds]
    #25871694 - 03/13/19 02:40 PM (10 days, 1 hour ago)

Quote:

stareatclouds said:
The currently accepted idea of our "sterilization" is just killing enough vectors for a "window" where mycelium can get a head start. Thus, we believe that even WITH proper "sterilization" for mycology, we're still dealing with SOME bad guys (and obviously there are people debating that concept now).



I have only ever seen that notion here. It is definitely not accepted in microbiology, food science, or pharmaceutical science. It is not a debate in those fields.


Quote:

If endospores survive your PC cycle, it's not likely they'll immediately germinate once it cools down to room temperature. As I said to Mateah, you don't open a bag of dry grains and puke from nasty bacteria stench. But if you soak your shit for 24 hours and leave it out a day or so, it reeks. Because those are conditions that catalyze the vegetative state (active bacteria), as well as endospore formation. So pointing to a dried out jar afterwards like, "See? Nothing!" is not indicative of everything being fine.



The jar can have enough moisture to support mycelial growth after several months. The drying (or non-drying) depends on relative humidity and mass transport of gas. And, if you kill vegetatively growing bacteria by heating to 100 C, you will see that endospores germinate quite quickly afterwards.


Quote:

Dropping a single grain on agar doesn't speak for the entire jar. Why would it? Why can't another grain be harboring bacteria that wasn't killed like others? No idea if this is optimal, but I suggested repeatedly to mix in water to cover the grains and leave it within activation/germination temperatures. I think this would be a better test than a grain on agar. Google for endospore activation/germination conditions.



So, you accept that individual grains can be sterilized? But not a bulk mass?


Quote:

No matter how many times someone repeats any success with this, we aren't closer to knowing at which point in any given jar is truly sterile (or even if they WERE sterile). Original endospore count, grain type, prep method, sterilization time, PSI used, steam vented, and 3753 other things all make my situation different than yours. Maybe your grains are sterile and your inoculant isn't? Maybe my sterile technique sucks and yours is good? Perhaps I'm using shitty SFDs? Both things muddy our results, but aren't the fault of the grain. And again, even if it works, viability is different from sterility.



Very good point, since apparently some people have contamination problems with (supposedly) sterilized jars. Clearly there are different opinions here. I believe most of the problems are on the operator side of the pressure cooker :smirk:


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Re: PC'd Grain - Life Span? [Re: Pinback]
    #25871718 - 03/13/19 02:49 PM (10 days, 1 hour ago)

Just a reminder to everyone in general. if you need to flame your argument is probably shit and even if it isn't it's still getting dumped


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Re: PC'd Grain - Life Span? [Re: Pinback]
    #25871725 - 03/13/19 02:54 PM (10 days, 1 hour ago)

Quote:

Pinback said:
Quote:

stareatclouds said:
The currently accepted idea of our "sterilization" is just killing enough vectors for a "window" where mycelium can get a head start. Thus, we believe that even WITH proper "sterilization" for mycology, we're still dealing with SOME bad guys (and obviously there are people debating that concept now).



I have only ever seen that notion here. It is definitely not accepted in microbiology, food science, or pharmaceutical science. It is not a debate in those fields.






It is definitely accepted in food science. Sterilization is a game of probabilities, only in food sciences you have the added constraint of maintaining taste.  This is why every relevant government agency suggests you limit how long you store canned goods for (e.g. https://www.cdc.gov/botulism/consumer.html), because no can is truly completely sterile.

And also in microbiology, you program your autoclave cycle against the most common expected contaminants. Every now and then sterilization fails 'cause you have something weird creep in, and you have to throw out your entire experiment, or you sequence something completely different.

For every time period, at temperature you experience a log reduction in the number of CFUs in the thing you are sterilizing.  Basic math tells you that you are unlikely to ever reach zero with a typical CFU load, but rather you take it down to an acceptable level without overly deteriorating the substrate.

It's also why you'll never ever find any sterilization method - beit filters or whatever - promising 100% sterilization rate. It's just not doable.

Something I've always wondered, but never tried or read: What actually happens when you sterilize grains for too long? Do they turn to literal mush?


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InvisibleMad Season
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Re: PC'd Grain - Life Span? [Re: teladi]
    #25871728 - 03/13/19 02:55 PM (10 days, 1 hour ago)

They dry out and go black


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Re: PC'd Grain - Life Span? [Re: Mad Season]
    #25871739 - 03/13/19 02:59 PM (10 days, 1 hour ago)

Even though it's all steam?


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Re: PC'd Grain - Life Span? [Re: teladi] * 1
    #25871740 - 03/13/19 03:01 PM (10 days, 1 hour ago)

:shrug: idk I've definitely left them in pc for way too long, and although it didnt run out of water, the grains were pretty much blackened af. Not like pure black, but pretty fuckin dark. Also yeah, they were much drier than they were going in.


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How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Invisiblestareatclouds
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Re: PC'd Grain - Life Span? [Re: Mad Season]
    #25871776 - 03/13/19 03:23 PM (10 days, 57 minutes ago)

Quote:

Pinback said:
I have only ever seen that notion here. It is definitely not accepted in microbiology, food science, or pharmaceutical science. It is not a debate in those fields.




Do you have links to sterilizing grains with 100% certainty in those fields or any other?


Quote:

So, you accept that individual grains can be sterilized? But not a bulk mass?




I am assuming that not all parts of the jar/bag are always accessed equally by the forces needed to kill all endospores. And not all grains are created equally when it comes to endospore count. From my research, there's clear differences in thermal death times for different species of endospores. Is it not possible certain bits of grain have one and not the other? Is it not definite that grains on the outer wall of the bag get "sterilized" faster than those in the middle? So pulling one of those grains does not equal every single grain. And we don't know what the minimum time is to kill everything, so what's the point?

I have never said a bulk mass can't be sterilized, by the way.


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InvisibleMateah
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Re: PC'd Grain - Life Span? [Re: Mad Season]
    #25871785 - 03/13/19 03:28 PM (10 days, 52 minutes ago)

The way we prep our grain makes it (impossible?) to know wether everything inside has been killed or not because even if it survived it doesn't mean it will germinate and even if it germinates that doesn't mean that it will be able to multiply and spread into threatening numbers. So...

(imo)
To be able to produce any kind of valuable info from testing 'sterility' inside grain would be
to create some kind of grain test jar that has both nutrient rich water and grain inside of it.
That way we can be sure that spores that are still viable will be in 'optimal' environment to germinate
and multiply in.


Any thoughts on what's faulty about this kind of test jar? :takingnotes:


Edited by Mateah (03/13/19 03:29 PM)


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