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InvisibleStipe-n CapMDiscord
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Re: Cultivation General Discussion [Re: Lenz]
    #27375728 - 07/05/21 10:10 PM (2 years, 7 months ago)

Quote:

Lenz said:
mycotoxins created by mold could be an issue.




Good call, I hadnt thought of that. It will be interesting to see what happens.

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Offlinegone-pear-shaped
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Re: Cultivation General Discussion [Re: Stipe-n Cap] * 1
    #27375730 - 07/05/21 10:29 PM (2 years, 7 months ago)

Quote:

p9hu7 said:
Another great test of endospore destruction would be to make an LC broth with a handful of different types of grain in each LC. If there are surviving endospores they should readily reproduce in the broth after about 8 hours, as endospore reactivation takes 8 hours when in a nutrientrich environment. They're killed too quickly in the broth alone because it's a liquid but should hypothetically be protected by the grain if the survival hypothesis is correct.  If they survive the broth will make for rapid expansion of the bacteria. 



I think it would be valuable if you put in some grains whole/dry versus powdered. It might tell us whether dryness is the reason grains tend to have microbes that are hard to kill.

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OfflineSpaceBaby
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Re: Cultivation General Discussion [Re: Stipe-n Cap]
    #27375796 - 07/06/21 01:25 AM (2 years, 7 months ago)

Quote:

p9hu7 said:
Yeah exactly, im just enthusiastic about cult talk, trying not to come across as an abrasive cunt because that's definitely not my intention..




I appreciate. your OMC persona, as well as the wealth of info.

just sayin


--------------------
SpaceBaby
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InvisibleTedsDead
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Re: Cultivation General Discussion [Re: SpaceBaby]
    #27375820 - 07/06/21 02:19 AM (2 years, 7 months ago)

Indeed.


Ya, the toxins ect... was what I was imagining.

Dont worry p9 your def not a dick to anyone. I was joking but maybe not so much about the unnamed😅


--------------------
weed gets you through times of no money better than money gets you through times of no weed...  -the fabulous furry freak bros
If you can buy it, you can burn it!



https://www.shroomery.org/forums/showflat.php/Number/25947396#25947396

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OfflineSpaceBaby
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Re: Cultivation General Discussion [Re: TedsDead]
    #27375851 - 07/06/21 03:42 AM (2 years, 7 months ago)

Quote:

TedsDead said:
Dont worry p9 your def not a dick to anyone. I was joking but maybe not so much about the unnamed😅




The greater the knowledge on offer, the less notice of dickheadedness there is, imo. The easily offended are pussies. And anyway, you're not a dick. The last few years have shown us all what a true dick is like.

More importantly [to me] I just put 30 cups of millet to soak for 10 jars of new-to-me grain spawn, à la your TEK. STOKED.
But I can't fucking find my fucking brewer's bag to fucking strain it with and those fuckers are teeny.

Do you reserve the soak water for agar or LC?

All I got for you p9 is :hatsoff:

Space

Post scriptum:  Anybody done a "Preserve Fruits in Honey TEK?'
Post post scriptum: I FUCKING LOVE CELLTREAT PETRIS


--------------------
SpaceBaby
SPACEBABY'S LAGM22 THREAD
MUSHBOY'S SHROOM TEA TEK

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Edited by SpaceBaby (07/06/21 03:43 AM)

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InvisibleJosex
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Re: Cultivation General Discussion [Re: SpaceBaby] * 1
    #27375857 - 07/06/21 03:52 AM (2 years, 7 months ago)

Can't wait to tell ya "I told you, dude", now that you've quit oats and are going to do millet. :hehehe:
All this talk about surviving endos in grain... all I now is that I've come across shit that I could not use, many times. Take that as you will.

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InvisibleJosex
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Re: Cultivation General Discussion [Re: Josex]
    #27375864 - 07/06/21 04:17 AM (2 years, 7 months ago)

I would like to openly challenge anyone (especially those that believe that the PC kills all endos in the grain) to do what I would call a proper test to finally dispel any doubts.
And the following is in my opinion a proper test:

1. Choose a grain of your choice, do your usual 'proper' prep that gives you success on the reg. Inoculate with tested LC. Leave out a control jar.

2. Use the same grain to do the worst possible prep (grain will be properly hydrated).
I suggest soaking the grain for 6 days until it smells like death, then strain and load wet so they finish hydration in the PC.
Want to add that it's perfectly possible and actually easy to do this prep and have properly hydrated grain, we don't want over-hydrated grain for this test. Inoculate with the same tested LC. Leave out a control jar.

My prediction is:

1. The grain prepped as per usual will fare well and grow healthy. Control jar will be fine too.

2. The second prep will give you some really bacterial spawn that will crap out as soon as you spawn it. However (and this is the interesting part), the control jar will be fine.

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Offlinegone-pear-shaped
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Re: Cultivation General Discussion [Re: Josex]
    #27375870 - 07/06/21 04:48 AM (2 years, 7 months ago)

Josex, shouldn't you modify the experiment so there is also a control jar based on prep #2? In any case, it's not a fair experiment because of all the metabolites and pH changes that occur as a result of the pre-fermentation in prep #2. The grains will probably be sprouted and turning acidic. It's not an apples to apples comparison.

Anyway, I just switched from millet to wheat. I'm tired of all my jars looking the same, and not being able clearly see mycelium characteristics. My millet jars all looked almost the same, and I couldn't easily tell which ones looked healthy or unhealthy.

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InvisibleJosex
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Re: Cultivation General Discussion [Re: gone-pear-shaped]
    #27375875 - 07/06/21 04:57 AM (2 years, 7 months ago)

Yes I also said to leave a control jar for prep #2.

I suggested that prep because the bacterial count will be very high at the end of the soak and you're going to end up with way more endospores than you started with.

A subset of the endospore forming bacteria will keep producing endospores regardless of an abundance of nutrition, this has been studied. It's also been my experience that prolonged soak times translates into more bacterial spawn.

I don't know about pH swings due to fermentation, all I'm saying is that if you do that prep I'm willing to bet you'll get bacterial spawn, the kind of thing you can put under a microscope and see it for what it is. If the PC kills everything and our inoculant is clean, that shouldn't be there, right?

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InvisibleJosex
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Re: Cultivation General Discussion [Re: Josex]
    #27375882 - 07/06/21 05:10 AM (2 years, 7 months ago)

Anyway, maybe 6 days was a lot lol.

Maybe a +72 hours soak for prep #2.

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Offlinegone-pear-shaped
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Re: Cultivation General Discussion [Re: Josex] * 1
    #27375886 - 07/06/21 05:12 AM (2 years, 7 months ago)

Okay, but that's not the way scientists typically test for contamination. It's a test for growing conditions (that may or may not coincide with lack of bacteria). A typical experiment should be more roughly like: take a one gram sample, macerate it with sterile water, filter and mix with buffer, glucose, and peptone, incubate for 8 hours, then make various dilutions and streak petri dishes to count the CFUs.

It's been a long since I read a paper like that, so I'm sure my memory is somewhat off. The point is that they don't use a proxy result (mushroom cult quality) when they can, with some effort, look at the direct outcome: how many live microbes are there?

I'm not saying your experiment has no merit. But it would tell us more about prep methods for cult, less about PC sterilization.

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InvisibleJosex
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Re: Cultivation General Discussion [Re: gone-pear-shaped]
    #27375891 - 07/06/21 05:16 AM (2 years, 7 months ago)

Who said I'm a scientist? I just know from experience prep #2 will give you bacteria and if that's the case that means the PC could not destroy all endospores. :shrug:

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InvisibleJosex
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Re: Cultivation General Discussion [Re: Josex]
    #27375898 - 07/06/21 05:29 AM (2 years, 7 months ago)

Quote:

Okay, but that's not the way scientists typically test for contamination. It's a test for growing conditions (that may or may not coincide with lack of bacteria). A typical experiment should be more roughly like: take a one gram sample, macerate it with sterile water, filter and mix with buffer, glucose, and peptone, incubate for 8 hours, then make various dilutions and streak petri dishes to count the CFUs.





Wrong, that's not the way to wake up a surviving endo in the grain. There's no better way to do that than through the action of the mycelial colony actually colonizing the grain. Sounds woo woo? Well I've seen it many times and it usually starts being more noticeable when the spawn is ~80% colonized.
We are no scientists here and we go by anecdotal evidence because that's what we have, not to say anecdotal can't kick some ass.

Fwiw, I know many respected members here that know that some endospores in the grain can recover from a cycle even if most are destroyed. These members ain't vocal about it cuz they know it's a touchy subject.

I also know a dude that ain't a member that grows more pounds per month than most culters here combined. He also knows this. That's the kind of people I trust.

But again, seen it too many fucking times myself.

Edited by Josex (07/06/21 05:46 AM)

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InvisibleStipe-n CapMDiscord
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Re: Cultivation General Discussion [Re: Josex]
    #27375930 - 07/06/21 06:30 AM (2 years, 7 months ago)

"Reactivation of the endospore occurs when conditions are more favourable and involves activation, germination, and outgrowth. Even if an endospore is located in plentiful nutrients, it may fail to germinate unless activation has taken place. This may be triggered by heating the endospore. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It is commonly characterised by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress.

Outgrowth follows germination and involves the core of the endospore manufacturing new chemical components and exiting the old spore coat to develop into a fully functional vegetative bacterial cell, which can divide to produce more cells"

Perhaps you notice endospore reactivation at roughly 80% colonization due to increased internal temps generated by the mycelium which incubates the endospore?


It seems reasonable that soaking for 24+ hours in warm nutrient rich water should be enough to reactivate endospores prior to to cycle which isn't  what we want, we want dormant endospores to sterilize, not vegetative bacteria, as vegetative bacteria is  ery easy to kill.


"While significantly resistant to heat and radiation, endospores can be destroyed by burning or by autoclaving at a temperature exceeding the boiling point of water, 100 °C. Endospores are able to survive at 100 °C for hours, although the larger the number of hours the fewer that will survive. An indirect way to destroy them is to place them in an environment that reactivates them to their vegetative state. They will germinate within a day or two with the right environmental conditions, and then the vegetative cells, not as hardy as endospores, can be straightforwardly destroyed."

Boiling for prep shouldn't  be sufficient conditions to reactivate nor kill a significant amount of endospore. So, boiling wheat as per usual to hydrate, then load/steilize, then incubate sterilized jar on a shelf with a heat pad for one week to allow any surviving endospore population to reactivate in a warm, nutrient rich environment should be the best course of action.

After incubation period remove individual grains via sterile lab tweezers to be cultured on agar, maceration process may contaminate the sample because I'm not a proper lab tech.

This seems like a good test. What do you think?

Edited by Stipe-n Cap (07/06/21 06:47 AM)

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InvisibleJosex
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Re: Cultivation General Discussion [Re: Stipe-n Cap]
    #27375934 - 07/06/21 06:46 AM (2 years, 7 months ago)

I know those quotes from memory, read them too many times.

All I know is that logic tells us (at least prior to actually experiencing) that soaking for prolonged periods should germinate all endos and therefore the now vegetative bacillus will be easy to kill.
The thing is that it's very likely you end up with more endospores in the grain after a long soak, as an important subset of the population still will snuff themselves out to produce endos even in an abundance of nutrition.

This is the theory, in practice I'd say hell yes this is true. The times I did a long soak I saw the worst results.

I didn't choose that prep for the made-up experiment randomly. I suggested it because I know that's the more likely way you'll end up with bacterial spawn even if you do a proper cycle and taking that your inoculant is perfectly axenic.

I really think most of the info regarding endospores out there is not even relevant to mushroom cultivation when it comes to grain spawn. I could tell you anecdotes to write a book that made it pretty obvious these things from hell can recover from a cycle. Most of them will be completely destroyed, some not.

Edited by Josex (07/06/21 06:57 AM)

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InvisibleStipe-n CapMDiscord
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Re: Cultivation General Discussion [Re: Josex]
    #27375944 - 07/06/21 06:54 AM (2 years, 7 months ago)

ok, well I'll run both scenarios just to be sure.

I have no desire to be correct here, I do however want to be in possesion of correct information. I  want to test our fast held beliefs in as controlled an environment as I can manage because I am genuinely curious about what is really going on. No matter what the outcome is it will be super fun to do and we can all have a deeper understanding of whats taking place through direct observation as apposed to anecdotal experience.

I will be super happy with any result.

Culturing the grains after running both scenarios should tell us a lot.

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OfflineA.k.aM
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Re: Cultivation General Discussion [Re: Stipe-n Cap]
    #27375947 - 07/06/21 06:58 AM (2 years, 7 months ago)

A while ago I was screwing around and poured like a spoonful of colonized wbs into an lc broth.

Totally forgot about it until months later and it tested clean. Looked super chunky though.


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InvisibleJosex
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Re: Cultivation General Discussion [Re: Stipe-n Cap]
    #27375952 - 07/06/21 07:10 AM (2 years, 7 months ago)

I've done a good fat amount of testing myself, just not documented. Most of all these last 6 months.

I've done sides by sides consisting of a shitty prep, a normal prep and a "hard" prep. And could observe the results from those three having inoculated with the same proven axenic LC. The "shitty" prep got real shitty on me...

Also left control jars from the three preps that grew nothing at all. Also 'inoculated' some jars with nutrient broth (not colonized) that grew nothing at all. Which made it more obvious my old presumption that the action of the myc colony colonizing the grain does contribute to making these surviving endos recover. I'm aware how this sounds and how this can make me look in the eyes of some, but I've experienced it many times to call it for what it is.

I also happen to have the shittiest source for grain out there, that really helps in deepening your understanding about endos, trust me :lol:

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Re: Cultivation General Discussion [Re: Josex]
    #27375953 - 07/06/21 07:15 AM (2 years, 7 months ago)

Well Im getting on this test today. This way nobody will ever have to say "trust me" again we can just refer to the data.

After removing grains in flow to be cultured, I will then inoculate the jars with agar wedge to see if a live culture was required to activate the bacteria. Spawn run will take place as the grains grow out on plates. This way every base should be well covered, and all conditions satisfied.

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InvisibleJosex
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Re: Cultivation General Discussion [Re: Stipe-n Cap]
    #27375955 - 07/06/21 07:23 AM (2 years, 7 months ago)

Hell I'm going to make some documented tests myself, was just too lazy to document it before.

Gonna start by putting some shitty grain to soak for some days to make it even shittier.
Will also do a standard prep and a hard prep. All will be noc'd up with tested LC.

Control jars included. Will also inoculate jars from the three preps with 20ml of nutrient broth.

Let's get the party started.
:etjesus:

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