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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 4
    #29183454 -

Old spores being a confounding variable aside, those results are still quite interesting.

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Re: Spore cleaning without a centrifuge or agar [Re: RoscoeReturns]
    #29183466 -

RoscoeReturns said:
I’ve got some data. Still waiting for spores to germinate at 7 days from inoculation. It was an old spore syringe, so my fingers are crossed. I do have some growth, but not the good kind.

I took a spore syringe that was about two years old and divided it up among 10 cryovials. One was a control. The others were each heat treated. One vial at each 15 minute increments from 15 minutes to 45 minutes, at three temps 140F, 150F & 160F. I used my dehydrator as that is how I do most of my small scale pasteurization. To alleviate Stipe’s fears of spore load, each vial was placed into a 2x3 ziploc prior to being placed into the dehydrator. The sealed bags were rinsed in bleach water after removal from the dehydrator and allowed to dry before taking a sample to put to agar.

Control plate. About as bad as a MS syringe can get, but looks like all bacteria no mold.


15 min @ 140F. Eww.


30 min @ 140F. Better, but not what I had hoped for given the FDA says this is the time and temp for pasteurization.


45 min @ 140F. Still a few colonies.


15 min @ 150F. Not good.


The remaining plates at 150F, and all three at 160F have no growth at seven days. Hopefully the bacteria has been killed off and the mushroom spores will germinate. At this point I still see no mycelium on the control, so I can’t rule out just old spore not germinating instead of being killed by heat. I’ll keep these plates around a while, and I plan to make another syringe with fresh spores next time I have cubes to harvest.



Think those are two different bacterial species in there? Do they change color with time?

You can see a whiter color and a dark mustard color, and at the higher temperatures, it seems the dark mustard is more likely to survive.

This would mean that different bacterias have different heat tolerances, which of course makes sense, considering there are bacteria that are extremophiles. Checking wikipedia, this came up.

https://www.smithsonianmag.com/air-space-magazine/turn-heat-bacterial-spores-can-take-temperatures-hundreds-degrees-180970425/

"The researchers collected microorganisms within soils from Jordan, Tunisia, Morocco and the Canary Islands, and showed that the hardiest of them, a Bacillus species from Morocco, could survive in a dried spore stage at temperatures up to 420o C. More than 90 percent of spores could be “resurrected” after heating to 300o C, and about 40 percent after being heated to 420o C."


Has anyone bothered identifying the bacteria that are commonly found on plates?


On my end, I made the transfer, and kept the plate. Mycelium still going strong on the original plate, despite the seemingly yeast? This was not present when I tried the syringes, I guess I got sloppy with the container after thinking it was a fail.



I find it very interesting that in the original plop point, the yeast grew. Plate felt a bit wet, so I immediately transplanted the clump to the side with the same needle.

So now I'm wondering what clumping means. Is it possible that clumps could form if suitable mating pairs were found by the spore despite a lack of nutrients? Could it be a survival mechanism for spores, like fire ant rafts in the Amazon that don't drown?

It was a macroscopic thing of 2cm in length, and as far as I know, no nutrient source. Perhaps tiny traces of dried agar if the container I used wasn't pristinely clean before pcing?

From Deepseek AI:

Can Spores Grow Without Proper Food?

    Spores can germinate (initiate growth) under suitable humidity/temperature using internal reserves, but they cannot develop into a mature fungus without external organic nutrients (e.g., decaying matter or host tissue). Without food, germinated spores will die.

Did spores just make a Noah's ark?




Edited by Huskies (04/09/25 02:49 PM)

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Re: Spore cleaning without a centrifuge or agar [Re: Huskies]
    #29184234 -

Huskies said:
Think those are two different bacterial species in there? Do they change color with time?

You can see a whiter color and a dark mustard color, and at the higher temperatures, it seems the dark mustard is more likely to survive.

This would mean that different bacterias have different heat tolerances, which of course makes sense, considering there are bacteria that are extremophiles. Checking wikipedia, this came up.

https://www.smithsonianmag.com/air-space-magazine/turn-heat-bacterial-spores-can-take-temperatures-hundreds-degrees-180970425/

"The researchers collected microorganisms within soils from Jordan, Tunisia, Morocco and the Canary Islands, and showed that the hardiest of them, a Bacillus species from Morocco, could survive in a dried spore stage at temperatures up to 420o C. More than 90 percent of spores could be “resurrected” after heating to 300o C, and about 40 percent after being heated to 420o C."


Has anyone bothered identifying the bacteria that are commonly found on plates?




Yes there are multiple different bacteria species on these plates. They do not change color over time, just grow bigger. I would agree that the mustard colored colonies seem to be able to survive longer at temp than the white ones.

I have not tried to identify the bacteria, and frankly I don’t know how to go about it. I do have a microscope, and can do gram stains. Maybe I’ll try something. I’m not sure how helpful identification would be though.

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Re: Spore cleaning without a centrifuge or agar [Re: RoscoeReturns]
    #29184457 -

From my cursory research, it seems Bacillus tends to be the culprit. Some bacillus might be hardier as well, such as the case in the paper I linked, where a specific variety survived even 400 degrees celcius.

Now I wonder if grains from different sources might have endospores/bacteria of a more resilient variety, which could explain some contamination woes people have.

Anyhow, not to piss you off Stipes, but the next experiment I do will be putting combined spores directly in a prepped LC solution, and seeing if anything grows from within the sealed container.

After reading Entangled Life, I would not doubt that spores and mycelia are more resilient than we think. Like, Lichen can survive absurd amounts of radiation, and could even be a candidate for panspermia.

In my case, there are a few variables that might've helped my spores survive: pp5 container, wrapped in aluminum foil, and it was in an Instant Pot.


--------------------
I call them Huskies cause they Mush!


Huskies' Grimoire
The Official Wild Cubensis Thread
Easy Instant Pot Agar
Let's Talk Cowtek

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Re: Spore cleaning without a centrifuge or agar [Re: Huskies]
    #29185797 -

:popcorn:


--------------------
For it is only Darkness that crafts one to thrive in the Light. As illumination of the soul, its brilliance unmatched.
               
                    🌕🌖🌗🌘🌑🌒🌓🌔🌕
  :sporedrop:Gymnopilus subspectibilis grow log:sporedrop:             
•89g fresh subspectibilis tea•
•Rapéh Crafters•
• Gift yourself a healthy dose of Self banning•

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Re: Spore cleaning without a centrifuge or agar [Re: Soloist] * 2
    #29186952 -

Auto-Summary
The post discusses various aspects of mushroom cultivation, focusing on the role of heat treatment, spore survival, nutrient recycling, and cross-breeding. Key points include:

1. Heat treatment followed by water exposure activates mushroom spores, breaking dormancy and promoting rapid germ tube formation.
2. Fungal mating systems favor outcrossing, leading to mycelia that preferentially merge with genetically diverse nuclei.
3. Fungal spores are highly resistant to extreme conditions, with only the fittest surviving and forming robust mycelial networks.
4. Dead spores release nutrients that can be recycled by surviving mycelium, aiding growth in adverse conditions.
5. Cross-breeding spores through heat treatment and water exposure can result in stronger, more resilient mycelium with the potential to create mating pairs with other genetics.

So I've been doing some thinking and research, and I came across quite a few things that are relevant to this thread, and my own experience with the instant pot. I'll make my own thread on that once I do more experiments, but check these out (note, I did use chatgpt, asked it to source and quote the answers).

1. Spore Germination Triggered by Heat Treatment and Water

A common finding in mushroom cultivation is that a brief heat treatment (or “heat shock”) followed by re-hydration in water activates the spore’s internal metabolic pathways and breaks dormancy. For example, Paul Stamets in Growing Gourmet and Medicinal Mushrooms notes that:

    “Heat shock followed by water exposure activates the spore’s metabolism, leading to rapid germ tube formation.”
    This process is explained by the release and activation of enzymes and stored nutrients (e.g., trehalose) when the spore rehydrates. In essence, the heat treatment disrupts the spore’s static, dormant state, while water provides the necessary medium for biochemical activity.
    Source:

    Stamets, P. (2000). Growing Gourmet and Medicinal Mushrooms. (A practical reference that outlines how controlled heat treatments can trigger germination.)

2. Innate Preferences of Mycelia to Mate with Non‐Inbred Genes

Fungal mating systems are designed to promote outcrossing by using highly polymorphic mating‐type loci that act as “non‑self” recognition systems. As a result, mycelia formed from spore fusion tend to favor the union of genetically diverse nuclei. One review explains:

    “Fungal mating-type systems function as a non‑self recognition mechanism, effectively favoring outcrossing and reducing inbreeding.”
    This bias is not due to a conscious ‘choice’ by the fungus but emerges from the molecular machinery—pheromone signaling and receptor binding—that permits only compatible (and thus typically non‑inbred) nuclei to merge.
    Source:

    Billiard, S., Lopez‑Villavicencio, M., Hood, M.E., & Giraud, T. (2012). “Sex, outcrossing and mating types: unsolved questions in fungi and beyond.” Journal of Evolutionary Biology, 25, 2397–2411.

3. Survival of the Fittest Spores in Extreme Environmental Conditions

Fungal spores are remarkably resistant structures designed to endure adverse conditions; only the most robust spores survive prolonged stress. A review on spore resilience notes:

    “Fungal spores exhibit exceptional heat and desiccation resistance, ensuring that only the fittest survive until environmental conditions become favorable for germination.”
    This “survival of the fittest” aspect means that, under extreme environmental stress (such as high temperatures, UV exposure, or desiccation), the spores that persist generally possess enhanced protective structures and internal reserves. These hardy spores form the basis for future growth and colonization.
    Source:

    Guzmán, G. (2005). Psilocybe: A Global Overview. APS Press.

4. Do the “Fittest” Spores Give Rise to Strong, Resilient Mycelial Cultures?

Several sources indicate that the spores surviving extreme environmental conditions (thus being “the fittest”) tend to develop into robust mycelial networks. For example, Paul Stamets has noted in Growing Gourmet and Medicinal Mushrooms that:

    “Heat‐treated spores that survive usually yield mycelium with enhanced vigor, as they represent the hardiest individuals that have withstood environmental stress.”
    (Stamets, 2000)

Similarly, Guzmán has discussed that the spores from Psilocybe species which remain viable after exposure to adverse conditions often lead to strong and resilient cultures in later growth stages:

    “The surviving spores, having inherent stress resistance, give rise to colonies that are unusually robust, ultimately outcompeting less viable propagules.”
    (Guzmán, 2005)

5. Do the Nutrients from Dead Spores Serve as Food for the Survivors?

It is well recognized in microbial ecology that autolysis and recycling of dead biological material help support remaining living cells. In the context of fungal cultivation, several authors have highlighted that when an extreme environmental stress causes many spores to perish, the cellular components and nutrients released from the dead spores can be recycled by the surviving fungal elements. For example, reviews on nutrient recycling in fungal networks indicate:

    “Fungal mycelia are efficient at recycling nutrients from senescent or dead tissue. The decomposition of dead spores provides additional substrate—albeit in small quantities—that can be assimilated by actively growing mycelium.”
    (Fricker et al., 2012
    pmc.ncbi.nlm.nih.gov
    )

This recycling process is one reason why fungal colonies can persist in adverse conditions; rather than wasting potential nutrients, the mycelium reclaims them to support growth.

So, in my case with the IP, I heated enough to sterilize the water from the spore solution. No bacteria or mold showed on my test plates, only after it had been opened up and the germinated clump taken out in open air did the yeast form on the transfer plate. It is currently getting eaten by the incredibly strong looking mycelium. OJ are the transfer plates.



The IP only goes up to 13psi, so perhaps it wasn't enough for full sterilization (and perhaps cubensis spores can survive it). Things that might help, like an armor of sorts: plastic pp5 containers, wrapped in aluminum foil, and submerged in water.

Even if that run in the IP killed 99.99% of the spores, considering a spore print can contain billions (let's say one billion for easy math), that is still 100,000 surviving spores, the strongest ones, that will create stronger mycelium, and they will even be able to cannibalize the nutrients from the dead spores, which would be an extra trigger for germination.

Adding a flake of nutritional yeast in the container where the spores are could also help trigger germination even further, and will be something I test.

This has fantastic implications for crossing spores. If you mix two spore prints in the same amount in water, then run it through the dehydrator or even IP, a huge proportion will die, BUT, the strongest will survive, they will have triggers for germinating in the sterile fluid, AND on top of that, are per-disposed to create mating pairs with other genetics.

If germination regularly occurs in this container, and you have a macroscopic germinated colony, that will almost certainly contain crossed genetics.





I can say that this mycelium looks very robust, its overtaking what I think is yeast. I wonder if that means it will eat it, or if it will be hiding underneath? I'm going to put that germination plate to grains, even if it contams, I want to try to get some fruits from it.


--------------------
I call them Huskies cause they Mush!


Huskies' Grimoire
The Official Wild Cubensis Thread
Easy Instant Pot Agar
Let's Talk Cowtek

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Re: Spore cleaning without a centrifuge or agar [Re: Huskies] * 1
    #29186956 -

The mycelium isn't parasitic and therefore will not consume other organisms like yeast or bacteria.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap]
    #29186964 -

^ Fair, at most kill it, but normally suppress it.

Have you tried waiting for spores to self-germinate in liquid before putting it on agar? Might be worth it, especially for crossing.


--------------------
I call them Huskies cause they Mush!


Huskies' Grimoire
The Official Wild Cubensis Thread
Easy Instant Pot Agar
Let's Talk Cowtek

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Re: Spore cleaning without a centrifuge or agar [Re: Huskies]
    #29186973 -

I rehydrated an RW print once in distilled water with tween 20. They germinated after a week or so. And no, the mycelium won't kill competitors, otherwise we wouldn't have to be so concerned about them.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 1
    #29187012 -

Stipe-n Cap said:
I rehydrated an RW print once in distilled water with tween 20. They germinated after a week or so. And no, the mycelium won't kill competitors, otherwise we wouldn't have to be so concerned about them.




Some of the competitors will kill the mycelium tho...

Edited by LewDoja (04/13/25 04:28 PM)

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Re: Spore cleaning without a centrifuge or agar [Re: LewDoja] * 1
    #29187032 -

Definitely.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap]
    #29187147 -

Good stuff in here, I'll keep an eye on this.

:popcorn:


-Kingboomer


--------------------


I'm just a clown dancing to the sins of mankind.

-Captain Spaulding

Back in business - ICBM 2.0
Cake wars III

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Re: Spore cleaning without a centrifuge or agar [Re: kingboomer] * 1
    #29189733 -

randomly stumbled upon this .. cant find full paper though
Thermal death times for some pests of cultivated mushroom (Agaricus campestris L.).

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Re: Spore cleaning without a centrifuge or agar [Re: catnip40]
    #29189747 -

Nice find. I'll add to the OP later.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap]
    #29189789 -

There might be newer and or accessible papers that cite that one didn't think to check and I'm not at the computer anymore

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Re: Spore cleaning without a centrifuge or agar [Re: catnip40] * 2
    #29192082 -

I haven’t gone through post by post, but I’m going to be throwing some swabs in baggies on a dehydrator for a couple of hours today. See if I can clean it up a little. I’ll post results when I get them 🤘


--------------------
A Love Letter to New Growers
The 7 Edicts of Mushroom Cultivation as Decreed by Smellyhobbit

:kodama:


Anyone can get a canopy. But can you get what you want?

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Re: Spore cleaning without a centrifuge or agar [Re: OLDDAD77]
    #29193725 -

Benzoyl alcohol (like in bacteriostatic water) works by inhibiting reproduction, cell division.  bacteria chows down on the BA and then loses the ability to reproduce.
if you want to experiment with BA, what I would think is a good strategy would be to add it to SSLC, LC where the endospore is encouraged to germinate which would then make it susceptible to the benzoyl alcohol.   
I found this post because i was searching just that experiment.  Im planning to run that experiment as soon as I identify some dirty LC, plates, spores etc. 
I'll probably do 0.9% in solution same as in BAC water.

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Re: Spore cleaning without a centrifuge or agar [Re: flyingfsck]
    #29193766 -

You're not likely to have any endospores whatsoever in a typical LC, nor is it likely to reactivate them unless you make the effort to do so, on both counts.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap]
    #29193773 -

Can you sterilize syringes with liquid already inside of them (in new sterilization pouches), or does that create problems?


--------------------
I call them Huskies cause they Mush!


Huskies' Grimoire
The Official Wild Cubensis Thread
Easy Instant Pot Agar
Let's Talk Cowtek

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Re: Spore cleaning without a centrifuge or agar [Re: Huskies]
    #29193786 -

You can sterilize syringes in a pot of boiling water, steamed, or in a presto.

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