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Mushrooms, Mycology and Psychedelics > Mushroom Cultivation
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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 7
    #29151260 -

Ok here I am a week later with some results to share.



Above is listed times and temperatures ran. Along with 2 of the 3 control plates. The third was too full of condensation to even bother sharing. They all looked the same regardless.

I got stingy when it came to testing each vial out after cooking them and only used 1 plate per centrifuge.

Plate C yielded enough good growth for me to potentially isolate away from contamination successfully. Plate D, although ran exactly the same hasn't yielded much but a tiny spot of bacteria.

Plate B is too contaminated to do anything with. The rest of the plates have provided zero growth. From here I'll dial in the process using the range I used for plates C and D.

Ideally it would be best to inoculate at least 5 plates minimum, when starting germination from wild spores, to increase your chances of success. I think this coupled with a cooler germination plate storage (55-60) will yield some phenomenal results.





In the meantime, I'm going to transfer from this dish C. I've been dying to get this culture rolling! Thank you Stipe :smile:

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Re: Spore cleaning without a centrifuge or agar [Re: PhenoDreamers] * 3
    #29151276 -

Excellent data. Attention to detail and and clear tracking. :rockon:
That's a very significant reduction. Very promising.

Once enough data has been collected, I'll pool it in the OP with credit to thw contributors.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 1
    #29152326 -

Update:

Both syringes didn't grow any mold, but did grow bacteria. No mycelium grew.

3 hrs @153 was enough to kill the mold, but not the bacteria it seems.


--------------------
I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: Spore cleaning without a centrifuge or agar [Re: PuffM4gicDr4gon] * 1
    #29152338 -

Did you submerge in a bath and remove all potential cross contamination vectors?

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 2
    #29152355 -

160f appeared to kill everything. Not sure the time, fell asleep.  But probably two hours.

Pasteurization method was to take a cap off of a fresh fruit and stuff it in a cryiovial of distilled water.  Fruit was pan cyan that likely had reduced spore load to begin with.

I will try again at a lower temperature, following Supa's lead and with a cap dried on low, so the spores are hardier.


--------------------
And if I only could
I'd make a deal with God
And I'd get him to swap our places
I'd be running up that road
Be running up that hill
With no problems

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Re: Spore cleaning without a centrifuge or agar [Re: tree frog] * 1
    #29152361 -

Stipe-n Cap said:
Did you submerge in a bath and remove all potential cross contamination vectors?


Yup, it was floated in water, and I turned the syringes every 30 mins or so.

You can see what I did here:

https://www.shroomery.org/forums/showflat.php/Number/29135984#29135984


--------------------
I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: Spore cleaning without a centrifuge or agar [Re: PuffM4gicDr4gon] * 2
    #29152860 -

Looks like Supa is leading the charge with results so far.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 2
    #29154106 -

update from the first test, as well next test.

Out of the five plates I inoculated they all eventually contaminated with no correlation between mold spore count and contamination time. All contaminated with bacteria with no signs of mycelial growth.

As a recap:
The syringes were submerged for an hour and held between 145-150f
I had five syringes with half a print worth of spores in each, each print was set under a plate contaminated with mold for different amounts of time.
in order I had a control, open air, shortest time, middle time, and longest time.
Sterile water was shot onto the print to disloge spores and then sucked into the syringe
Took around a week for any signs of bacterial growth

Next experiments:


5 syringes filled with sterile water, same as last time, keeping the method of contaminating prints the same as well.

What I will be changing is the method of getting spores in a syringe, I will be using some modified plates I created that have injection ports in them instead of the glass jars I used last time. The injection ports are blobs of silicon.

First:
For the first experiment I will be doing one hour at 145f submerged (not floating incase that causes improper pasturization)

The change is the different method of getting spores into the syringe. My previous method I used was reckless and not good sterile technique imo.

Second:
For the second experiment I will be doing one hour at 160f submerged


final thoughts:

I have inoculated a plate with mold spores to reduce variables.

this will take some time for me, I have classes starting back up so I expect to inoculate the first plates on wednesday.

As I mentioned before, this is new to me. please let me know if you see any errors in my methodology and what you would do to improve it, should you feel compelled to do so.


--------------------
Yummy!


LAGM 2025
https://www.shroomery.org/forums/showflat.php/Number/29174693#29174693

Edited by syllable (03/11/25 04:40 AM)

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Re: Spore cleaning without a centrifuge or agar [Re: Axman] * 1
    #29155932 -

Axman said:
Update 3-12-2025:



Dirty sample:







Pasteurized sample:


Just a checkup on these as I figure this might be useful info. The dirty samples both grew mycelium, and one of them overpowered the contamination. But there is no mycelium present in the pasteurized plates.

I ran another set of 2 syringes in the water bath and did 1hr, and 1.5hrs, nothing grew, not even on the control plates. So I'm writing that experiment off as a loss.


 

Figured this was relevant enough to share.


--------------------
The Hitchhikers Guide to the Shroomery
:sporedrop:L.A.G.M. 2025:sporedrop:
Tico


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Re: Spore cleaning without a centrifuge or agar [Re: Axman] * 1
    #29155987 -

Oh wow. I just stumbled upon this, instantly what comes to mind is now being able to prepare large grain bags safely inoculated by injection port, using SS instead of LC. If it can just eliminate the bacteria risk that alone makes it viable using carefully prepared spore prints;

I wish i knew about this 2 years ago, i lost a few cool species and strains because the aussie heatwaves made the spores go dormant, they did wake up eventually but not  until a dozen other organisms had been having their way with the plate for over a week.
Actually this is just straight up a recurring problem here that sustained temps over 30 seem to knock out mushroom spores harder than the mold and bacteria.

Incidentally i found a culture incubator called a "shake n stack", thermofisher i think, out behind the old abandoned bio-lab, so this is perfect, it goes up to 100C and claims to be ridiculously precise/even heating, other than a sous vide i think this is THE hardware youd want for doing this, i can try reviving some of my old not quite yet dead prints that suffered from not being clean, a few were wild and were tricky to clean when they were fresh.

One other thing. for spores that have gone good and dormant, would this pasteurization by chance snap them out of their dormant state quicker?

-edit-
After re-reading through everything i have 4 things to add:

1: why dont you guys use partitioned plates? you could run so many more experiments then :p

2: about peroxide treatment; you can control peroxides half-life by adding catalysts that decompose it. solids like manganese dioxide would be biologically inert while limiting how long peroxide can exist in a given solution.

3: is there any particular reason why the spores of mushrooms would be more resilient to pasteurization than mold spores or bacterial spores?

4: certain things are known to dehydrate spores, and my understanding here is you want spores in an inactive dehydrated state during pasteurization. to that end, could there exist a safe controlled protocol for exposing a sample to well above 70% isopropyl alcohol? and, what about the impact of salinity of your spore water?
At one time ive seen someone explain that medical saline is not viable for spore syringes as it prevents the spores from germinating or hydrating in some critical way, but this then makes me wonder, what if you did the same thing as here, and only approach that threshhold. My sleep deprived hallucinating brain envisions possibly some sort of salinity modulated thermal cycling like the original premise of pasteurization-sterilization, exploiting the fact that organisms are vastly more heat sensitive once they germinate from spore/endospore. but then, if you can inhibit this reversibly for mushroom spores but not bacteria, or mold, then you might be able to increase the scope of what can be killed off and thus how clean you can make the spores. Also especially if salty water allows for higher temperatures before denaturing occurs.
Something tells me that mold and bacteria are probably more tolerant of salinity than mushrooms, especially those who naturally grow on rain-leeched substrates like cubensis.

Edited by DrMushroom (03/13/25 06:41 AM)

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Re: Spore cleaning without a centrifuge or agar [Re: DrMushroom] * 5
    #29156802 -

Check this shit out! :rockon:


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Re: Spore cleaning without a centrifuge or agar [Re: PhenoDreamers] * 1
    #29156810 -

Aw fuk yeah :rockon:

Same protocol as C?

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 3
    #29156877 -

This is a T1 from the C plate. I have probably tried a good 5 times before you mentioned your theory here to get clean growth from this variety. I have always come back empty handed 😂 so this is awesome. I have a lot of other ones to try it with as well.

I have to mention with the initial start of this experiment, I used one single spore print across 100ml of water. Which is feel diluted it way too much even though I used the entire print. (I had them in bulk so I didn't care.) But on the next go. Maybe I'll use a whole print across only 10ml of water. I'm thinking that's why I just didn't get growth on some of the plates. So it's definitely worth doing over again.

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Re: Spore cleaning without a centrifuge or agar [Re: PhenoDreamers] * 3
    #29156895 -

Keep up the good work, brotha. That transfer is exactly the sort of results we're after. Great proof of concept.

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Re: Spore cleaning without a centrifuge or agar [Re: Stipe-n Cap] * 3
    #29157430 -

Ps cyan, 45 min @ 60 *C. Plate inoculated with mss 03-05, spread crudely with inoculation loop. Germination spotted 03-12. I am not 100% convinced this is cyan, but if it turns out to be, it indicates cyan (clade?) can survive pasteurization.



Edit: oh yeah if it's cyan it's yours @Lew


--------------------
No nation, no border
Fight law and order


Species grown so far

Edited by Churpee Choozle (03/14/25 08:23 AM)

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Re: Spore cleaning without a centrifuge or agar [Re: Churpee Choozle]
    #29158241 -

:hatsoff:

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Re: Spore cleaning without a centrifuge or agar [Re: catnip40]
    #29158277 -

spores are very very resilient.  there's no reason for me to believe that they die when frozen.  and in my experience, even the mycelium, which is much more fragile, can somewhat endure freezing.

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Re: Spore cleaning without a centrifuge or agar [Re: dinosaurs]
    #29158419 -

Have you personally tested these theories across a wide range of species and varieties? Because I definitely beg to differ

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Re: Spore cleaning without a centrifuge or agar [Re: PhenoDreamers] * 1
    #29158463 -

Stipe gave a much better response to catnip in the post right after anyway regarding spores :shrug:

But yeah, I have cultures that don't even survive in my refrigerator.  So I don't suggest people start freezing their mycelium to check dinosaurs hypothesis.


--------------------
And if I only could
I'd make a deal with God
And I'd get him to swap our places
I'd be running up that road
Be running up that hill
With no problems

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Re: Spore cleaning without a centrifuge or agar [Re: tree frog]
    #29158494 -

My 2 survived and colonized but still bacteria infected.

I will slurry spawn it today.  Pins in 7 days if it will fruit.


--------------------
The better guide to grow mushrooms.
https://www.shroomery.org/forums/showflat.php/Number/8553541

Beware the power of one.  The power of two. The power of many.

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Shop: Sporeworks.EU Spores for European Microscopy   MagicBag.co Certified Organic All-In-One Grow Bags   Myyco.com Isolated Cubensis Liquid Culture For Sale   Original Sensible Seeds Bulk Cannabis Seeds   Unfolding Nature Unfolding Nature: Being in the Implicate Order   North Spore Bulk Substrate   Mushroom-Hut Liquid Cultures

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