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Spores and Cultures: A Comprehensive Guide * 22
    #29020482 -

EDIT: THERES A LOT OF OUTDATED INFORMATION ON THIS THREAD. ILL BE REDOING THIS..... eventually

Spores and Cultures: A Comprehensive Guide

Here I'll highlight various techniques for germinating, cleaning wild and domestic spores, and preparing wild and domestic spore prints and swabs.


Section 1: Materials

-Amazon

Amazon Associate Link

-Local
Hydrogen Peroxide 3% (brown bottle from the store)
Brown rice flour
Pressure cooker capable of 15 PSI
FFU or Still Air Box
Torch
Measuring cups, tablespoons, and teaspoons
Weighing and mixing containers

Section 2: Obtaining Spores

- Printing and swabbing wild mushrooms

- Printing and swabbing domestic spores

Section 3: Germination

- Verifying spore load

- Making and Pouring Agar and Gellan Gum


NOTE: VENT THE PC. I WAS WRONG ABOUT THIS AND WILL BE MAKING A NEW VIDEO AT SOME POINT.
I made this video for another group of people so some pieces are irrelevant and I trimmed it too soon. Put your agar on a stir plate before cooking for a bit and after cooking for bit. It's less important to use a stir plate for gellan but comes out extremely well if you do. Pressure cook at 15 PSI for at least 10 minutes. Extremely long agar cooks are unnecessary as long as your solids are fully dissolved before cooking.
I mentioned in the video later on using .5% for nutrient plates. This is for exotics. If you're working with cubensis or aff. natalensis, I suggest 1 to 1.5% (although nutrients plates are irrelevant for this thread.)

Edit: if using gellan gum, I've come to realize it is extremely important to add .1% epsom salt to help solidify!



- Serial Dilution

Serial Dilution 1

Serial Dilution 2



Not all wild spores require serial dilution or water agar. These are serial diluted spores on .5% nutrient plates that I definitely could have gotten away with using grab and drag. Shout-out to Nichrome for this very clean wild print!





Another set of, not as clean, spores. Circled in red is clean germination.

On .5% from serial dilution:




On water agar from serial dilution:




It's safe to let them sit a few more days and make sure it's clean and what I'm desiring before making transfers. If it grows too fast, it's usually and indicator, in certain species, that it's not clean growth.
I should have did a grab and drag, for these particular spores, to water agar for further comparison.

- Streaking



A new method: Streaking via Nasopharyngeal swabs
With nasopharyngeal swabs, spores (good and bad) easily break away from the swab tissues. As a result, colonies are more easily dispersed. This has recently become my most favorite method of wild spore inoculation to agar.



- Reviving old spores

Experimental Process*
I've heard of sugars and, of course, excess hydration being of great help to revive stubborn spores. I don't yet have conclusive results but trying to a method told to me by Basidium Equilibrium.
Germinate spores in 1% sugar water in the fridge. In a 2ml centrifuge I have spores soaking in a solution of polysorbate and 1% dextrose. I have one stored in the fridge and one stored at room temperature. This could take several months for all I know. Results pending...

Another method.. Difco R2A
I recently had failure germinating wild natalensis spores via serial dilution and streaking with cotton tipped applicators.
Here I've tried streaking via nasopharyngeal swabs onto .5% nutrient, 1.2% nutrient, and R2A plates. The results are phenomenal! Although some contamination is clearly present, which was expected, I have what appears to be, some clean growth to work with on the r2a plate!





Section 4: Culture Cleaning

-Bacteria contaminations

There are various methods that work best against bacteria including agar trenches, brf pucks, and agar sandwiches. While these methods will not work as well against molds and other contaminations, they work well for bacteria in particular.

BRF Puck

Cabin Sequestering Text

Cabin Sequestering Video

-All other contaminations

Other contaminations can be trickier but with the aid of hydrogen peroxide, I have found great success cleaning up the worst of cultures.

Storing germination plates at a cooler temperature (55-60F) can give you a great jumpstart on molds!

-Reviving stalled cultures and getting around pigmentation

I've had great success with dealing with most difficult of cultures with Difco R2A It's a premixed agar medium usually used for testing potable water. However, when used with stalled cultures, I have found a 95% success rate currently! Not only that but it has provided faster, more vigorous growth than ever before!




I tried using a cheap R2A off Amazon before going with Difco. The results were not great for me. I owe credit to Scapo for introducing this to me along with nasopharyngeal swabs!

If you don't have R2A and don't want to get it. Simply just switch your primary agar ingredient. For instance, if you use light malt extract, try using potato dextrose. I've had great results getting away from stalls by doing this. Currently I switch back and forth on every time I pour!

Edited by PhenoDreamers (07/24/25 08:19 AM)

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Re: Spores: A Comprehensive Guide [Re: PhenoDreamers] * 8
    #29051946 -

Cabin sequestering has me fully converted, currently running pretty much everything I have through it after seeing preliminary result with nasty pan cyan Bangalore post sequester.

Before:


After:


Will update later but also testing it for cloning, small mushrooms are always a pain to clone. I’ve taken 4 samples of whole stipe sections from zapotecorum and placed entire sections in the cabin. Samples of clone tissue were handled with bare hands and cut with non sanitized scissors eg handled in a very dirty way without regard. They were placed within the cabins with sterilized tweezers however, just trying to convey I took no care to ensure tissue samples were clean in any regard, rather tried to make them dirtier. All samples are now starting to show initial faint threads emerging out the cabin, no bacteria can be seen yet. Will update with pictures once things are more visible for pictures.

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Re: Spores: A Comprehensive Guide [Re: kirkeng] * 2
    #29053974 -

Thanks for the feedback Kirkeng 😁 curious to try this wild gill frags as well.

I updated the original post body with nasopharyngeal swabs and Difco R2A. A lot more work to be done on this thread but it's getting there.

Edited by PhenoDreamers (12/12/24 06:47 AM)

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Re: Spores: A Comprehensive Guide [Re: PhenoDreamers] * 1
    #29055364 -

Very exciting! Thanks for introducing me to the cabin sequestration technique - giving it a try today.

Edited by sadiman (12/13/24 09:40 AM)

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Re: Spores: A Comprehensive Guide [Re: sadiman] * 2
    #29055375 -

Following so I'm not bugging you on Signal about this stuff :lol:


--------------------
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: Spores: A Comprehensive Guide [Re: tree frog] * 4
    #29056729 -

Well the clone cabins were not strong enough for the nastiness within the clone sample. Some biofilm is seeping from the edges. Noticeable in first picture not so much in second.




That being said it still seems the cabin was effective in separating them with the film seeping from the seams of the cabin, meanwhile atop the roof appears healthy mycelium so far. I’ve transferred shallow cuts from the cabin roof to new plates and await to determine cleanliness, I did not move them to a second cabin though I will if they’re still dirty. Ideally though one cabin was enough.

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Re: Spores: A Comprehensive Guide [Re: kirkeng] * 2
    #29066947 -



Also getting my cabin on. And I appreciate your post kirkeng.  Following your lead and transferred off the roof of the cabin.  This is bisporus, so hard to tell from the pictures.

I took two transfers to a clean plate and dropped a wedge into an aio jar.  The cabin tops definitely looked clean and one of those had very nice radial growth one transfer away from the germ plate.


--------------------
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: Spores: A Comprehensive Guide [Re: tree frog] * 1
    #29074629 -

:camping:
Probably going to have to use some of this soon.


--------------------
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: Spores: A Comprehensive Guide [Re: Soloist] * 5
    #29074722 -

Trying out the cabin sequestering.  Hope this is ok to post in here.  I understand this is your guide thread.. maybe agar envy would be better?

Originally I was gonna try and clone a semp with the tip of a needle.. I got it pulled apart and the inside was a wet mess due to daily misting so I scratched that idea and instead opted for a peroxide soak of stipe section to hopefully clean enough of the outside to get some clean myc through transfers.  Also just trying to get my chops with agar work so this is good practice.  (I also want to try out other techniques like the trap door and maybe the roof method etc eventually)

Turned out exactly as expected


Thats actually the second plate I put the tissue to.  After there was some regrowth on the first plate..


I thought maybe I could pull the stipe in half and bottom would stay stuck to the plate and expose cleaner tissue on the inside.. kinda worked, mostly didn’t lol

Anyway.. when I got some aerial growth off the tissue, I picked it up again off the second plate and laid that aerial growth down toward clean agar and after some growth on the plate, that’s where I grabbed the transfer for the cabin


The cabin building felt pretty clumsy so we’ll see how that goes.  That little piece of agar in there bugs me.. kinda takes the sequester part out of it.  I also used the scalpel to move the roof and had a hard time with that as you can see, I put quite a few slits in it.. do you guys use a forceps to move that?

Some ideas for what I’m going to do differently next time I try to clone a full stipe (any thoughts on these is more than welcome):

After the peroxide soak, instead of going directly to nutrient agar, I’m thinking of doing a serial rinse in 2-3 4oz jars of sterile water and then going to water agar first.  Once I get that aerial growth, then move it to nutrient agar and lay the aerial myc down like I did above to give that myc a running start and hope the rinses were good enough to wash away most of the surface bacteria and that the myc didn’t lift any off the surface as it grew.

Thoughts?

I’ll keep this posted on how the CS is doing/did. 

Nice thread!

Edit: Actually, after looking at this pic with the backlight, a lot of that “myc” is mold myc?  Are all those dots mixed in spore bundles?


Nvm.. that’s condensation from the lid :lol:  whew.. got pretty bummed for a minute there

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Re: Spores: A Comprehensive Guide [Re: fahtster] * 1
    #29074732 -

Nice write up Supa!

:popcorn:

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Re: Spores: A Comprehensive Guide [Re: fahtster] * 1
    #29074846 -

We're all thrilled to have you post here faht! 😎

Taking it back to water agar instead of nutrient is a good idea for the bacteria. Man if you ever want a challenge, try cleaning up psilocybe pelliculosa on agar. Single handedly the most difficult species I've ever clean up. Cabin sequestering and water 🌊 agar is what gave me the best results. I'll have to post some pictures later.

I'm glad you said that was condensation because I was going to say the dogs were definitely mold. If it were mold. I've found good results by spraying the whole dish with hydrogen peroxide and letting it sit a minute and then transferring a tiny spot as far as possible. And then taking an early transfer again on the following plate immediately when you see good growth. Mold can't be the biggest pain

Edited by PhenoDreamers (01/02/25 06:20 AM)

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Re: Spores: A Comprehensive Guide [Re: PhenoDreamers] * 1
    #29074859 -

Nice work!
:noargument:


--------------------
This hobby has taught me many things.
Most of those things are patience.

The Hitchhiker's Guide to the Shroomery
The Official Shroomery Cultivation Museum
Hobbit’s Secondhand Mycology Challenge
Round 2

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Re: Spores: A Comprehensive Guide [Re: Scemilope] * 2
    #29074953 -

Supa
I admire your dedication and follow-through with this piece. keep them coming. the comprehensive touch is nice.


--------------------
----<><><>----
⁂ tail-lights, back-pockets, rearview mirrors ⁂

⁂ guides | grows | supreme air box | sporeaddict ⁂
bring back ῥͳϻ

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Re: Spores: A Comprehensive Guide [Re: fiddle_head] * 3
    #29074979 -

Thanks guys! I have more tricks up my sleeve! Just need definitive results before I publish them

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Re: Spores: A Comprehensive Guide [Re: PhenoDreamers]
    #29075642 -

SupaThaRipper said:
Thanks guys! I have more tricks up my sleeve! Just need definitive results before I publish them


Tricks are for whores

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Re: Spores: A Comprehensive Guide [Re: LewDoja] * 2
    #29075643 -

It’s an illuuuuusion, Michael

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Re: Spores: A Comprehensive Guide [Re: fahtster] * 6
    #29084433 -

My cabin is looking pretty good… I think.  Especially with what I started with.

Gonna give it a few more days before I pull a leading edge but it shouldn’t be hard to get clean myc

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Re: Spores: A Comprehensive Guide [Re: fahtster] * 14
    #29089215 -

Got a handy little bonus tip to add to the cabin sequestering toolkit.
As some of you know I found a cool wild fruit and cloning wild finds is something I’m fairly confident with to the point of cockiness, well colour me humbled, probably gone through almost 30 plates trying to clean up this thing transferring away from the most obnoxious bacteria I’ve ever encountered till I was ogling my last patch of surviving myc and realised that the bacteria’s spread is highly dependent on gravity, next transfer I cabin sequestered then leant the plate on its side and prayed it survived, check it

When I made the transfer into the cavity I made sure the myc was touching  one edge of the agar inside that hole and memorised the position so that after it was capped and the dish sealed I leant it so that side was up in the hope it got an advantage over the bacteria which grew and dripped downwards, had to slightly rotate occasionally as the myc grew so it was always at 12 o’clock as it spread under the sandwich of agar but now 7 days later it’s grown out onto open agar and even grown through the top, doing this again just in case but with any luck I’ve saved this thing 🤞


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

Clean MS from fresh fruits every time.
How I prep brown rice

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Re: Spores: A Comprehensive Guide [Re: 3.A.M]
    #29090879 -

Genius move!

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Re: Spores: A Comprehensive Guide [Re: PhenoDreamers] * 9
    #29097039 -

Trying some stuff here.. doing a whole stipe clone of pan mib.

First one is the one I’m most hopeful.  I did a 30 second peroxide soak.  Dipped most of the fruit in the h2o2 (cap first) and kept the end out that I cut to release the fruit from the sub.  Then took hot scissors and cut a small section out of the soaked stipe.  Then did a 3 jar serial rinse in sterile water.  The hope there was to get as much of the h2o2 off and as much bacteria off as possible before going to agar.

My original thought process was to then split the sample in half exposing the inner tissue with two pieces.  One went to straight to a normal nutrient plate and the other, I wanted to try a combo of a trap door and a trench.  The idea with the trap door was to have the outside of the stipe laying against the bottom of the bare Petri and the inner tissue half facing up against the agar slip.. so the outer tissue doesn’t get any nutrients to grow contamination.  Woulda went great but I ran out of hands and forceps :lol: and then the sample all turned the same color so I kind of lost which side was which…


I put a small piece of agar on top of the slip so I can just grab it when it gets colonized and put it immediately into a cabin sequester.. that way I don’t have to deal with cutting or scraping anything in the moment, I can just yoink it off the slip.

This one I just put the other half of the rinsed sample to a plate.  I had good luck with the semp I did with just peroxide so I think this one will be even less work having done the serial rinses


Now this one is completely an experiment.. don’t crucify me here :lol:


I’ve labeled it S.E. for “scorched earth” because I used a torch on the outside of the stipe.. very briefly but enough that I thought it to be sufficient to maybe kill enough or all of the surface contamination but leave enough inner tissue alive to possibly expand to the agar.  I got the idea when I was cloning my semps a couple weeks ago.  When I pulled the semp stipe apart, the inner tissue was soaked because I had been misting daily.  This gave me the idea that maybe the tissue being soaked might insulate it enough from the outside heat to keep the inner most tissue alive and it’ll grow out into the agar.

Pan don’t have the same soaked inner tissue so I’ll be repeating this again with semps to test it because it was incredibly fast and easy to do.  So if it works (at least enough to get good growth for a decently clean transfer to a cabin) it’ll be cool.

I will say nothing has grown so far in the 24 hours since doing it so I think that’s a good sign.. when I did my whole stipe semps, I could definitely see bacterial growth after 24 hours.  I just need some of that inner tissue to have survived and break in outta there :lol:

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