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what do you do for culture storage?


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#1 orangutan

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Posted 05 March 2017 - 11:57 AM

Right now I make a liquid culture with karo and store them in jars in the fridge.

 

This is starting to take up space!

 

I remember reading about storing in sterile water but can't find the thread.

 

What's this thing with vacutainers?

 

No method too simple or complicated!  Freeze drying?  Normal freezing? 

 

I want to have a refrigerator full of little vials of culture, not a bunch of pint jars.  I have housemates and only get one shelf of the refrigerator that I need to use for food and mushroom stuff.  It's tight.



#2 BigTexas

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Posted 05 March 2017 - 12:07 PM

Buy a mini fridge, my girl hates me putting bullshit in her fridge, lol just get one thats an actual freon type, or maybe one of those electric style ones if its efficient, cheap ones don't stay cold very well.



#3 peacefrog

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Posted 05 March 2017 - 12:50 PM

Personallly I like agar plates which will keep for a couple or so months depending on the species, how deep the agar is poured etc. for short term storage. But using them can start to get pretty crowded as well after a while.

For long term storage, slants are my go to. They don't take up much space at all really. There are other possibilities out there of course but those are my preferred ways.
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#4 MLBjammer

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Posted 05 March 2017 - 02:19 PM

Prints are quite easy to store as well.

 

And I have seen folks take a little colonized grain and store it in sealed jars.


Edited by MLBjammer, 05 March 2017 - 02:20 PM.

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#5 fahtster

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Posted 06 March 2017 - 12:29 AM

Prints are quite easy to store as well.

And I have seen folks take a little colonized grain and store it in sealed jars.

I used these..

https://mycotopia.ne...-test-of-time/.

**the picture missing in #2 (link takes you to home page for some reason) is this one:

0A9C136A-B148-437C-81F0-F2DCC5D2DEF3-1395-000002112910672D_tmp.png

Basically using a lid that you can manipulate the FAE from 95% covered (for grain colonization) to 50% to allow said grain dry out with the early stage myc; freezing it in the prefruiting stage. Then you can reanimate at will. They've lasted more than a year.. you can hydrate and re-dry every six months to keep the culture from going bone dry.. and you can use the myc water immediately and securely. Pretty proud of that one :)

Faht

Edited by coorsmikey, 07 March 2017 - 09:05 PM.

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#6 MLBjammer

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Posted 06 March 2017 - 04:20 AM

Yes, I love that method!  I am surprised more folks didn't try it.


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#7 Microbe

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Posted 06 March 2017 - 01:30 PM

Plates can be stored for 1 year if you pour thick enough and is the best way to maintain working inoculate.

Agar slants are excellent for storing cultures long term and can be kept for many decades with some annual maintenance.

Steralized distilled water storage is something i am venturing into thanks to needles and tenderfoot but this method requires a nutrient free and oxygen free environment. When done properly you dont even need cold storage and the cultures can be stored at room temp.

I have been experimenting a little with this method but have not yet attempted to store cultures this method. As i continue to acquire master cultures and go to slants that i have made and are ready to go, i will make 2 other copies using the distilled water method.

I will be using vacutainers and 4 ounce mason jars. The vacutainers are specimen collection tubes for general purpose so the are not sprayed with any anticoagulant agents which used for collection of blood. I did make the mistake of buying some polypropylene ones that were sprayed with EKTA i think it is.

Anyway harvest the mycelium from a plate making sure to leave behind any nutrient media, a small amount is ok, but it needs to be minimal.

Place this mycelium into a jar of steralized water with screws so that you can blend it up them draw it into a syringe.

Once the mycelium is drawn into the syringe then puncture the cap of the vacutainer and the vacuum will pull the solution out of the syringe. The minimal amount of dissolved O2 and or nutrients will be depleted quickly and then the mycelium will stop all metabolic activity.

The second method im going to make my 4 ounce jars air tight with 2 SHIPS and steralize the jars with a little bit of distilled water in them along with some broken shards of a Crown Royal bottle.

Once the jars are steralized, a vacuum will be created and then fill a syringe with some blended mycelium water (distilled) puncture the SHIP and the contents will empty.

I placed 2 ships so when i go to extract the mycelium im not jamming a airport sharp in the same hole as my working sharp. That sounded inappropriate lmao.

Jammer is right about spore prints but keep in mind gene recombination may leave you with different phenotypes.

This talks about several methods including use of cryoprotectants similar to what i was doing in my randoms thread but abandoned it at the moment because i added propylene glycol to a sugar lc and again, even this method requires a nutrient free media because the mycelium will suffocate. If there is O2 and no nutrient then the mycelium will starve.

This is fairly new to me so do your own research but i can say with certainty, agar slants are a sure way to store master cultures and plates are great for working inoculate.

Several methods including Distilled Water.

https://www.ncbi.nlm...les/PMC3209878/

Distilled Water supported by scientific data.

https://www.ncbi.nlm...cles/PMC186689/

I will be documenting my culture storage and will attempt to recover ay 6 month, 1 year, 18 months, and finally 2 years. If i can successfully recover after 2 years of distilled water storage, i will eliminate agar slants. I believe in that last link i posted they revived 97% of the cultures. Some species cant be stored this way as effectively as others.

Hope this helps. I want to go into more detail but my wife is mean mugging me, we are at the furniture store lol

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#8 Arathu

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Posted 06 March 2017 - 01:57 PM

This place is literally the new Alexandria..........................

 

Awesome information and trips down the endless wormholes of data...........

 

A


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#9 fahtster

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Posted 06 March 2017 - 02:04 PM

Plates can be stored for 1 year if you pour thick enough and is the best way to maintain working inoculate.
Agar slants are excellent for storing cultures long term and can be kept for many decades with some annual maintenance.
Steralized distilled water storage is something i am venturing into thanks to needles and tenderfoot but this method requires a nutrient free and oxygen free environment. When done properly you dont even need cold storage and the cultures can be stored at room temp.
I have been experimenting a little with this method but have not yet attempted to store cultures this method. As i continue to acquire master cultures and go to slants that i have made and are ready to go, i will make 2 other copies using the distilled water method.
I will be using vacutainers and 4 ounce mason jars. The vacutainers are specimen collection tubes for general purpose so the are not sprayed with any anticoagulant agents which used for collection of blood. I did make the mistake of buying some polypropylene ones that were sprayed with EKTA i think it is.
Anyway harvest the mycelium from a plate making sure to leave behind any nutrient media, a small amount is ok, but it needs to be minimal.
Place this mycelium into a jar of steralized water with screws so that you can blend it up them draw it into a syringe.
Once the mycelium is drawn into the syringe then puncture the cap of the vacutainer and the vacuum will pull the solution out of the syringe. The minimal amount of dissolved O2 and or nutrients will be depleted quickly and then the mycelium will stop all metabolic activity.
The second method im going to make my 4 ounce jars air tight with 2 SHIPS and steralize the jars with a little bit of distilled water in them along with some broken shards of a Crown Royal bottle.
Once the jars are steralized, a vacuum will be created and then fill a syringe with some blended mycelium water (distilled) puncture the SHIP and the contents will empty.
I placed 2 ships so when i go to extract the mycelium im not jamming a airport sharp in the same hole as my working sharp. That sounded inappropriate lmao.
Jammer is right about spore prints but keep in mind gene recombination may leave you with different phenotypes.
This talks about several methods including use of cryoprotectants similar to what i was doing in my randoms thread but abandoned it at the moment because i added propylene glycol to a sugar lc and again, even this method requires a nutrient free media because the mycelium will suffocate. If there is O2 and no nutrient then the mycelium will starve.
This is fairly new to me so do your own research but i can say with certainty, agar slants are a sure way to store master cultures and plates are great for working inoculate.
Several methods including Distilled Water.https://www.ncbi.nlm...les/PMC3209878/
Distilled Water supported by scientific data.https://www.ncbi.nlm...cles/PMC186689/
I will be documenting my culture storage and will attempt to recover ay 6 month, 1 year, 18 months, and finally 2 years. If i can successfully recover after 2 years of distilled water storage, i will eliminate agar slants. I believe in that last link i posted they revived 97% of the cultures. Some species cant be stored this way as effectively as others.
Hope this helps. I want to go into more detail but my wife is mean mugging me, we are at the furniture store lol
Sent from my LGLS992 using Tapatalk


Nice. Man, my gf would have thrown my phone at the wall. Lol.

@Orangutan Obviously, slants and the lot Microbe is talking about is the superior way to store cultures.. I was giving an example of what Jammer was referring to.

Faht
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#10 Microbe

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Posted 06 March 2017 - 02:21 PM

Plates can be stored for 1 year if you pour thick enough and is the best way to maintain working inoculate.
Agar slants are excellent for storing cultures long term and can be kept for many decades with some annual maintenance.
Steralized distilled water storage is something i am venturing into thanks to needles and tenderfoot but this method requires a nutrient free and oxygen free environment. When done properly you dont even need cold storage and the cultures can be stored at room temp.
I have been experimenting a little with this method but have not yet attempted to store cultures this method. As i continue to acquire master cultures and go to slants that i have made and are ready to go, i will make 2 other copies using the distilled water method.
I will be using vacutainers and 4 ounce mason jars. The vacutainers are specimen collection tubes for general purpose so the are not sprayed with any anticoagulant agents which used for collection of blood. I did make the mistake of buying some polypropylene ones that were sprayed with EKTA i think it is.
Anyway harvest the mycelium from a plate making sure to leave behind any nutrient media, a small amount is ok, but it needs to be minimal.
Place this mycelium into a jar of steralized water with screws so that you can blend it up them draw it into a syringe.
Once the mycelium is drawn into the syringe then puncture the cap of the vacutainer and the vacuum will pull the solution out of the syringe. The minimal amount of dissolved O2 and or nutrients will be depleted quickly and then the mycelium will stop all metabolic activity.
The second method im going to make my 4 ounce jars air tight with 2 SHIPS and steralize the jars with a little bit of distilled water in them along with some broken shards of a Crown Royal bottle.
Once the jars are steralized, a vacuum will be created and then fill a syringe with some blended mycelium water (distilled) puncture the SHIP and the contents will empty.
I placed 2 ships so when i go to extract the mycelium im not jamming a airport sharp in the same hole as my working sharp. That sounded inappropriate lmao.
Jammer is right about spore prints but keep in mind gene recombination may leave you with different phenotypes.
This talks about several methods including use of cryoprotectants similar to what i was doing in my randoms thread but abandoned it at the moment because i added propylene glycol to a sugar lc and again, even this method requires a nutrient free media because the mycelium will suffocate. If there is O2 and no nutrient then the mycelium will starve.
This is fairly new to me so do your own research but i can say with certainty, agar slants are a sure way to store master cultures and plates are great for working inoculate.
Several methods including Distilled Water.https://www.ncbi.nlm...les/PMC3209878/
Distilled Water supported by scientific data.https://www.ncbi.nlm...cles/PMC186689/
I will be documenting my culture storage and will attempt to recover ay 6 month, 1 year, 18 months, and finally 2 years. If i can successfully recover after 2 years of distilled water storage, i will eliminate agar slants. I believe in that last link i posted they revived 97% of the cultures. Some species cant be stored this way as effectively as others.
Hope this helps. I want to go into more detail but my wife is mean mugging me, we are at the furniture store lol
Sent from my LGLS992 using Tapatalk

Nice. Man, my gf would have thrown my phone at the wall. Lol.

@Orangutan Obviously, slants and the lot Microbe is talking about is the superior way to store cultures.. I was giving an example of what Jammer was referring to.

Faht
I have stored colonized grain jars for 9 months before. I wrap the entire jar in shrink wrap and once a month i bring it out and and remove the shrink wrap and allow it to get to room temp and allow for it to get some GE. I call it exercising for my cultures. Once the exercise is complete it gets wrapped up again and placed back into cold storage. I noticed after 90 days my cultures did loose vigor but were still very much viable.

I love that lid. Where did you get it? Im assuming you placed that over tyvek or a sythetic filter disc?
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#11 Microbe

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Posted 06 March 2017 - 02:50 PM

You can take tubes and make what i like to call grain shots. Inoculate the tube with a drop of lc or a single agar plug or another piece of colinized grain.

The tubes i started off with were glass but had polyethylene push on caps which couldnt be steralized but i soaked them in bleach after drilling holes in them.

Once i inoculated the tubes i took the cap out of the bleach and wiped down with a 70% iso soaked paper towel to remove the remaining bleach residue then place it on the tube. I packed the hole with a little bit of polyfill and cover with a layer of micropore tape.

Once colonized i place down into deli quart containers and place into cold storage. I think i was able to store 15 90 ml tubes in a single container.

I called them grain shots because i would take it out and inoculate a single quart of grain with it.

I dont do this anymore but after reading through the posts again, you did specify not complicated and i assure you harvesting mycelium without getting any agar is complicated IMHO and limited experience.

I probably should have edited my last post as it is related to grain instead of creating another post instead of post padding huh?! ;)

Edited by Microbe, 06 March 2017 - 02:55 PM.

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#12 fahtster

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Posted 06 March 2017 - 06:34 PM

You can take tubes and make what i like to call grain shots. Inoculate the tube with a drop of lc or a single agar plug or another piece of colinized grain.

The tubes i started off with were glass but had polyethylene push on caps which couldnt be steralized but i soaked them in bleach after drilling holes in them.

Once i inoculated the tubes i took the cap out of the bleach and wiped down with a 70% iso soaked paper towel to remove the remaining bleach residue then place it on the tube. I packed the hole with a little bit of polyfill and cover with a layer of micropore tape.

Once colonized i place down into deli quart containers and place into cold storage. I think i was able to store 15 90 ml tubes in a single container.

I called them grain shots because i would take it out and inoculate a single quart of grain with it.

I dont do this anymore but after reading through the posts again, you did specify not complicated and i assure you harvesting mycelium without getting any agar is complicated IMHO and limited experience.

I probably should have edited my last post as it is related to grain instead of creating another post instead of post padding huh?! ;)

 

haha... you talkin' ta me?  yeah, I made the lids... kite makers tyvek sandwiched between two postal... half is silicone for a SHIP on the top and bottom (directly underneath each other) and the other half you leave open and cover with tape the desired amount.  I imagine that you can use a filter disc no problem, too--just put tape over until you want to dry out the myc..  the reason I like the tyvek is that while there is a seal around the rim of the jar, there's also a seal between the metal ring and the threads of the jar; extra protection... you don't get that with filter discs.  The two types of tyvek is because it can stretch in the PC and I felt safer with the three layers being that they sat for so long and were me masters.

 

faht


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#13 MLBjammer

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Posted 06 March 2017 - 07:40 PM

That kite tyvek is the best there is--straight advice from H3, I think?  Brings back old memories, my friend.

 

Anyway, I think your tek has been greatly undervalued, and it was the first way I learned to store cultures, and it is very newb friendly: https://mycotopia.ne...ge-tek-revised/

 

Great comments, Uncle Microbe.  I am all out of likes, but I will light you up later, lol.


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#14 peacefrog

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Posted 07 March 2017 - 06:39 AM

@Microbe,
I love the "exercising your culture" bit.

Cubes and oysters are very forgiving in cold storage and can last a while on agar plates/jars. Pans on the other hand do not tolerate it as well, IME. I have never successfully revived a plate over 3 months of cold storage before. The mycelium just turns blue and deflates way before dehydration of the agar and will not grow again once transferred. And the same goes with slants. 12 months is my maximum wait time before rejuvenating my slants with pans. Now you can use the wood trick and get more mileage, but I rarely use it. But I will if I think it might be a while before I will take um back out. I probably should do it more these days, as life at Froggy's house is still sometimes hectic and I am not as good at keeping up my library of prints and cultures lol.

I have read a lot about the sterile water storage and it seems very interesting. I have just never attempted it personally. But perhaps I should one day to see for myself.

RogerRabbit used to post about using dried fruit tissue as well for storage and then rehydrating and inoculating onto to agar. I have never attempted that either personally.

@faht,
That grain tek is pretty awesome. I often thought about using dehydrated grain from a known culture similarly but have never attempted it. Although I must say, your way seems a lot better than what I had in mind. I will have to give your tek a try, brother man.
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#15 Microbe

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Posted 07 March 2017 - 07:07 AM

@peacefrog thanks for the info on the plate storage advice on pans. The same can be said about distilled water storage. Some species may not tolerate it and is exactly why i will maintain agar slants also until it is without a doubt i can store species X long term in distilled water.

The wood stick is what i saw and read about and is the only reason why i use it. Fortunately i never had to resort to pulling and reviving from the stick but came very close. In my most recent agar thread i had a slant that had very little agar left and literally the stick would roll around in the slant. It had a very thin layer of agar that was colonized by a sickly looking culture that i was able to revive. My point is even with the stick, routine maintenance is demanded at least once a year but twice a year is better.

Once i get done playing with this China strain im going to work with
Panaeolus tropicalis, my first Copelandia strain. Time to learn something new ;)


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#16 peacefrog

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Posted 07 March 2017 - 07:42 AM

I hear you. Yes the wood trick is a very good practice and should be used. I would always highly recommend it. I'm just lazy sometimes and used to be very good at taking mine out once every year or so to rejuvenate. Now a days my HI pan culture is the only one I really keep up with. My oysters and lions mane cultures are going on 3 years now and my only cube culture sat in there for 6 years. I though it was dead, but alas, I have revived it after a few nail bitting transfers. Haven't fruited it yet, but it is in the works. I hope it still produces as well as it did back then. We will see.

And no problem on the Pan advice. That's just been my experience with um. Yours and others might vary. I have grown cyans, cams, and Pan bisp and they always acted the same for me in the fridge. Could be just me who the hell knows lol. I once kept a pint of grain colonized by pans for 4 months and the mycelium had completly changed from white to translucent. I let it sit at room temp for 3 days and it never recovered. I never took it to agar, grain or sub so I really don't know if it was still viable. I threw it away. Now I did not "exercise" them, so that clever trick of yours might have saved it. That might be the way to keep such cultures alive, might have to steal that one, bro. See I love learning new things!
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#17 Microbe

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Posted 07 March 2017 - 10:46 AM

I hear you. Yes the wood trick is a very good practice and should be used. I would always highly recommend it. I'm just lazy sometimes and used to be very good at taking mine out once every year or so to rejuvenate. Now a days my HI pan culture is the only one I really keep up with. My oysters and lions mane cultures are going on 3 years now and my only cube culture sat in there for 6 years. I though it was dead, but alas, I have revived it after a few nail bitting transfers. Haven't fruited it yet, but it is in the works. I hope it still produces as well as it did back then. We will see.

And no problem on the Pan advice. That's just been my experience with um. Yours and others might vary. I have grown cyans, cams, and Pan bisp and they always acted the same for me in the fridge. Could be just me who the hell knows lol. I once kept a pint of grain colonized by pans for 4 months and the mycelium had completly changed from white to translucent. I let it sit at room temp for 3 days and it never recovered. I never took it to agar, grain or sub so I really don't know if it was still viable. I threw it away. Now I did not "exercise" them, so that clever trick of yours might have saved it. That might be the way to keep such cultures alive, might have to steal that one, bro. See I love learning new things!

Did you keep the grain jar airtight? My only experience with a pan species is a spore germination on agar but i will say that the mycelium does not blanket the agar surface like cube myc. It is more similar to mexicana which i also only have experience on agar with it and never put either to grain yet.

I suppose it does the same in grain jars so i can see how grain storage would have a realistic shelf life of 1 to 2 months. You seem to know your pans or any other mushroom you grow really so i doubt it was operator error.

Grain will dry out quickly in the fridge if not air tight and the mycelium will suffocate without O2 if it has colonized a nutrient source which 99% of storage methods require a nutrient source but in cold storage metabolic or cellular respiration is decreased significantly so one can keep the jar air tight for a certain amount of time. I settled on 30 days, im sure i have gone longer but i try to at least once a month allow the culture to warm up and get back to full metabolic function and cellular respiration. It gets to eat a little and grow before being stunted again.

This method may not work for pans and im dont use glc like i used to and this the only reason whybi kept grain masters was for this purpose. Now any glc use is from a 4 oz or 8 oz jar and its a 1 time use.

Now i do keep steralized grains around for weeks wrapped up in shrink wrap waiting to be inoculated but that is a whole other subject. Anyway i look forward to your guidance when i finally graduate to a more difficult species to grow. Cubes are simple and are the carp of actives, its hard to kill them and they can thrive in less then ideal conditions.
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#18 peacefrog

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Posted 07 March 2017 - 11:48 AM

I don't use grain like that very often. I like to either spawn or case mine while it's still fresh. I just had too much going on at the time and then got busy and forgot about it. And it was left in a ziplock baggie with the same filtered lid it was grown in.

I have done the same for cubes before and put a few grains onto agar and all was fine after 8 months of storage like that. Like you said, the grain will dry out after certain amount of time and I prefer drying out verses suffocation. The pan jar just died, or appeared to have died way faster than cubes.

For my slants and agar jars, they have the lids loose wrapped in parafilm and placed in a special section of my fridge.
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#19 MLBjammer

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Posted 07 March 2017 - 05:28 PM

I have seen some pan myc recently that was anything but wispy. And it was Froggie's genetics. I will throw up a pic when I get home. Crazy stuff.
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#20 peacefrog

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Posted 07 March 2017 - 07:23 PM

The genitcs may have come from me, but you have surely taken those spores and added your own spin on them to make them better.
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