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OfflineEarShot
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Confused about isolate
    #28885697 - 07/24/24 04:32 PM (5 months, 21 days ago)

(I'm a returning grower who never did agar/isolates and have questions)

Several days ago I inoculated 6 tiny petri dishes with spores in my SAB, got another 14 clean. There's cottony blobs of growth in all 6, with only one visible contamination although it's really hard to tell as there's condensation. The dishes are wrapped with cling film (btw I don't understand how that doesn't stifle growth).

My question really is what next? Am I right in thinking I should pull some of the bits of growth out to other dishes in the hope of creating an isolate?

Should I only pick the best looking? None look particularly interesting, let alone rhizomorphic. I think I read that doesn't happen until you transfer a few times? (if so, could someone explain why?)

Do I wait until I have a good rhizomorphic growth before transferring to grain?

I think my biggest point of confusion is that this would use up a lot of petri dishes (and I don't have that many) and I could potentially end up with multiple good looking strains on agar, and would then need to run them all through grain and tub to see if they even fruit, and retain the one which does best.

Is that what people do?

I'm thinking that sounds like a lot of effort compared to inoculating multiple small jars of grain (or brown rice petris) accepting I'll get some contaminate from the spored, growing the uncontaminated ones out as quickly as possible, and then cloning the fruit from one that did well (I've done that procedure a few times). At least I end up with a strain that grows well on my substrate, at my temperatures, and which actually fruits.

If the clone has a contaminant, I would just isolate that out through repeat agar transfers, which feels like the more logical time to do that, rather than before knowing that I have something worthwhile.

I'm no doubt missing something, so please do enlighten me :-)


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Invisiblenotthatsorry
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Re: Confused about isolate [Re: EarShot]
    #28885760 - 07/24/24 05:16 PM (5 months, 21 days ago)

I don't think you're really missing anything.

What next?
Yes, take a couple of transfers from your germination plate to new plates. You're not looking to create an 'isolate', you're just looking to take some germinated spores from the initial plate and give them room to grow out. This will help you ensure that what you have is healthy mycelium.

Should I only pick the best looking?
You want to take growth that looks organized and vigorous. It doesn't necessarily have to be rhizomorphic, but that's a good indicator. You only need to take transfers until you're confident that you have a healthy culture. Somewhere between 1 and 3 transfers is typically enough unless your culture is nasty, IME.

On using lots of petris and non-fruiting cultures.
In general, unless you're hunting for fun phenotypes or working with wild spores, you only need to use as many petris as it takes to get a single clean culture. My understanding is that its pretty rare to get a culture that doesn't fruit when working with commonly cultivated species.
Plenty of people DO take multiple transfers from their germination plates and make multiple transfers of those transfers, but it's not necessary.

On inoculating multiple small jars directly with spores and cloning fruits.
What you're looking for here is known as PF Tek and is specifically designed for direct inoculation of spores. If you don't want to fuck with agar, this is a perfectly reasonable route.

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OfflineA.k.aM
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Re: Confused about isolate [Re: notthatsorry] * 1
    #28885913 - 07/24/24 06:52 PM (5 months, 21 days ago)

Take transfers as small as possible, you can even use a syringe tip. Search for josex poke.

I like to take 3-4 transfers from the germination plate and put them all on one plate. Then a couple days later I’ll pick whatever looks best and transfer again.


If you’re planning on doing consistent growing it’s worth learning all this, but if you just want to grow occasionally spore syringes are fine just knock up a few jars like you said.


There’s lots of detailed threads about agar work and genetics in some of the sticky’s.


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OfflineEarShot
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Re: Confused about isolate [Re: A.k.a]
    #28886281 - 07/25/24 04:38 AM (5 months, 20 days ago)

Quote:

A.k.a said:
Take transfers as small as possible, you can even use a syringe tip. Search for josex poke.





I was actually thinking of using the syringe tip! Either that or I'll hammer a paper clip and melting the other end into a toothbrush handle to make a ghetto scalpel.

Quote:

A.k.a said:
I like to take 3-4 transfers from the germination plate and put them all on one plate. Then a couple days later I’ll pick whatever looks best and transfer again.





That's maybe the bit that's missing. That would definitely use up fewer plates. Although it might increase risk of contam until I get my SAB technique down.


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OfflineDERRAYLD
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Re: Confused about isolate [Re: EarShot] * 2
    #28886283 - 07/25/24 04:41 AM (5 months, 20 days ago)

Limit your number of plates and force yourself to work within clear boundaries, you'll notice an ability to learn faster when you are focused rather than having 200 plates that have low value.

Edited by DERRAYLD (07/25/24 04:55 AM)

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OfflineEarShot
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Re: Confused about isolate [Re: notthatsorry]
    #28886294 - 07/25/24 05:08 AM (5 months, 20 days ago)

Quote:

notthatsorry said:
On inoculating multiple small jars directly with spores and cloning fruits.
What you're looking for here is known as PF Tek and is specifically designed for direct inoculation of spores. If you don't want to fuck with agar, this is a perfectly reasonable route.




Thanks. I'm aware that PF Tek is more resilient to contamination due to the fact it doesn't move (but possibly other factors?). I don't plan to go down that route partly because I don't have that kind of jar, nor do I want that type of fruiting chamber. I want to do trays, which have worked for me in the past. But I think there's a way to obtain the benefit of PF Tek cakes without making the cakes, simply by cooking up a load of very small tubs (think take away sauce tubs) with something like brown rice, which is what Violet does here (great read btw):
https://www.shroomery.org/forums/showflat.php/Number/19035259/fpart/1/vc/1

I was thinking that could be done with the actual grain I'll be growing on to select a strain that does well on that, but I've made some brown rice ones (just repaired my PC and it needed a test run) so will experiment with that.


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OfflineEarShot
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Re: Confused about isolate [Re: DERRAYLD]
    #28886295 - 07/25/24 05:09 AM (5 months, 20 days ago)

Quote:

DERRAYLD said:
Limit your number of plates and force yourself to work within clear boundaries, you'll notice an ability to learn faster when you are focused rather than having 200 plates that have low value.




Fair point.


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OfflineMycCycle
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Re: Confused about isolate [Re: EarShot]
    #28886316 - 07/25/24 05:44 AM (5 months, 20 days ago)

Quote:

I'll hammer a paper clip and melting the other end into a toothbrush handle to make a ghetto scalpel.




Ghetto scalpel?
That’s full on penitentiary mode 😂

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OfflineBigdogg
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Re: Confused about isolate [Re: EarShot]
    #28887195 - 07/25/24 07:47 PM (5 months, 20 days ago)

Quote:

EarShot said:


Quote:

A.k.a said:
I like to take 3-4 transfers from the germination plate and put them all on one plate. Then a couple days later I’ll pick whatever looks best and transfer again.





That's maybe the bit that's missing. That would definitely use up fewer plates. Although it might increase risk of contam until I get my SAB technique down.




I am also new to agar work and was given the same advice. It is no different than taking any other transfer.

Turn your plate so the transfer sector is facing about 4 o'clock. Lift the lid, take your transfer, set the lid back on, put it in the new plate, put the lid back on. Rotate both plates. Repeat.

That's assuming your right handed. That's the comfortable angle for me anyways.

You got it.

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OfflineEarShot
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Re: Confused about isolate [Re: MycCycle]
    #28888158 - 07/26/24 04:05 PM (5 months, 19 days ago)

Quote:

MycCycle said:
Quote:

I'll hammer a paper clip and melting the other end into a toothbrush handle to make a ghetto scalpel.




Ghetto scalpel?
That’s full on penitentiary mode 😂




I have two goals:

1. Grow some mushrooms.
2. Be the most reprehensible ghetto cheapskate the site has ever seen.


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OfflineEarShot
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Re: Confused about isolate [Re: Bigdogg] * 1
    #28888166 - 07/26/24 04:13 PM (5 months, 19 days ago)

Quote:

Bigdogg said:
Quote:

EarShot said:


Quote:

A.k.a said:
I like to take 3-4 transfers from the germination plate and put them all on one plate. Then a couple days later I’ll pick whatever looks best and transfer again.





That's maybe the bit that's missing. That would definitely use up fewer plates. Although it might increase risk of contam until I get my SAB technique down.




I am also new to agar work and was given the same advice. It is no different than taking any other transfer.

Turn your plate so the transfer sector is facing about 4 o'clock. Lift the lid, take your transfer, set the lid back on, put it in the new plate, put the lid back on. Rotate both plates. Repeat.

That's assuming your right handed. That's the comfortable angle for me anyways.

You got it.




Thanks. I should really look at some videos. My work felt sloppy AF!! I did some wedge transfers leaving the source dish open the whole time as I figured constantly putting the lid on and of would be more likely to draw up a spec of dust with air movement, and it's also more activity over the plate. Plus I'd be putting the lid down somewhere. Maybe that's the wrong way to go? I'm assuming the source dishes have a fair chance of being contaminated now.

Bear in mind I'm working with tiny 1" diameter plastic plates, and it's really finicky lifting the lids off cleanly. I feel like I'd be working differently with regular size plates.

Either way, I did a few transfers from the 3 dishes that had reasonable growth, and those are fluffing up nicely in their new dishes so far, so fingers crossed.


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OfflineBigdogg
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Re: Confused about isolate [Re: EarShot]
    #28888190 - 07/26/24 04:32 PM (5 months, 19 days ago)

If you just have tiny plates probably 1 transfer each. I had some 60mm plates and did 1 to each then got 90mm and put 3 on each.

Inside the SAB should have a damp towel and all plates should be on a wire rack. I put an alcohol soaked paper towel down and put the lid on that while I'm cutting. Then I pull the transfer section out as I place the lid back on. The clean tray I just tilt the lid and slip the transfer in.

There are some good videos here if you look at some agar teks.

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OfflineA.k.aM
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Re: Confused about isolate [Re: Bigdogg]
    #28888194 - 07/26/24 04:35 PM (5 months, 19 days ago)

One inch plates??

You gotta find some real ones, 60mm at least.

The condiment cups are prob about an inch and they’re pretty useless for culture work compared to real plates. I use them to test syringes or i colonize them with a good culture and then pop the whole puck into the grain, they’re perfect for that. Especially if you dice up the agar first.


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Offlinealienascii
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Re: Confused about isolate [Re: EarShot]
    #28888613 - 07/27/24 12:39 AM (5 months, 18 days ago)

I think we need a thread for reprehensible ghetto cheapskates to show off their ingenuity.  Love seeing that stuff.

Don't be afraid to slip your lids on and off it's a key part of my technique.

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OfflineEarShot
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Re: Confused about isolate [Re: alienascii]
    #28888714 - 07/27/24 05:36 AM (5 months, 18 days ago)

Quote:

alienascii said:
I think we need a thread for reprehensible ghetto cheapskates to show off their ingenuity.  Love seeing that stuff.





There was a recent (2023?) competition thread on that themes but I can't find it for shit. Maybe we should start one.


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OfflineEarShot
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Re: Confused about isolate [Re: A.k.a]
    #28888738 - 07/27/24 06:29 AM (5 months, 18 days ago)

Quote:

A.k.a said:
One inch plates??

You gotta find some real ones, 60mm at least.

The condiment cups are prob about an inch and they’re pretty useless for culture work compared to real plates. I use them to test syringes or i colonize them with a good culture and then pop the whole puck into the grain, they’re perfect for that. Especially if you dice up the agar first.




Yeah I just ordered some ready made plates as I didn't trust myself, and didn't realise they'd be that size! I'll order some proper ones and make my own agar in the coming weeks.

I happen to have plenty PP5 condiment tubs, and given my spore syringe seems pretty clean (the only contaminated plate was from agar seeping over the side) I'm thinking I can just fill them with left over rice and shoot spores and see what grows, then case them. I obviously wouldn't use them to inoculate grain jars though.


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OfflineA.k.aM
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Re: Confused about isolate [Re: EarShot]
    #28888760 - 07/27/24 06:57 AM (5 months, 18 days ago)

Why wouldn’t you knock up jars with them?? That’s what they’re for :lol:


Especially if you’ve already put some on agar and it’s clean, you’re good to go. My first 20 or so tubs were syringes to quarts of wbs.


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OfflineEarShot
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Re: Confused about isolate [Re: A.k.a]
    #28891670 - 07/29/24 05:09 AM (5 months, 16 days ago)

Quote:

A.k.a said:
Why wouldn’t you knock up jars with them?? That’s what they’re for :lol:


Especially if you’ve already put some on agar and it’s clean, you’re good to go. My first 20 or so tubs were syringes to quarts of wbs.




Well I'm now really not sure the spores are that clean, I can't tell cobweb from myc!

I do have grain bags (bought sterile) which I inoculated with the spores running in parallel while I fuck around with agar.


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