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InvisibleDoctor Mario
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26923922 - 09/07/20 10:08 PM (3 years, 5 months ago)

:discorex:


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Invisiblemaxmush
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Registered: 06/13/20
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Doctor Mario]
    #26924482 - 09/08/20 10:54 AM (3 years, 5 months ago)

Great thread. Been looking for a step by step clean up with pics. Very helpful and good job :smile:


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: maxmush]
    #26926275 - 09/09/20 12:42 PM (3 years, 5 months ago)

Quick update: Three tracks running simultaneousy...w00t!

Tubs after 1.5 days - Colonizing pretty fast and evenly, except for 2A -The top one is our suspect 2A. It looks almost normal, but we've seen the trail leading to this so this should be educational.



Jars from 3rd xfer plates - Moving along OK. I shook the jars pretty well after dropping agar wedges, most leap off has been from the wedges. Just seeing smatterings of leap off from the shake.



Plates from 4th xfer after 2 days - looking pretty vigorous, but not digging the "bald" spots...



That's it for now.

Gonna try and whip up some LME agar and do plates soon.

Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


Edited by WunFunGai (09/10/20 03:54 AM)


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26927537 - 09/10/20 03:45 AM (3 years, 5 months ago)

Update: Only 2 days and strands have popped on 1A - it's usually taken much longer for that on my shoebox runs. Interesting...



Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26929876 - 09/11/20 12:01 PM (3 years, 5 months ago)

UPDATE: Meanwhile back at the ranch...

Tubs moving along decent for 3 days in. Less progress on suspect 1A tub that's at forefront of 1st picture. Second one is just the good kids.


1A wants to win the race...


Jars moving along, a little bit of leap off from A2G shake, but thinking they'll be due for a shake this weekend:


4th transfer plates grew out quick - put into cold storage for now.


Gonna rustle up my first try at LME agar - anyone know approximately how long PC'd agar takes to cool down to 120f after PCing?

My last batch I didn't have time so I let it completely cool, put in the fridge until the weekend and did a microwave thang to get it liquid, but I poured a bit hot and got the condensation blues.

Hope these are helpful for those following...comments, suggestions welcome.

Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


Edited by WunFunGai (09/11/20 05:32 PM)


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InvisibleDoctor Mario
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26930031 - 09/11/20 01:40 PM (3 years, 5 months ago)

Using that same recipe, I let it cool down to 120 and still had too much condensation. Next batch it cooled to 115 and was almost perfect. I had the digital thermometer set to the recommended setting for glass.

Fuck waiting for it. I have to crunch time because I can't really do anything until my kids go to bed. That gives me a couple of hours to work and get my ass to sleep.

I put a pot in the sink and fill it with cold water. Place the bottle in the pot. Wait a minute, swirl the bottle in the cold water and take a temp reading. Empty half of the pot fill it up again and repeat until its cool enough to use. This cools the bottle to usable temps within minutes.

Make sure to wipe the water off the bottle before you check the temp or it will give you a false reading.


Edited by Doctor Mario (09/11/20 01:41 PM)


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Doctor Mario]
    #26930434 - 09/11/20 05:32 PM (3 years, 5 months ago)

Quote:

Doctor Mario said:
Using that same recipe, I let it cool down to 120 and still had too much condensation. Next batch it cooled to 115 and was almost perfect. I had the digital thermometer set to the recommended setting for glass.

Fuck waiting for it. I have to crunch time because I can't really do anything until my kids go to bed. That gives me a couple of hours to work and get my ass to sleep.

I put a pot in the sink and fill it with cold water. Place the bottle in the pot. Wait a minute, swirl the bottle in the cold water and take a temp reading. Empty half of the pot fill it up again and repeat until its cool enough to use. This cools the bottle to usable temps within minutes.

Make sure to wipe the water off the bottle before you check the temp or it will give you a false reading.




Thanks for the tip! I may give that a try.

For me, no kids (that I know of), and I may do it just to get a benchmark timing on temps and time to cooldown. Plus I just got an IR Temp gun I've been itching to use to play with other than to see how cranked up my GPU gets while playing video games.

Based on past shoebox runs, I'm guesstimating full colonization mon-wed, then first fruits possibly by next weekend...

Will try and post Tub/Jar progress or LME agar updates and share later.

As always, comments, experiences, suggestions welcome.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


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InvisibleDoctor Mario
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26930740 - 09/11/20 08:36 PM (3 years, 5 months ago)

Great job! It's moving along quickly. My plates are moving real slow. The spore plates went fast. I think I screwed up the agar revipe for the second set of plates because after transferring, the mycelium is moving real slow and doing weird things. Its going inside of the agar and traveling out instead if moving across the top. I'm not too sure why. Some of the plates started showing contamination and i transferred them again to a plates made with a different batch of agar and they're moving steady but slow. 🤷‍♂️


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Offlineseand04
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Doctor Mario]
    #26930793 - 09/11/20 09:03 PM (3 years, 5 months ago)

Just wanted to chime in. 🤗 I love to do agar. It is the least waste if it gets messed up.  Here is a 2nd Pf classic transfer.


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: seand04]
    #26931026 - 09/12/20 12:22 AM (3 years, 5 months ago)

Quote:

seand04 said:
Just wanted to chime in. 🤗 I love to do agar. It is the least waste if it gets messed up.  Here is a 2nd Pf classic transfer.




Noice! Those are cool containers you are using. What are those?

I'm totally digging the petri's and kicking myself in the ass for not sucking it up sooner and jumping in...like every TC ever has suggested.

I can see where having Pasty Plates or small agar plates could be useful for propagating A2G or just quickly pushing a strain to test viability.

Agar seems complicated and scary at first but it's just another process...lesson learned. Explore processes individually and find what works for you. By not trying other processes you are limiting yourself. If it don't work, no problemo, you've just found a way that doesn't work for you...move on.

"Just because something doesn't do what you planned it to do doesn't mean it's useless." - Thomas Edison

If you can embrace that, you can get further down the rabbit hole...I'm working on it. :smile:

Thanks for adding to the stone soup! :thumbup:

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26931353 - 09/12/20 08:05 AM (3 years, 5 months ago)

I used to be intimidated by agar,  until I started doing the pasty plates. Big ups to Pastywhyte 💪💪💪. I just ordered  those lil plates off of ebay. Just searched small sealable containers.  They were like 5 bucks for 10. But yea agar is super fun, like I said super low risk as well cause if they get messed up,  its just a little spores and agar. I wish I would have started with agar, live and learn 🙏🙏🙏


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OfflineMartinsapin
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26931359 - 09/12/20 08:15 AM (3 years, 5 months ago)

For condensation,I let cool my agar for 2 hours inside the PC then pour.

Then split the stack in 2 stacks of 10 and put a water glass/small jar full of hot water on top of the stacks it works great. I think thats the way RR does it in his DVD


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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Martinsapin]
    #26931429 - 09/12/20 09:14 AM (3 years, 5 months ago)

I just checked this plate. It is is a pe 2nd transfer from an original PE tissue sample. When I put the transfer i moved it around to the middle,  I hope thats why the lil white dots are there🙏🙏🙏


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InvisibleDoctor Mario
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: seand04]
    #26931536 - 09/12/20 10:21 AM (3 years, 5 months ago)

Quote:

seand04 said:
I just checked this plate. It is is a pe 2nd transfer from an original PE tissue sample. When I put the transfer i moved it around to the middle,  I hope thats why the lil white dots are there🙏🙏🙏




Thats very possible. Just keep an eye on them. You dont want them. Here's an example of a very contaminated plate that recently started showing dots.



I think the mold sporulated and the "dots" are new growth.

Here's another cool pic of contam. Lots of pretty colors in this one. All bacterial, possibly different types of yeast? The plate was intentionally contaminated on 9/1/20. I opened the plate and breathed on it. This shit originated from my mouth...🤮

Pretty gross....



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Offlineseand04
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Doctor Mario]
    #26931917 - 09/12/20 02:18 PM (3 years, 5 months ago)

I tossed it. I have a few really good looking plates I am gonna transfer onto new ones.


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: seand04]
    #26932305 - 09/12/20 06:09 PM (3 years, 5 months ago)

Update: These guys looked like they could use a shake so they got shook...



Got busy and whipped up not, one, but two bottles of LME Agar:


This is my first time trying LME vs PHA agar. Hoping to get some clear looking plates without the flake sediment I get with PHA - purely for aesthetics. If it doesn't perform as well, I'm going back to PHA.

Gonna PC those bad boys and see if they are cool enough to pour some tonight.

Tubs seem to be working hard today, will see if it's worth an update later.

Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26936202 - 09/15/20 12:23 AM (3 years, 4 months ago)

Update 09/14: Busy weekend, managed to whip up two 500ml bottles of LME agar ala D3, pour 20 plates of green and lower plate condensation rate, shake jars w/ good recover, and mist tubs.

Agar pour: So happy they turned out pretty clear and much less condensation. Started pour at 125f with ample time to pour without issues.


Update:09/15 - After 24hrs all the condensation has gone away. Will let them sit for 48hrs to observe for contam then store away.



Question: Experienced guys...would you wrap plates now that condensation is gone or after a day or two?


Jars after shake: Looks like they recovered well from the shake. Gonna have to prep for firing up more subs and tubs...


Tub Action - As suspected 2A is fail - Mean Green showed up and is getting tossed tomorrow - if you scroll back through the thread, it started with a agar wedge fumble/fiddle shit when transferring, and that tiny little start just messed up and stunted the whole thing...


For those of you experienced folks that might be watching my awkward progress. I haven't done shoeboxes for awhile, and I'm really struggling with balancing a microclimate.

I have a number of 6qt shoeboxes from the past, some with holes, some unmodified that I was going to put on top once I see pins. I'm reading and rereading the forums, and Shapers SFF has caught my attention, but would appreciate any suggestions, comments as I stumble through this again.

NOTE: I'll update this post later with some timing/temp data on the agar pour as I have time.

Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


Edited by WunFunGai (09/15/20 01:18 PM)


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26942050 - 09/18/20 09:08 AM (3 years, 4 months ago)

Update: Tubs are looking pretty spiffy and moving along. First pins on all tubs but waiting till they progress a bit more before snagging some more pics.

Here's where they were at as of 09/16:




Close up of the new neighbors...inside conditions look pretty good based on pics of seen of preferred conditions. It's far better than what I can recall in the past so leaving things alone as advised lending weight to the adage "less is more".



Enjoy.

WFG :smile:


--------------------
The Official TNF Reccomended Teks & Methods
Bod's Simplified Cultivation Methods <-- Awesome!!!
D3monics Perfect Transfers and Agar Tek

No matter where you go, there you are... - Buckaroo Banzai

You gotta be here, if you're not all there...


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Re: Trying isolation with agar and petri dishes - thoughts? [Re: WunFunGai]
    #26942606 - 09/18/20 02:50 PM (3 years, 4 months ago)

rhyzo asf


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OfflineWunFunGai
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Re: Trying isolation with agar and petri dishes - thoughts? [Re: Martinsapin]
    #26945115 - 09/19/20 11:25 PM (3 years, 4 months ago)

Update: Rub a dub dub, pinning and spawning tubs...

Some pics of the kids. Conditions looking pretty decent as far as I can tell, and have tried to just leave em alone to do their thang.

Without further ado...

NOTE: Orientation of image sequence is Left, Front, Top, Frontwards. Sorry for the quality of some images, trying to get better at consistent angles and process

The Kids together:



1A Observations: Doing pretty well, not much in the way of side pins, but they are there. Climate conditions look good, haven't touched anything and the microbeads of water are present on the surface and condensation on lid looks decent, but not drippy.


2B-1 Observations: This one also looks pretty good aside from a case of the side pins. I varied my process here somewhere and I'm thinking it was a spawn time and how I tamped spawn, and then top level. I did try to put a bit more pressure along all the edges this time though after reading ShaperDreamings SFF Shoebox tek...question to anyone reading: How tight do you tamp it down? I'm usually too chickenshit to deviate too much from what I've done, which is a gentle leveling and light hand pressure to level but that probably makes it hard to see any postive/negative effects...blergh...


2B-2 Observations: This one has led the pack from the start and it has not dissapointed. #1 seed for xfer to those sexy new glass plates poured a few days back. Conditions look good, microbeads on surface holding but might be slightly less. Lots of new neighbors today...Condensation are on wider flat portions of tub, corners seem to be drier. Watching the directions of the "boys" and it seems they are seeking a way out...

While that's all going on...the culls from second transfer are trying to bust out of their jars...

Second Transfer Jars 2A1A/2B1A-1/2B1A-2/2B2A:



2Xfer Observations: These jars were seemed faster and more aggressive in growth. No signs of contams, and much earlier and prolific appearance of rhyzo strands. They want out, so Happy Spawning Day to them! :rockon:

Whew...that was a shitload more work and clean up organizing of images and info than I expected. If you've survived the wall of text and my mindless drivel this far...Thank you. :bow2:

I doubt many TCs or vets are following this since it may be noob ground they've seen before and have gotten tired of seeing. However, I have one favor to ask, if you've tread this path before and have any suggestions or see anything important I may have missed or should be aware of going into home stretch, any tips or sharing experiences of "what works for you" are welcome and encouraged!

  • Do conditions inside look normal? Are dry corners OK?
  • What's up with side pins? Conditions look similar on all Tubs. Didn't pack down enough on edges?
  • I have old tubs both with and without holes. As they got taller was going to flip a tub and put it over. However, has anyone else just unlatched the cover and let the fruits push their way up till completion?

Side pins make me feel like my evil twin - UnFunGai :frown:

This was a big post, but I hope it was worth your time getting this far.

Enjoy.

WFG :smile:


Edited by WunFunGai (09/20/20 10:42 AM)


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