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OfflineGrinchGrower
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GG Agar Test
    #27008268 - 10/28/20 12:16 PM (3 years, 3 months ago)

Messing around with agar for the first time:
:agnosticism:

-(10/23/2020 AM) Day One: Preparation
Prepared MEA plates...

  • 1) In a low form 1000ml boro. beaker, added 600-900ml (total amount unknown I meant to pour off the excess and got distracted) of deionized H2O.
  • 2) Added 8g of store bought MEA powder and microwaved for 5-6 minutes while stirring occasionally with a glass rod until boiling and mixed well.
  • 3) Removed the solution from microwave and stirred vigorously. Beware of superheating!
  • 4) Added cheesecloth to 100mm boro. funnel and placed on first 250ml boro. media bottle.
  • 5) Filled two bottles to 200ml capacity, and one to ~230ml with hot MEA solution filtered through cheesecloth. Then added three drops of violet food coloring to each, capped, and shook vigorously to mix.
  • 6) Loosened cap 1/16 turn (just until I felt the material lining the inside of cap was not creating a seal with glass lip).
  • 7) Added foil shrouds for each bottle reaching halfway down with crimped necks to prevent any moisture from entering during sterilization.
  • 8) Placed media bottles into 23qt Presto PC with 3.5qt H2O (along with a "pre-sterilized" grain bag with rye berries someone sent me as an experiment...more on that in a different log soon), and ran for 30 minutes @ 15-17 PSI.


:beercat:


So no one got hurt and nothing broke! Hold my beer...
Once the pressure in my PC dropped to 0 PSI and steam had cleared, I removed everything and set it on the counter to cool.
Patience is a cultists most valuable asset, yet attempting to apply this sage advice to the real world is so fucking difficult.

  • 1) Proceed to obsessively monitor temperature of cooling media bottles with infrared thermometer gun until they have dropped to ~135°F. I assumed that it would cool significantly more while prepping for SAB work. MISTAKE: WAIT LONGER!!!!
  • 2) Placed two 200ml full bottles in fridge and wiped ~230ml full bottle with 70% ISO before placing in SAB on rack.
  • 4) Wiped outside of plastic sleeve and opened sterile polystyrene petri dishes. Separated into three stacks five dishes high (15 total) in SAB on rack.
  • 5) Carefully removed the foil shroud from media bottle and loosened cap.
  • 6) Lifted stack of five petri dishes with left hand (starting from bottom dish lid) and media bottle with right hand, removing cap with fingers.
  • 7) Smoothly and deliberately poured first stack of five plates. Moved the first stack to back of rack and slid second stack forward.
  • 8) Here is where things get kind of goofy....Only enough solution left to fill 2-3 plates in second stack.  :shakingfist:
  • 9) Not wanting to waste sterile petri dishes, I slowly grabbed one of the two 200ml full media bottles from the fridge. This one ended up being perfect temperature.
  • 10) Filled remaining dishes, but had excess solution so one was filled very full close to the edge.
  • 11) I used parafilm to seal 5 of the plates for later use. They were still quite warm and the agar had not settled completely, so it ended up splashing all over the inside of the lids at this point. 4x ruined plates trashed once I let them settle and realized it was gooped all over. One very low-filled plate was salvaged for later use.
  • 12) The other ten plates stayed sealed in my SAB while I let them cool. I considered sealing them at this point to work with later, but wanted to get a jumpstart on isolating some cultures.


-(10/23/20 AM) Day One: Inoculation
Another step where I should have been more patient....

  • 1) I waited about 15-30 minutes after pouring plates and returned to SAB.
  • 2) Wiped down two pre-sterilized grain bags (including the experimental bag from PC), and placed into SAB with plates. Repeated process for one Albino Cambodia MSS and one PF Classic MSS, including individually wrapped needles.
  • 3) Installed needles on syringe in SAB and flame sterilized the PF Classic MSS needle outside on butane burner.
  • 4) Back in SAB, squirted some spore solution on floor to cool needle.
  • 5) Lifted stack of five petri dishes with left hand (starting from bottom dish lid) and attempted to place one drop of spore solution in each dish. Resulting amount of spore solution introduced with this technique varied a lot. Once all five dishes were inoculated and closed, I sealed them with parafilm (some were still not completely set and almost all of them had heavy condensation) and moved stack to back of rack in SAB. Set MSS aside in SAB for now.
  • 6) Repeated steps 3-5 above with Albino Cambodia MSS and second stack of five petri dishes.
  • 7) Flame sterilized PF Classic MSS again with butane burner and wiped down SHIP on pre-sterilized grain bag (experiment from PC) in SAB.
  • 8) Injected 1-3cc spore solution into bag and resealed the MSS for storage.
  • 9) Repeated steps 7-8 above with Albino Cambodia MSS and non-PC'd pre-sterilized grain bag.
  • 10) Moved inoculated plates (and grain bags) to closet shelf for incubation.


-(10/25/20 PM) Day Two: Post-Inoculation
Some small colonies forming on each of the plates visible with backlight. Still too early for identification or transfers to begin... Pictures coming soon!

-(10/28/20 AM) Day Five: Growth
Noticeable growth on all ten plates that appears to be coming only from areas where spore solution was applied...

I have some concerns regarding the viability of all A.C. plates for transfers, because the growth is so very scattered.
It looks like tons of tiny colonies forming together en masse.
By contrast, the PFC plates all appear to be proceeding with uniform growth in isolated areas.
I am not certain if this is due to improper inoculation (I definitely used too much solution on some), or just a difference in genetics between the two strains.

Pictures for comparison:


An overview of all ten plates (upside down). Left: A.C. / Right: PFC



The questionable A.C. plates.



A closeup of one of the A.C. plate colonies.



Comparison of A.C. (left) to PFC (right). The A.C. colonies appear disorganized / crowded.



Another comparison showing the clearly organized growth on PFC plates (right) compared to the A.C. plates (left).

:awewtf:

Some input on whether the A.C. plates are worth attempting a transfer would be much appreciated!
Also curious about which colonies look like the best candidates from each strain, and when I should consider making a transfer to fresh agar plates.
I know the pictures may be a little unclear so I will try to post updated/numbered plate images in another update.

-(10/29/20) Day Six: First Transfers Tomorrow?
Growth looks promising on the PFC plates, but absolute garbage mess of unorganized colonies for the most part on A.C. plates...

First off, is it normal for mycelium to eat up the food dye (violet) used in agar?
Some of the colonies on two of my plates have pink tinged streaks or the centermost point/nipple is slightly pink...
Worried it could be contam, but I wouldn't mind losing these based on the growth patterns tbh.

Here are the plates in question:


Kind of blurry, but most of the "nipples" are pink from the bottom.


Pink streaking visible.


While I've got you here, please let me know which of these PFC plates looks the most viable candidate for transfer tomorrow morning.
Or is it best practice to take each to new plates for the second round?

Pictures are from the bottom due to condensation on my noob plates, but they all show organized clean growth so far:



-(10/30/20) Day Seven: First Transfers
Completed transfers of 5x PFC and 5x AC samples to new plates using BOD's Comprehensive Agar TEK...

:goodnews:


PF Classic sample transfer on new plate with reheated and freshly poured MEA solution.


Albino Cambodia sample transfer ready to blossom into a beautiful rhizomorphic culture.


Edited by GrinchGrower (11/02/20 03:58 PM)


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OfflineGrinchGrower
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Re: GG Agar Test [Re: GrinchGrower]
    #27008296 - 10/28/20 12:37 PM (3 years, 3 months ago)

-(11/02/20) Day Ten: T-1 Growth [3 Days]
All ten plates are showing encouraging signs of early growth...


PFC and AC transfer cultures are expanding faster than MSS, but too early to see real organization.
I think my agar wedge transfers were a little too large because the growth is quite tall in the center of each plate.
I don't think it will hit the lid, though I am worried that it will increase chances of contamination if it does.


Photos are the only way to hold on to what you knew, because the moments they show never change when the mycelium in them do.



-(11/09/20) Day Seventeen: T-1 Growth [10 Days] / T-2 Growth [3 Days]
T-1 plates looking surprisingly healthy. T-2 starting to show growth...


Some of these look so good I am considering taking them straight to grain. One of the PFC plates grew right over the transfer hole and recovered perfectly!

T-1 plates I did not take transfers from:



T-1 plates I did take transfers from:



T-2 plates:



I probably took too many T-2 plates, but I am getting a better understanding of the process this way.

:learning:

I also broke out some sterile plastic inoculation loops I had and started a couple more germ plates from MSS.



I wasn't really sure of the exact steps for using the loops, and the packaging was kind of hard to open with gloves.
I just cut the packaging open with a sterile scalpel in my SAB and set it down on rack.
Then sterilized my MSS needle, and removed the loop from packaging in SAB.
I squirted a few drops on the end (this is harder than it sounds if your hands are even slightly unsteady), and streaked the plate. It felt way more awkward than dripping directly to the plate!

-(11/16/20) Day Twenty-four: T-2 Growth [10 Days] / Germ Growth [7 Days]
Some quick updates with ongoing transfer isolations...

I am not 100% sold on the cleanliness of these transfer plates, so they may end up getting moved again when/if they show anything funky.

I kind of butchered a few with my scalpel (unsteady hands) before switching back to D3 Perfect Transfer Tek pucks.

One of each variety has a T-2 plate that looks ok to fall back on if everything goes to hell!


PF Classic plates


Albino Cambodia plates

I went ahead moved Pink Buffalo & Escondido germ plates over to T-1 as well.

B+ and Golden Teachers aren't showing shit yet though... :sad:



-(11/22/20) Day Thirty: T-3 Growth / T1 Growth [6 Days]
Transfers are starting to look a little better...

These cultures are showing more healthy formations, I think:






And the other varieties need more work, but look okay so far:


Most plates are showing some small clear bacterial growth near the edges.

So I need to transfer them again soon, unfortunately.


Edited by GrinchGrower (11/23/20 12:10 AM)


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OfflineGrinchGrower
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Re: GG Agar Test [Re: GrinchGrower]
    #27010927 - 10/29/20 06:15 PM (3 years, 3 months ago)

<reserved>


Edited by GrinchGrower (10/30/20 07:12 PM)


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OfflineGrinchGrower
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Re: GG Agar Test [Re: GrinchGrower]
    #27012691 - 10/30/20 07:09 PM (3 years, 3 months ago)

No feedback, so I've been forging ahead! Updated OP w/ a little info on T-1 transfers completed today.


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OfflineGrinchGrower
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Re: GG Agar Test [Re: GrinchGrower] * 1
    #27017550 - 11/02/20 01:42 PM (3 years, 3 months ago)

-(11/02/20) Day Ten: T-1 Growth [72 Hours]
All ten plates are showing encouraging signs of early growth...


PFC and AC transfer cultures are expanding faster than MSS, but too early to see real organization.
I think my agar wedge transfers were a little too large because the growth is quite tall in the center of each plate.
I don't think it will hit the lid, though I am worried that it will increase chances of contamination if it does.


Photos are the only way to hold on to what you knew, because the moments they show never change when the mycelium in them do.



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Offlinefraki58
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Registered: 11/20/19
Posts: 246
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Re: GG Agar Test [Re: GrinchGrower]
    #27017691 - 11/02/20 02:51 PM (3 years, 3 months ago)

Looking good!


--------------------
If any of the said above is incorrect, please correct me! Still learning here and constructive criticism is the best :smile:


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OfflineGrinchGrower
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Re: GG Agar Test [Re: fraki58]
    #27017757 - 11/02/20 03:45 PM (3 years, 3 months ago)

Thanks fraki58! Trying to put the work in without rushing it.


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OfflineSingularFusion
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Re: GG Agar Test [Re: GrinchGrower]
    #27018585 - 11/03/20 01:24 AM (3 years, 3 months ago)

looks good from here GG

:borat:


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿


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OfflineGrinchGrower
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Re: GG Agar Test [Re: SingularFusion]
    #27030873 - 11/09/20 10:39 PM (3 years, 3 months ago)

-(11/09/20) Day Seventeen: T-1 Growth [10 Days] / T-2 Growth [3 Days]
T-1 plates looking surprisingly healthy. T-2 starting to show growth...


Some of these look so good I am considering taking them straight to grain. One of the PFC plates grew right over the transfer hole and recovered perfectly!

T-1 plates I did not take transfers from:



T-1 plates I did take transfers from:



T-2 plates:



I probably took too many T-2 plates, but I am getting a better understanding of the process this way.

:learning:

I also broke out some sterile plastic inoculation loops I had and started a couple more germ plates from MSS.



I wasn't really sure of the exact steps for using the loops and the packaging was kind of hard to open with gloves.
I just cut the packaging open with a sterile scalpel in my SAB and set it down on rack.
Then sterilized my MSS needle, and removed the loop from packaging in SAB.
I squirted a few drops on the end (this is harder than it sounds if your hands are even slightly unsteady), and streaked the plate. It felt way more awkward than dripping directly to the plate!


Edited by GrinchGrower (11/09/20 10:48 PM)


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OfflineSingularFusion
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Re: GG Agar Test [Re: GrinchGrower]
    #27030892 - 11/09/20 11:01 PM (3 years, 3 months ago)

I feel you on the dripping mss to loop

tricky, especially when you are high

I put a drop to the plate then streak it to spread it out

but yes man it looks like you are well on your way over there, you can't have too many plates in the beginning. You learn a lot from watching the ones that fuck up too

:rockon:


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿


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Offlinenewbieatx
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Re: GG Agar Test [Re: GrinchGrower]
    #27030974 - 11/10/20 01:11 AM (3 years, 3 months ago)

Quote:

GrinchGrower said:
-(11/09/20) Day Seventeen: T-1 Growth [10 Days] / T-2 Growth [3 Days]
T-1 plates looking surprisingly healthy. T-2 starting to show growth...


Some of these look so good I am considering taking them straight to grain. One of the PFC plates grew right over the transfer hole and recovered perfectly!

T-1 plates I did not take transfers from:



T-1 plates I did take transfers from:



T-2 plates:



I probably took too many T-2 plates, but I am getting a better understanding of the process this way.

:learning:

I also broke out some sterile plastic inoculation loops I had and started a couple more germ plates from MSS.



I wasn't really sure of the exact steps for using the loops and the packaging was kind of hard to open with gloves.
I just cut the packaging open with a sterile scalpel in my SAB and set it down on rack.
Then sterilized my MSS needle, and removed the loop from packaging in SAB.
I squirted a few drops on the end (this is harder than it sounds if your hands are even slightly unsteady), and streaked the plate. It felt way more awkward than dripping directly to the plate!




Thank you so much for posting your progress on this. Helps people like me that are learning to watch someone else's efforts.  :cheers:


--------------------
Noob here, so no words of wisdom, just a gal trying to figure this shit out.


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OfflineGrinchGrower
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Re: GG Agar Test [Re: newbieatx]
    #27042783 - 11/16/20 07:30 PM (3 years, 2 months ago)

Quote:

Nef said:
tricky, especially when you are high







Quote:

newbieatx said:
Thank you so much for posting your progress on this. Helps people like me that are learning to watch someone else's efforts.  :cheers:




No problem dude!

I'm glad some people are finding my noobish musings and misadventures helpful...


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OfflineGrinchGrower
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Re: GG Agar Test [Re: GrinchGrower]
    #27042794 - 11/16/20 07:35 PM (3 years, 2 months ago)

-(11/16/20) Day Twenty-four: T-2 Growth [10 Days] / Germ Growth [7 Days]
Some quick updates with ongoing transfer isolations...

I am not 100% sold on the cleanliness of these transfer plates, so they may end up getting moved again when/if they show anything funky.

I kind of butchered a few with my scalpel (unsteady hands) before switching back to D3 Perfect Transfer Tek pucks.

One of each variety has a T-2 plate that looks ok to fall back on if everything goes to hell!


PF Classic plates


Albino Cambodia plates

I went ahead moved Pink Buffalo & Escondido germ plates over to T-1 as well.

B+ and Golden Teachers aren't showing shit yet though... :sad:



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OfflineGrinchGrower
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Registered: 10/02/20
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Re: GG Agar Test [Re: GrinchGrower]
    #27053225 - 11/22/20 11:44 PM (3 years, 2 months ago)

-(11/22/20) Day Thirty: T-3 Growth / T1 Growth [6 Days]
Transfers are starting to look a little better...

These cultures are showing more healthy formations, I think:






And the other varieties need more work, but look okay so far:


Most plates are showing some small clear bacterial growth near the edges.

So I need to transfer them again soon, unfortunately.


Edited by GrinchGrower (11/23/20 12:07 AM)


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OfflineKOTAthefriend
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Registered: 11/19/20
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Last seen: 3 years, 4 days
Re: GG Agar Test [Re: GrinchGrower]
    #27053239 - 11/23/20 12:18 AM (3 years, 2 months ago)

Dude, this is awesome! I'm brand new to this (forum and growing shrooms) and am setting up my own involved experiment here soon and I have a feeling we're gonna be buds haha. I would love for you to look over my experiment once I get the procedure on paper, it seems like you have quite a bit more experience in this stuff than myself

Also I gotta learn how to insert hilarious gifs lmaooo:grin:

I posted a little thing about it in my journal if ya want a sneak peek, the idea is to test substrates by growing in a petri dish and weighing/measuring the growth and fruits


--------------------
I live inside my head-you don't think I've made improvements?


Edited by KOTAthefriend (11/23/20 12:20 AM)


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OfflineGrinchGrower
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Re: GG Agar Test [Re: KOTAthefriend]
    #27065641 - 11/30/20 11:59 PM (3 years, 2 months ago)

-(11/30/20) Day Thirty-eight: T-4 / T-3 / T-2 / Germ Growth


One of my T-3 plates is looking okay a few days after transfers were taken:





The rest are in various stages of early growth:








Finally got these to germinate with the last of my MSS too:



Edited by GrinchGrower (12/01/20 12:11 AM)


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