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OfflineD3monic
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D3's Perfect transfers and agar TEK. * 19
    #26628234 - 04/25/20 10:35 PM (4 months, 21 days ago)

Perfect Transfers and Agar TEK


I'm frequently asked how I get my agar so clear, the colors and how I get such perfect transfers. The following below is a detailed explanation of the process. There's many ways to achieve similar results but this what works for me. Feel free to experiment with different recipes and nutrient levels.



Tools Required






Petri Dishes




By far Polystyrene and Glass will give the best clarity. I use polypropylene for streaks or other circumstances where I don't expect to show off samples. Nothing beats the cheap disposable dishes though. I've tried no pours, deli cups, glad mini rounds.. none where for me. The particular brand I'm using right now is Nest Scientific. I'm open to others suggestions on brands. Right now a case is averaging me around $90 for 500 count.

The Agar


I've tried several brands of agar agar and premade mixes. Most left fibrous bits in my petris detracting from the overall appearance. Ultimately I have found that this brand gives consistent results



For nutrients i'm using Maltoferm light malt extract it disolves clean and clear.



For coloring, Misty turned me onto this brand. Wilton Neon Gel it has given best results and color doesn't break down in the PC.



Ingredients


The following recipe will produce about 40 petri dishes. You can reduce by half if pouring only one sleeve of 20. I've had best results so far with this amount of nutrients, feel free to experiment with more or less levels of LME. Lower the nutrients the more likely you will get rhizomorphic growth, higher nutes more tomentos. Of course genetics and other environmental conditions come into play as well.

  • 20g agar agar
  • 16g LME
  • 1000ml distilled water


Directions


1. Add distilled water to pot and begin heating to light boil.
2. While water is heating up, measure out dry ingredients.
3. Once water is beginning to lightly boil, whisk in dry ingredients and reduce heat to prevent boil over.
4. If you have a stick blender, blend for 30-45 seconds thoroughly.



5. Continue to lightly boil agar for 2-3 minutes stirring to prevent foaming and boil over.
6. Add food coloring to media bottles. Personally I use 3 bottles for a 1000ml batch. 3 drops food coloring or 1 drop per 100ml per media bottle. Have fun, experiment with mixing colors.
7. Funnel agar mix into media bottles distributing evenly among bottles. Swirl to distribute food coloring, cap loosely and cover top with foil.



8. Add enough water to PC for a 20 minute run. I tend to fill just over the rack without using jar rings to raise rack. This is sufficient for doing a agar run.



9. Follow the manufacturer instruction for purging and bringing PC up to a boil prior to adding the weight. I use the modified weight with 2 quarters tapped to it. This reduces rattle and allows the pressure cooker to reach pressures above 15psi.
10. Bring the PC up to 15-17psi and set timer for 20 minutes. Continue to monitor pressure don't allow it to exceed 17psi for safety sake.
11. Once time is up, turn off heat and allow pressure to release naturally, use this time to shower, prep SAB if using one, wipe down your prep surfaces ect.
12. In pot large enough to hold media bottles, heat several inches of water to 140f (60c) or if you own a sous vide this is even better.
13. Carefully remove media bottles from PC, place into pot of water ( this serves two purposes, one it helps cool the bottles down to handling temps and helps keep bottles warm to prevent from setting up while pouring other bottles) move your pot and media bottles to where you will be performing pours.
14. Using a laser temp probe measure the media bottles temperature. I begin pouring once temperature reaches 135-140f (57-60c)

Once all plates are poured leave unwrapped in SAB with arm holes covered for several days or several hours in front of flowhood to allow condensation to subside.

 

The end result is a crystal clear plate with no condensation




Taking Transfers


This method of taking transfers can be performed within a SAB or in front of a flow hood. For obvious reasons a flow hood is much easier to work in front of. My technic has room for improvement but you will get the gist.



Tools Needed


The main focus of the TEK utilizes a hole punch to act as a cookie cutter making perfectly round holes in your sample for transfer. The method has been floating around on the groups for some time now. You can make one these out of brass tubes, metal straws what ever. You're only limited by your imagination. I personally use either 5/16th or 1/4" punches. I have a custom tool being made right now that I will share once it's received.



  • Leather hole punch
  • Flowhood or SAB
  • Scalpel
  • Torch or alcohol burner
  • Parafilm or cling wrap... what ever you prefer wrapping your plates with
  • Petri dishes transferring from.
  • Poured petri dishes for transferring to.
  • Spare dish for cooling heated tools ( I use a ugly dish, one that had sediment or perhaps agar started to gel toward end of pours)



Directions


1. Select the cultures you will be transferring from.
2. Prep you work surfaces as usual, wipe down with iso ect. I personally don't spray any chemical cleaners in my lab.
3. Prep your sending and receiving dishes, remove parafilm, put receiving dishes in stacks relative to amount of transfers you intend to take ect.
4. Flame sterilize your scalpel and place into sterile glass container blade up.
5. Flame sterilize your leather punch or transfer tool. You will not be able to get the steel red hot without burning yourself and not being able to handle the tool. Just gently flame the tip and slot opening for a few good seconds like shown in video above.
6. Cool the leather punch / transfer tool in the spare petri dish by gently depressing into agar several times until it stops sizzling.
7. Use the tool to cut perfect circles into the leading edge of your myc





8. Using your previously flame sterilized scalpel, gently poke into the side of your transfer disc and lift. This may take a little practice but it's extremely easy once you have done it a couple times. i recommend practicing on a blank petri until you get the hang of it.





I like to place the transfer Myc face down, this helps speed up the growth and sandwiching any contams between the agars giving the myc a chance to get a head start on it.



By gently pressing the disc onto the recieving plate it should stick and allow you to slide your scalpel out. Sometimes if your scalpel slipped too far into the disc you may have a little difficulty getting it free.

You're left with a perfect little transfer disc in the center of your agar.



The end result after a few transfers are beautifully symmetrical dishes.





Hope this little tutorial helps you elevate your agar and transfer game. I'll update periodically with better videos and pictures.

Thanks, D3


Edited by D3monic (06/26/20 05:36 PM)


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OfflineSockadin
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Re: D3's Perfect transfers and agar TEK. [Re: D3monic] * 1
    #26628238 - 04/25/20 10:36 PM (4 months, 21 days ago)

YES!

Great TEK. Well written and lots of detail, nice one!


Edited by Sockadin (04/25/20 10:51 PM)


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Invisiblewoofwoof
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Re: D3's Perfect transfers and agar TEK. [Re: Sockadin] * 1
    #26628260 - 04/25/20 10:48 PM (4 months, 21 days ago)

Nice job!


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Re: D3's Perfect transfers and agar TEK. [Re: Sockadin] * 1
    #26628276 - 04/25/20 10:56 PM (4 months, 21 days ago)

:rockon:


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Offlinemushhead
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Re: D3's Perfect transfers and agar TEK. [Re: Subnet Mask] * 1
    #26628283 - 04/25/20 10:58 PM (4 months, 21 days ago)

I've been waiting for this specific write up!


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Offlinepureshrooming
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Re: D3's Perfect transfers and agar TEK. [Re: mushhead]
    #26628310 - 04/25/20 11:09 PM (4 months, 21 days ago)

They're so pretty :smile: Definitely gonna give this a try


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OfflineD3monic
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Re: D3's Perfect transfers and agar TEK. [Re: pureshrooming]
    #26628326 - 04/25/20 11:19 PM (4 months, 21 days ago)

I need to fix some the picture orientations. Looked better on the laptop


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Offlinehallmaker
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Re: D3's Perfect transfers and agar TEK. [Re: D3monic]
    #26628382 - 04/26/20 12:00 AM (4 months, 21 days ago)

Nice right up duuuuude. Gonna definitely use the food coloring and leather punch tips. Couple quick questions.
1. In a earlier post, I seen you cross breed using liquid streaks. Would putting multiple agar punches from 2 different strains do the same? Also, I'm gonna use the marker on the petri dish lid to trace the streak tip.
2. Do you do anything special with your camera for clearer shots. Both my camera and phone blur just before the shot
3. Is your leather punch particularly extra sharp or will a decent set from Amazon be ok?

Thanks for the help


--------------------
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Offlineorange771
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Re: D3's Perfect transfers and agar TEK. [Re: hallmaker]
    #26628423 - 04/26/20 12:21 AM (4 months, 21 days ago)

Ive been using a cork borer, cheap red one off of amazon about $7 and it works great. Basically does what the leather punch and scalpel do but with one tool.


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OfflineD3monic
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Re: D3's Perfect transfers and agar TEK. [Re: hallmaker]
    #26628454 - 04/26/20 12:36 AM (4 months, 21 days ago)

Quote:

hallmaker said:
Nice right up duuuuude. Gonna definitely use the food coloring and leather punch tips. Couple quick questions.
1. In a earlier post, I seen you cross breed using liquid streaks. Would putting multiple agar punches from 2 different strains do the same? Also, I'm gonna use the marker on the petri dish lid to trace the streak tip.
2. Do you do anything special with your camera for clearer shots. Both my camera and phone blur just before the shot
3. Is your leather punch particularly extra sharp or will a decent set from Amazon be ok?

Thanks for the help




Any set should be fine, there’s also other options like dermal punches. I got a tool being made for me right now that has a custom made handle of either wood or acrylic. Look forward to trying it out.

I’ve never used liquid inoculant for streaks only spores. I traced my streaks out and labeled them before hand to just help
Me plan and remember what was streaked where since I was doing so many different cross attempts.



As far as the sandwiching agar pucks for crosses I’m not sure. Probably a better question for the more experienced folks over on the strain crossing thread.


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OfflineD3monic
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Re: D3's Perfect transfers and agar TEK. [Re: orange771]
    #26628459 - 04/26/20 12:41 AM (4 months, 21 days ago)

Quote:

orange771 said:
Ive been using a cork borer, cheap red one off of amazon about $7 and it works great. Basically does what the leather punch and scalpel do but with one tool.




I just looked up the cork borer, interesting tool. Does the puck stick inside it when you do the punch then you just pop it out with the t-handle? If so curious as to how it stays inside. I think I’ve only had it stick like once with the leather punch. Would like to see it in action.


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InvisiblefahtsterM
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Re: D3's Perfect transfers and agar TEK. [Re: D3monic]
    #26628505 - 04/26/20 01:14 AM (4 months, 21 days ago)

Very well put together!  Man.. those punches make for some beautiful growth.

Faht


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Offlinefungi4us
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Re: D3's Perfect transfers and agar TEK. [Re: D3monic]
    #26628510 - 04/26/20 01:15 AM (4 months, 21 days ago)

TY for the info


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Re: D3's Perfect transfers and agar TEK. [Re: D3monic] * 1
    #26628518 - 04/26/20 01:17 AM (4 months, 21 days ago)

That's some really good looking growth 👌
D you havent had any problems with contams dropping of your hand onto the open plate or the piece being transferred?
I get contams everytime I accidentally pass my gloved hand over a plate. Especially when I'm pouring. I always know which sleeve is going to have a bad plate in it. I see the pic where you are cutting the disk and it scares the shit outta me lmao. I mean it seems to be working good for you but it makes me uncomfortable for some reason.


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Offlineorange771
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Re: D3's Perfect transfers and agar TEK. [Re: D3monic]
    #26628519 - 04/26/20 01:17 AM (4 months, 21 days ago)

Mine has 3 different size diameter T handle tubes, then there’s a skinny rod that sticks down the length with a flat piece of plastic at the handle end for your thumb. You use that to pop the piece that’s in the tube out of there. It takes a little practice to get it to stay in every time but it works great when you want to do a bunch of transfers quickly.


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OfflineD3monic
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Re: D3's Perfect transfers and agar TEK. [Re: gizmo1]
    #26628537 - 04/26/20 01:25 AM (4 months, 21 days ago)

Quote:

gizmo1 said:
That's some really good looking growth 👌
D you havent had any problems with contams dropping of your hand onto the open plate or the piece being transferred?
I get contams everytime I accidentally pass my gloved hand over a plate. Especially when I'm pouring. I always know which sleeve is going to have a bad plate in it. I see the pic where you are cutting the disk and it scares the shit outta me lmao. I mean it seems to be working good for you but it makes me uncomfortable for some reason.




The only contams I’ve gotten where from spore streaked plates. I don’t even wrap the cooling plate and have never had it contam. I’ll leave the plate out for a few weeks or until it’s hideously scarred from me cooling tools in it. Not sure if that’s a testament to how clean my lab is or what. I do frequently squirt some iso on my gloves and wipe them down good if I touch things like my phone. If it wasn’t for how dry iso makes my skin I’d probably not even wear gloves. I also typically do my agar work first thing after I’ve showered making sure to not pet the dogs or go outside beforehand.


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Re: D3's Perfect transfers and agar TEK. [Re: D3monic] * 1
    #26629336 - 04/26/20 10:43 AM (4 months, 21 days ago)

Grateful for your artist approach.


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Invisibleeatyualive
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Re: D3's Perfect transfers and agar TEK. [Re: Hobbit GDF] * 1
    #26629438 - 04/26/20 11:29 AM (4 months, 21 days ago)

Beautiful! :thumbup:

And I like the music.


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Edited by eatyualive (04/26/20 11:34 AM)


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OnlineCrackatoa
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Re: D3's Perfect transfers and agar TEK. [Re: Hobbit GDF] * 1
    #26629450 - 04/26/20 11:36 AM (4 months, 21 days ago)

:leocheers:


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Re: D3's Perfect transfers and agar TEK. [Re: Crackatoa] * 1
    #26629591 - 04/26/20 12:26 PM (4 months, 21 days ago)

always a fan of your work:cheers:


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