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OfflineoO_wombat_Oo
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Newbie agar question...
    #26968485 - 10/04/20 02:35 AM (3 years, 4 months ago)

Apologies if this seems dumb, I've never worked with agar before.

I'm wondering, why sterilize the agar and then pour? Why not pour the agar and then steralize the dishes?

Assuming there's a good reason for the first thing, what's the most effective way to do this sans HEPA flow hood? I have a small, home-made glove-box, but to be honest, it feels pretty clumsy and I never have a good idea if I've sterilized it properly or not (I basically just spray a lot of alcohol-mist in there, then close up the lid and leave it for a bit).


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OfflineMrTheseThings
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26968492 - 10/04/20 02:56 AM (3 years, 4 months ago)

People sterilize there agar then pour because  the dishes are sterile in the packedge. They also won't hold up to heat sterilization in our pressure cookers. There are different types of dishes and methods to do agar though.  I highly recommend you look into some of the no pour agar teks.  Seams right in line with what you are wanting to do .

Your glove box set up sounds stressful.  Maybe try a real SAB I made mine using the easy as fuck method and taught myself to use it through reading here . It's pretty damn stress free , easy , cheap and efficant! 

Hope this helps ya out a bit !


--------------------
These things infected me... and now I have to  grow them.


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OfflineForresterM
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Re: Newbie agar question... [Re: MrTheseThings]
    #26968533 - 10/04/20 04:14 AM (3 years, 4 months ago)

Quote:

MrTheseThings said:
Maybe try a real SAB




:whathesaid:

Ditch any box with attached gloves and make a proper SAB, pour agar plates inside that or use a no-pour method (tons of easily searchable write-ups on these) and you're good to go.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: Forrester]
    #26970012 - 10/05/20 12:46 AM (3 years, 4 months ago)

Thanks guys, I appreciate all the advice.

I have read write-ups on SOB's and watched some YouTube videos but I still have some questions.

It seems to me to be just a box with holes cut in it for hands. Some use a wet towel underneith and others do not.

I haven't found one that explains how to properly use it, apart from "move deliberately and quickly and don't flame sterilize inside the box".

Some seem to allude to spraying the ways of the box with water and a drop of soap, but don't explain why or if this is necessary. Do you also spray the "roof"? Wouldn't that make it hard to see? And if you don't, wouldn't it then defeat the purpose of spraying the walls?

If someone can explain in a bit more detail, that would be great.


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OfflineForresterM
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26970083 - 10/05/20 02:49 AM (3 years, 4 months ago)

Just spray everything with ISO alcohol, not soap and it will not linger or create a film.  You can move regular speed just deliberately.  All you're doing is trying not to stir up air currents and knock spores off of things :smile:

You'll get it down, just give it a shot!


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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OfflineCegda
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Re: Newbie agar question... [Re: Forrester]
    #26972008 - 10/06/20 11:48 AM (3 years, 4 months ago)

I use pre pour dishes my friend.

I specifically like the target brand twist locks.. they stack easier than the ziploc brand and are a whole lot easier to wash.  Pour, stack in pc, and 20 minutes later they're ready


--------------------
“What senses do we lack that we cannot see or hear another world all around us?”

-Orange Catholic Bible,
    -from Frank Herbert's Dune


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: Cegda]
    #26976400 - 10/09/20 02:59 AM (3 years, 4 months ago)

This is how I've done it. These are heat-resistant up to about 120 C, so might be pushing it in the pressure cooker, we'll see.

I drilled holes in the side and covered with micro-pore tape for air exchange. I play to pour the agar, have the lids on loosely, put in the pressure cooker for 90 minutes post-pour, let the PC cool down, then snap the lids on tightly as I remove them one by one.

Sound like a plan?




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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26976631 - 10/09/20 08:30 AM (3 years, 4 months ago)

Well, I went ahead with this despite there being no replies. From everything I can tell, they turned out perfect. :smile:


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Offlinepolaritymind
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26977194 - 10/09/20 03:21 PM (3 years, 4 months ago)

Ok so you can SAB pretty much very easily by just working with gloves within your SAB and thats it. The idea is that the benefit compared to a glove box is, that pressure changes can be equalized better and with a glove small pressure changes from the arm/hand movements actually suck in dirty air, and with an SAB not cause no "under-pressure" can build up.
Some people like to wipe down with alcohol before or sterilize hands in gloves in alcohol. Some people like to wasch their hands in gloves with soap and wipe down the walls with soap water and place the soapy towel at the bottom. This supposedly makes the contaminants stick and not bounce off the walls. No matter the explanation, from experience, it works, and better than without but not neccesary.
Remember that the air is still "dirty" and your freshly sterilized plates or grain jars clean, so try not to leave them open longer than neccesary. To transfer spores or mycelium onto or from the plate use sterilized tools, which is to say flame sterilize outside and then take the knife inside. Remember that spores are heat sensitive so either wait for the knife to cool or cool in target plate (sterilized).
Consider: Airflow is the enemy. So close windows and stop fans, and dont breathe into the armholes but insted more ontop of the box.

I can tell you I also do pre peur and then PC, for me it works since I use my old small PF Tek jars that arent heat sensitive. I heard of people not even making any holes for gas exhange so any small jar with an unmodified lid works.


--------------------
"to affirm life is to also affirm death"
-Albert hofmann


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OfflineCegda
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Re: Newbie agar question... [Re: polaritymind]
    #26977222 - 10/09/20 03:42 PM (3 years, 4 months ago)

I dont have any holes in my twist lock containers.  For a press on lid I would add a few


--------------------
“What senses do we lack that we cannot see or hear another world all around us?”

-Orange Catholic Bible,
    -from Frank Herbert's Dune


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OfflineDoDecaPus
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Re: Newbie agar question... [Re: Cegda]
    #26977262 - 10/09/20 04:15 PM (3 years, 4 months ago)

Congrats on your success. Just to echo what's been said...

It sounds like you want to pour then sterilize. This means you want a no-pour tek. I don't use them, but theyre out there.

As to the still-air-box, you can get a tote for 10-15$ and use a large can (coffee, mine was peaches) heated on the range to melt the holes into it. Cheap and easy.


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: DoDecaPus]
    #26977892 - 10/10/20 02:46 AM (3 years, 4 months ago)

Thanks guys for those replies, I read them all, great info, thanks.

As far as making the SAB goes ... I'll just take the gloves off my glovebox! ... or am I missing something? lol.


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OfflinePearl
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26978505 - 10/10/20 01:08 PM (3 years, 4 months ago)

Nope, SAB's are stupid simple things :smile:


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: Pearl]
    #26989487 - 10/17/20 02:32 AM (3 years, 3 months ago)

I have several different wild species on agar now, but none of them seem to be actually growing any mycelium after a few days. They're just getting sort of slimey.

Two of the jars (out of about 15) look contaminated, as there's a very thin layer of something white developing over the entire surface of the agar.

Am I doing something wrong? Does it need to be kept really warm or something? (the species are cold-weather species).


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OfflineForresterM
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26989534 - 10/17/20 04:08 AM (3 years, 3 months ago)

No, if they're cold weather species I don't think you want to keep them warm, as it will only give the bacteria (which loves it warm) a bigger advantage.

If they are not showing any growth but slimy-ness I'm guessing the bacteria is just winning, but it's hard to say without pictures.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: Forrester]
    #26989623 - 10/17/20 06:12 AM (3 years, 3 months ago)

Just the mushroom flesh that I put in there starts to look a bit slimey. I guess because it's pretty moist in there and it's getting old. Doesn't look much like anything growing there though.

How long should it usually take from mycellium to begin forming from the bit of mushroom flesh?

I must be doing something wrong I guess, I have about 20 of these jars with various types of mushrooms and not one looks like it is working. :frown:


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OfflineForresterM
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26989653 - 10/17/20 06:59 AM (3 years, 3 months ago)

Not sure what species you're working with but can you get a drier tissue sample?  Are you taking it from inner flesh or just putting a piece of stipe?


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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OfflineoO_wombat_Oo
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Re: Newbie agar question... [Re: Forrester]
    #26989743 - 10/17/20 08:44 AM (3 years, 3 months ago)

I'm taking it from the inner flesh. Pulling the mushroom apart and then cutting out a wedge. I think the agar dishes are just pretty moist from the pressure cooking process. There's lots of condensation inside them after I'm done.


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OfflineForresterM
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Re: Newbie agar question... [Re: oO_wombat_Oo]
    #26989946 - 10/17/20 11:25 AM (3 years, 3 months ago)

Yeah that's almost certainly not helping.  It sounds like your cloning method is good so I'd just try to get that agar a little drier and you should be good!


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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OfflineCegda
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Re: Newbie agar question... [Re: Forrester]
    #26989994 - 10/17/20 11:59 AM (3 years, 3 months ago)

Try storing your agar upside down, and then shaking excess moisture from the lid while in the SAB


--------------------
“What senses do we lack that we cannot see or hear another world all around us?”

-Orange Catholic Bible,
    -from Frank Herbert's Dune


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