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InvisibleNecroMyce
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Registered: 05/12/13
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Attempt at agar.
    #19385884 - 01/07/14 11:41 PM (10 years, 23 days ago)

So ive read around a lot recently the last two days on agar. And i just want to make sure im getting everything right and the route i want to take is logical.

So about a year ago i had a great job and $ and bout almost everything you need for a grow from BRF, Rye, Agar, polyfill, everything. So i got a good amount of resources and i checked my chest the other day and found i still have like 4 packs of telephone brand Agar agar and Light Malt Extract.

I read up on these sites.
http://www.alohaculturebank.com/media/media-list.html
http://www.shroomery.org/9393/FastFreds-Media-Cookbook
http://www.shroomery.org/8504/Peroxide-Agar-Tek


and basically what i got from all that is the procedure is this.. (Atleast in my situation)

Ive read a lot 10g agar, 10g LME, 500ml water. But i don't always want to make that many plates. Therefore i assume you can reduce it by half 5g A, 5gLME, 250ml. And reduce/raise to your liking depending on how many plates i want. ive also read 3g A, 3g LME, 150ml water.

Step 1. Sterlize your pour jar and mix your agar mixture up.

Step 2. Put your agar mixture in its pour jar in the PC with your plates to sterlize.

Step 3. Let it cool(or put a thermometer inside and make sure its under 140F so you can add peroxide if you wish to do so.)

Step 4. Pour your agar plates 1/4inch or 1/8inch.(Im assuming inside a SAB for protection.)

Step 5. Grab your fruit you want to clone. (Optional) Break it down the stipe and cut some tissue ina  carving motion.

Step 6. Transfer to your agar plates, then wrap in parafilm or saran plastic wrap.

Step 7. Let colonize and complete more transfers to other plates throughout colonization.

Seem legit?
--------

Well i have a few questions i couldn't find.

      1. Is peroxide that beneficial to contam resistant feature it adds to your mixture?
Ive read that.... "Many people seem a bit confused as to what peroxide can and can not do. Hydrogen peroxide will kill bacteria and fungal spores and inhibit the growth of yeasts. Unfortunately, mushroom spores fall under the category of fungal spores, so they will not germinate on peroxidated agar because they are killed on contact. The good part about this is that mold spores, such as spores from the dreaded green mold, fall under this category as well and are also killed upon contact with the agar surface. What will grow on peroxidated agar is existing fungal colonies, such as your living mushroom mycelium, because their cells can produce enzymes that will cause the peroxide to be broken down into water and oxygen. The downside, however, is that existing colonies of some molds, such as green mold, are also capable of producing these enzymes, so peroxide will not guard against mold spores that have already germinated. But perhaps the best attribute of peroxide is that it will kill all bacteria, so you will never have a problem with bacterial contamination on peroxide enriched agar."

    2.What is the temperature or environment that i should provide for my plates?
I've read that for long term storage for culture slants you want it in 39-50F i think it said, 50F is nice if i remember. And you want to use a wine type mini fridge with a fan inside for constant fresh air. Even though they will be in parafilm. Regular minifridges do not hold constant temp and fluctuate. I eventually would like to have a Culture slant i can take wedges from rather than doing agar for every grow or just constantly. I currently cant afford one right now and dont trust my fridge for sterile conditions let alone constant temps. I dont have to have master cultures on slants yet, but i would love to attempt agar if i have the materials in the time being for learning so when i have enough saved up i will be some what experienced.

    3. I also have read you can use regular half pint jars as your petri dishes instead of buying glass petri dishes, well i bought plastic ones way back before i realized the troubles it can cause. Are they still usable of some sorts?

    4. I also read a few ways to make agar lids, and they were using whatamans or whatever those are called. is there anyway to do it with SFD's? i cant find any other teks or posts without whatamans? i know they are somewhat cheap, but free is cheaper :laugh: i have 85 SFD's left and theyre gonna last a looooong time hahhah
Im not sure if there is something important that i forgot to add in this post. If there is any links that will be benefitial to my learning and research or any tips and tricks or proper methods/ideas i should be aware of please feel free to post so i can create some awesome prints of clones to send your ways!

thank you!

-casp


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OfflinecronicrFacebook
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Re: Attempt at agar. [Re: NecroMyce]
    #19385911 - 01/07/14 11:46 PM (10 years, 23 days ago)

check pasty's out bro much easier, mix pour pc cool innoc, sfd's poly tyvek micropore!
i'll be back to go through more of your post but right now i need a blast! but glad to see you making the leap!


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I'm tired do me a favor


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InvisiblePirateSwazey
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Re: Attempt at agar. [Re: cronicr]
    #19385969 - 01/08/14 12:00 AM (10 years, 23 days ago)

Plastic petris are fine.

You can always pour extra dishes and store them in the fridge until you're ready to noc em up.

Keep them stacked after you pour so the condensation will rise to the top plate :thumbup:

Doc T also has a nice no pour agar tek if your interested in taking that route.


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BULK RYE PREP  -  MY FAVORITE THREADS  -  BUILD A FLOWHOOD



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OfflinecronicrFacebook
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Re: Attempt at agar. [Re: PirateSwazey]
    #19385993 - 01/08/14 12:05 AM (10 years, 23 days ago)

Quote:

CaspuuuR said:

Step 1. Sterlize your pour jar and mix your agar mixture up.

Step 2. Put your agar mixture in its pour jar in the PC with your plates to sterlize.

Step 3. Let it cool(or put a thermometer inside and make sure its under 140F so you can add peroxide if you wish to do so.)

Step 4. Pour your agar plates 1/4inch or 1/8inch.(Im assuming inside a SAB for protection.)

Step 5. Grab your fruit you want to clone. (Optional) Break it down the stipe and cut some tissue ina  carving motion.

Step 6. Transfer to your agar plates, then wrap in parafilm or saran plastic wrap.

Step 7. Let colonize and complete more transfers to other plates throughout colonization.

Seem legit?


-casp




Quote:

PirateSwazey said:
Plastic petris are fine.

You can always pour extra dishes and store them in the fridge until you're ready to noc em up.

Keep them stacked after you pour so the condensation will rise to the top plate :thumbup:

Doc T also has a nice no pour agar tek if your interested in taking that route.



:thumbup:
i only used peroxide agar once for cleaning my wild fruits up
and for storing you can wrap them in a towel or clothe or something and put them in a lunch box or something of the sort in the fridge to stabilize the temps


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It doesn't matter what i think of you...all that matters is clean spawn

I'm tired do me a favor


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Offlinemushrume man
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Re: Attempt at agar. [Re: cronicr]
    #19386010 - 01/08/14 12:10 AM (10 years, 23 days ago)

Looks like you got it down, I like my agar on the thicker side because its easier to get on the scalple

Your plastic plates work fine if the bag is not torn or punctured.

The jars can be poured then pc'd for ease, just dissolve ingredients in bottle, pour, let solidify then PC. You make it sound like you want to pour to jars in sab? This can be done but is more efficient with 20 plastic dishes then a dozen jars. If you only want a dozen, make forty! They can be stored for later use, I just leave them on my shelf


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InvisiblePirateSwazey
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Re: Attempt at agar. [Re: cronicr]
    #19386013 - 01/08/14 12:11 AM (10 years, 23 days ago)

Ya make sure to store sterile.

I wrap mine in parafilm but saran wrap or ziplocks work too.

Never messed with peroxide... never even heard of using it :lol:


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OfflinecronicrFacebook
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Re: Attempt at agar. [Re: PirateSwazey]
    #19386021 - 01/08/14 12:12 AM (10 years, 23 days ago)

yeah don't bother, even with wild fruits i found it unnecessary


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I'm tired do me a favor


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InvisibleNecroMyce
Mostly Ghostly
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Re: Attempt at agar. [Re: PirateSwazey]
    #19388047 - 01/08/14 12:53 PM (10 years, 22 days ago)

Quote:

mushrume man said:Your plastic plates work fine if the bag is not torn or punctured.

The jars can be poured then pc'd for ease, just dissolve ingredients in bottle, pour, let solidify then PC. You make it sound like you want to pour to jars in sab? This can be done but is more efficient with 20 plastic dishes then a dozen jars. If you only want a dozen, make forty! They can be stored for later use, I just leave them on my shelf




By the plastic bag not being torn part, do you mean the plastic bag they came in? Basically they're still sterile inside the bag they came in if they're medical grade and or store bought ones.


------------------------------

Quote:

PirateSwazey said:Doc T also has a nice no pour agar tek if your interested in taking that route.




I really like this write up, as i have the same materials and it seems like the pour step is kinda pointless anyways. So does this mean i have to pre sterilize my jars before pouring the agar mixture inside them? Use a PC? soap and water?

-------------------------------

Fuck peroxide for now, ill just stick with the regular recipe and if i get crazy contam problems, then ill look into it.

-------------------------------

And how long do they normally last for? whats the best way for me to test the temp flucts in my fridge? i think ima just go get a lunch box like you and put my hygro meter til i figure out the right temp needed.

What is the optimal temps for agar cultures. Both storage and colonization?

What i mentioned above about the wine cooler.. Is that necessary? the FAE 24/7 fan they provide?


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OfflinecronicrFacebook
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Re: Attempt at agar. [Re: NecroMyce]
    #19388063 - 01/08/14 12:56 PM (10 years, 22 days ago)

with the no pour theres no need to pre-sterilize the jars just mix pour and pc
and temps are the same for everything else, room temp is fine and i've never used a wine cooler:shrug:


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It doesn't matter what i think of you...all that matters is clean spawn

I'm tired do me a favor


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InvisiblebodhisattaMDiscordReddit
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Re: Attempt at agar. [Re: cronicr]
    #19388071 - 01/08/14 12:58 PM (10 years, 22 days ago)

I would imagine peroxide would decompose during the sterilization process anyway.


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OfflineCamtaro420
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Re: Attempt at agar. [Re: cronicr]
    #19388076 - 01/08/14 12:59 PM (10 years, 22 days ago)

Since your mix will be steaming when you pour it in, let it cool a little before topping it and pressure cooking it and this will prevent some of the hard to see through condensation after the PC.


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Offlineblueconfusion
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Re: Attempt at agar. [Re: bodhisatta]
    #19388105 - 01/08/14 01:06 PM (10 years, 22 days ago)

If you're worried about bacteria get some tetracycline from your local pet store just add a small amount I have an agar tek in my sig you can use LME in place of potato.


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InvisibleNecroMyce
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Re: Attempt at agar. [Re: cronicr]
    #19388132 - 01/08/14 01:12 PM (10 years, 22 days ago)

Quote:

Camtaro420 said:
Since your mix will be steaming when you pour it in, let it cool a little before topping it and pressure cooking it and this will prevent some of the hard to see through condensation after the PC.




Does it always have to be cooked up a little? and whats the reason for it coming to a boil? ive read tons of teks that are telling me to mix then bring to a boil. Never telling me what this accomplishes?

Quote:

cronicr said:
with the no pour theres no need to pre-sterilize the jars just mix pour and pc
and temps are the same for everything else, room temp is fine and i've never used a wine cooler:shrug:




So temps are the same for everything that means i can just open a drawer in my desk and clean it in there and set up?

The only reason im worried about temps is cuz my house doent stay "room temp" it stays at like 70 during the day maybe a little more/less. then at night itll drop to under 60 sometimes. always under 70 at night tho. So im thinking if my grow room has a little room heater and is kept at 70-75F  24/7 maybe i can just stick my plates in there? Buy a little plastic bin for them to keep them a little consolidated and to keep temps a little more secure inside.

The reason i choose to put it in a plastic bin, was because ive always tried to keep my colonized grains away from my colonizing/uncolonized grains to prevent contams...

With the idea i have is it logical? the last thing i would want is a fully colonized mono tub fruiting in a room and then find a contam in one of my agar plates, then it somehow get into my monotub. :frown:


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Invisiblebootster
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Re: Attempt at agar. [Re: cronicr]
    #19388136 - 01/08/14 01:13 PM (10 years, 22 days ago)

I'm using a kegerator that hasn't been cleaned in about 4 years (no more kegs in it). It's had over 50 kegs in it during that time and has never been cleaned out, just wiped with a soap and water rag. All my stuff goes into a separate container that is wiped with iso on its way in, and out, and I've never had a problem.

Just make sure all of the things in it are in ziplock bags and wiped down with iso.

When I heard "Lunchbox", I was thinking of Scooby Doo or Banana Splits metal ones. :rofl2:


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OfflinecronicrFacebook
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Re: Attempt at agar. [Re: NecroMyce]
    #19388144 - 01/08/14 01:15 PM (10 years, 22 days ago)

Quote:

CaspuuuR said:


Does it always have to be cooked up a little? and whats the reason for it coming to a boil? ive read tons of teks that are telling me to mix then bring to a boil. Never telling me what this accomplishes?

:frown:



it dissolves all the ingredients
http://www.shroomery.org/forums/showflat.php/Number/19208976
and with no pour teks the contams won't be jumping out on ya:wink:


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I'm tired do me a favor


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OfflineCamtaro420
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Re: Attempt at agar. [Re: cronicr]
    #19388160 - 01/08/14 01:19 PM (10 years, 22 days ago)

Quote:

cronicr said:
Quote:

CaspuuuR said:


Does it always have to be cooked up a little? and whats the reason for it coming to a boil? ive read tons of teks that are telling me to mix then bring to a boil. Never telling me what this accomplishes?

:frown:



it dissolves all the ingredients
http://www.shroomery.org/forums/showflat.php/Number/19208976
and with no pour teks the contams won't be jumping out on ya;)




^ :whathesaid: once you work with the ingredients you will see how hard it is to get that shit to mix.

I grind every ingredient again to powder even if it comes pre-ground.

I use my hotwater maker to get it steaming before I put it in the pot and bring it to a simmer then start slowly mixing it in.

Then mix vigorously and filter through a mesh strainer twice, then poured into the jars.

Get PP5 plastic if u can though.


--------------------


Edited by Camtaro420 (01/08/14 01:19 PM)


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InvisiblePirateSwazey
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Re: Attempt at agar. [Re: Camtaro420]
    #19388945 - 01/08/14 04:10 PM (10 years, 22 days ago)

This is the agar I use.

It's super simple... weigh powder, add warm water, stir, add more water, stir again, PC 45 min, cool ~2 hours, pour.

I'm pretty sure this is the exact same thing RR is using in his videos.

If you stick with Doc T's method just follow his tek exactly... mix agar, pour, sterilize, cool, inoculate.

You got it man it's not hard :thumbup:


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Invisiblebootster
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Re: Attempt at agar. [Re: PirateSwazey]
    #19389635 - 01/08/14 05:48 PM (10 years, 22 days ago)

"Qoute:"
"it seems like the pour step is kinda pointless anyways."

Super super super bad info. Disregard in all fashions, methods, and comprehension. Stricken it. The "pour method" is the only way to go (for me anyways). I've always done it that way and will continue to do so. 

No flames please but I am vehement about my stance on this.

Sincerely,
bootster


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Re: Attempt at agar. [Re: bootster]
    #19389646 - 01/08/14 05:49 PM (10 years, 22 days ago)

Quote:

bootster said:
"Qoute:"
"it seems like the pour step is kinda pointless anyways."

Super super super bad info. Disregard in all fashions, methods, and comprehension. Stricken it. The "pour method" is the only way to go (for me anyways). I've always done it that way and will continue to do so. 

No flames please but I am vehement about my stance on this.

Sincerely,
bootster



y? whats your base on this? i'll discuss this with you civilly:evil:


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I'm tired do me a favor


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InvisiblebodhisattaMDiscordReddit
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Re: Attempt at agar. [Re: bootster]
    #19389663 - 01/08/14 05:53 PM (10 years, 22 days ago)

Quote:

bootster said:
"Qoute:"
"it seems like the pour step is kinda pointless anyways."

Super super super bad info. Disregard in all fashions, methods, and comprehension. Stricken it. The "pour method" is the only way to go (for me anyways). I've always done it that way and will continue to do so. 

No flames please but I am vehement about my stance on this.

Sincerely,
bootster




I've tried 3-4 different no pours, some of them work well but nothing works as well as a poured dish IMO, The way the dish handles the size and shape the control etc.. all better than working out of a jar not made to be used as a petri just to skip the "scary" pour step. Just get good at it, no one starts off good, Practice is the hard part, but everyone is afraid of failure...


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Invisiblebootster
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Re: Attempt at agar. [Re: bodhisatta]
    #19389693 - 01/08/14 05:58 PM (10 years, 22 days ago)

Thank you sir, you are correct.


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OfflineCliftonGK1
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Re: Attempt at agar. [Re: bodhisatta]
    #19389724 - 01/08/14 06:02 PM (10 years, 22 days ago)

Quote:

bodhisatta said:
I've tried 3-4 different no pours, some of them work well but nothing works as well as a poured dish IMO, The way the dish handles the size and shape the control etc.. all better than working out of a jar not made to be used as a petri just to skip the "scary" pour step. Just get good at it, no one starts off good, Practice is the hard part, but everyone is afraid of failure...




That's exactly it for me, too.  Having grown up in and around clin micro labs (my Dad was a lab director, go figure that I went on to do the same thing)  I don't see how you're supposed to properly streak an agar jar without scoring the media since you have to work at such a steep angle.  I think it's easier to section a shallow petri, especially since I don't take a chunk all the way to the plastic, I cut my sectioning lines and then slice in shallow underneath it like a scoop so I'm less likely to have a tall transfer that might contact the lid of the plate.


--------------------


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InvisiblebodhisattaMDiscordReddit
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Re: Attempt at agar. [Re: bootster]
    #19389726 - 01/08/14 06:02 PM (10 years, 22 days ago)

You cant argue with what works though, some people swear by no pour, if it is working for them :thumbup: awesome.


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Re: Attempt at agar. [Re: CliftonGK1]
    #19389738 - 01/08/14 06:04 PM (10 years, 22 days ago)

my pp5's are the same size and depth as a petri:shrug: i love petri's as well, same with cakes, i've been growing for 10 years and still like to grow them:shrug: variety is awesome, learning new tools is great!


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I'm tired do me a favor


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Re: Attempt at agar. [Re: CliftonGK1]
    #19389743 - 01/08/14 06:05 PM (10 years, 22 days ago)

Quote:

CliftonGK1 said:
Quote:

bodhisatta said:
I've tried 3-4 different no pours, some of them work well but nothing works as well as a poured dish IMO, The way the dish handles the size and shape the control etc.. all better than working out of a jar not made to be used as a petri just to skip the "scary" pour step. Just get good at it, no one starts off good, Practice is the hard part, but everyone is afraid of failure...




That's exactly it for me, too.  Having grown up in and around clin micro labs (my Dad was a lab director, go figure that I went on to do the same thing)  I don't see how you're supposed to properly streak an agar jar without scoring the media since you have to work at such a steep angle.  I think it's easier to section a shallow petri, especially since I don't take a chunk all the way to the plastic, I cut my sectioning lines and then slice in shallow underneath it like a scoop so I'm less likely to have a tall transfer that might contact the lid of the plate.




I do all of my work in a SAB, using no pour jars of various sizes always hindered my sterile technique. I would bump shit or touch shit inadvertently. I also like to be super stoned when I'm doing this shit which doesn't help my hand movements. Petri's have always given me a better outcome.

Chron what PP5's do you use for no pour? if they're so shallow doesn't that shit boil all over and make the whole inside of the plastic covered?


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Re: Attempt at agar. [Re: bodhisatta]
    #19389799 - 01/08/14 06:13 PM (10 years, 22 days ago)

to be fair there a wee bit bigger but not by much, i use to use them for the pour style but tried the no pour and lone behold no boil over:rockon:  took them from the hospital they use them for boob pads

thats not actualy agar but it worked with agar no prob:thumbup:


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Re: Attempt at agar. [Re: cronicr]
    #19389809 - 01/08/14 06:14 PM (10 years, 22 days ago)

but like i said i'll use it all if i have it handy


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Re: Attempt at agar. [Re: cronicr]
    #19390429 - 01/08/14 08:24 PM (10 years, 22 days ago)

y? whats your base on this? i'll discuss this with you civilly:evil:

Not a problem at all my friend.


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Re: Attempt at agar. [Re: bootster]
    #19390454 - 01/08/14 08:29 PM (10 years, 22 days ago)

:rockon:


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