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OfflinefirstTIMER420
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MEA and PDA agar questions
    #21917347 - 07/08/15 10:34 PM (8 years, 6 months ago)

hey guys, I have purchased the ingrediants to make MEA as well as pre mixed PDA. I have also purchased petri dishes

I just have a few questions.

everyone has been telling me to use a no pour agar tek, and all of the ones I have seen don't include using petris, so I am wondering if someone could link me to a tek that does, either pour or no pour im not picky.

Since I am going to be using petris, I have noticed that the mycelium need GE to grow, so how does that work inside a petri?

what is the best way to prepare the pre mixed PDA powder?

thank you, as you can tell this is my first time attempting agar, and I want to get it right.


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InvisibleNumeroEno
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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21917408 - 07/08/15 10:54 PM (8 years, 6 months ago)

Okay. First of all, you need to pour your plates inside your SAB if you have petri dishes. No pour agar is done with either polypropylene storage containers or PF style jars, and if you wanna do no pour it's a completely different process. Here's how to use your petris:

Get a bottle that you can put through the PC, mix up your agar by mixing it with the proper amount of cold water in a small saucepan, and heat it slowly while stirring. Constantly. Once the agar mix is dissolved, pour/funnel it into your bottle, and PC it with the lid on the bottle loose and covered in foil at 12-15psi for 30-45 minutes. Bring the pressure up slowly and down slowly to minimize your agar boiling out of the bottle. 

Once you can open your PC, place your bottle of hot agar and your sleeve(s) of petris in the SAB. You want to pour your plates once the bottle is cool enough that it isn't painful to hold in your gloved hand.

Open your sleeve of petris inside the SAB and pour agar into each plate. The deeper/fuller you pour your plates, the longer you can store them.

Stack your poured plates to minimize condensation, and inoculate them once they're cool. After you inoculate them, wrap them with saran wrap, cling wrap, or parafilm. Alternatively, you can keep them shut with tape. The lid of the petri dish is designed to allow gas exchange.


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21917429 - 07/08/15 10:58 PM (8 years, 6 months ago)

I invested in a 1000 mL Sterile Polycarbonate Erlenmeyer Flask. 
Before owning that i was successful with other various short glass bottles with skinny necks.  Make sure you find one that can sit upright in your PC.  It just makes things easier. 

I have always used a scale to weigh out the agar ingredients, but it is not necessary. 

I use bottled water and mix the ingredients until they are broken down.   

Fill the PC so the water covers the liquid agar medium in the flask.  Bring the PC to 14psi slowly.  PC for 30minutes.  Be careful not to let the weight on the PC shake violently.  When 30 minutes has passed, slowly bring the psi back to 0. 

I let the PC cool inside an SAB for no more than 1hour 45minutes. 

Agar is fun.  Good luck.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21918116 - 07/09/15 05:39 AM (8 years, 6 months ago)

Quote:

shroomyaxn said:
I invested in a 1000 mL Sterile Polycarbonate Erlenmeyer Flask. 
Before owning that i was successful with other various short glass bottles with skinny necks.  Make sure you find one that can sit upright in your PC.  It just makes things easier. 

I have always used a scale to weigh out the agar ingredients, but it is not necessary. 

I use bottled water and mix the ingredients until they are broken down.   

Fill the PC so the water covers the liquid agar medium in the flask.  Bring the PC to 14psi slowly.  PC for 30minutes.  Be careful not to let the weight on the PC shake violently.  When 30 minutes has passed, slowly bring the psi back to 0. 

I let the PC cool inside an SAB for no more than 1hour 45minutes. 

Agar is fun.  Good luck.




Why let it cool inside an SAB? The air inside is pretty still if the lid is on.
But just so you know the PC sucks in air as it cools, the inside of a PC isnt sterile. This is why anything sterilized needs to be wrapped up or inside jar with filtr etc

But yea that would work if you took it immediatly off a stove straight in front of a large flow hood, but since the air in the still air box is not sterile it will suck in contaminated air, defeating the purpose of that step.


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21918238 - 07/09/15 06:51 AM (8 years, 6 months ago)

jesus, I think I might faint. Someone that actually answered every question I just asked, so thank you very much

should I re-wrap the plates back into the sleeve they came in after pouring and stacking?


also, what should I do if the plates get a lot of condensation, so much so they start pooling/dripping?

any idea of how to mix  MEA agar?  Im thinking I mix the water and malt extract evenly, then I add the agar and stir the entire time on the stove so it mixes evenly again.  I just need to know the ratios, as well as what kind of heat to use(if I boil, or simmer, when to do each or both)

since I have SFD, I think ill cut a hole in a cap nd place a small piece of SFD in the cap and then put foil over it when PCing.  This is going to be my first time using a PC so thank you for the info about it boiling over, as I have seen people complain about the agar boiling over and they all have different suggestions but no solid info.


To MUSH

so after the PC is cool enough to open I take the agar and place it in the SAB?


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Edited by firstTIMER420 (07/09/15 06:52 AM)


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21918462 - 07/09/15 08:36 AM (8 years, 6 months ago)

No problem :cheers:

I leave my poured plates inside the SAB until ready to use. The only time I wrap blank plates is if I know I'm not gonna use them for a while.

Waiting until your agar cools down and stacking the plates will keep the condensation out of all but the top couple plates. If you still have condensation, you can leave the plates unused for a week or so and the condensation will mostly go away.

I mix up MEA at about 10-12 grams of both malt extract and agar powder into 500ml of water.

Yeah, go ahead and put the SFD on the cap. If you're gonna use a liquor bottle make sure to take any foam or soft plastic out of the lid.


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

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What shall we say, shall we call it by a name
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Re: MEA and PDA agar questions [Re: mushpunx]
    #21918559 - 07/09/15 09:12 AM (8 years, 6 months ago)

Quote:

mushpunx said:
But yea that would work if you took it immediatly off a stove straight in front of a large flow hood, but since the air in the still air box is not sterile it will suck in contaminated air, defeating the purpose of that step.




The air in my SAB is cleaner than the air in my kitchen.  Also, the SAB is located in my "lab room" that is cleaned way more often than the kitchen.  There is also a constant draft in my kitchen with the windows being open.  There is little to no draft in the room where the SAB is located. 

Maybe placing it in the SAB to cool does nothing helpful, but it sure is peace of mind.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21919236 - 07/09/15 10:50 AM (8 years, 6 months ago)

Yea I hear you on peace of mind haha. If you leave the lid on your PC it should be same as your SAB tho save you some work :lol:


I use a GE filter on my agar, I use a liquor bottle with a hole drilled in the cap, SFD underneath. Its easiest to pour from for me but maybe not in shorter SABs. Mason jars work fine too. As long as you have a lid on your bottle doesnt matter if you leave it in PC untill you use it or take it out as soon as you can open it.

Wipe your agar bottle down well with ISO especially the neck. And your sleeve of plates before you cut it open etc..

I found if you let the agar cool down alot.. longer than I usually would let it go the condensation is minimal. Almost non existent!


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Re: MEA and PDA agar questions [Re: mushpunx]
    #21919363 - 07/09/15 11:04 AM (8 years, 6 months ago)

Pour your agar at 47C.


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Re: MEA and PDA agar questions [Re: bodhisatta]
    #21919522 - 07/09/15 11:35 AM (8 years, 6 months ago)

Quote:

bodhisatta said:
Pour your agar at 47C.





What do you use one of those thermometer guns? I found a sweet spot but its really hard to get it everytime. But when I pour at this temp I get no condensation, not even on the top plate. Its just hot enough still that it another few minutes itll start to thicken


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Re: MEA and PDA agar questions [Re: mushpunx]
    #21919568 - 07/09/15 11:44 AM (8 years, 6 months ago)

Some SAB's arent level at the botom, depending on how you're using the SAB. this causes uneven pooling on the petri. I have found that adding a cutting board wrapped in foil and sterilized with 91% iso al makes a better surface to work on. Also, be careful when you're moving or stacking your plates. You want to be as gentle as possible as to cause minimal turbulence so that the agar wont splash to the top of the plate and out the edges. Splashing the agar increases the likely of contamination.


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Re: MEA and PDA agar questions [Re: shellzenone]
    #21919693 - 07/09/15 12:06 PM (8 years, 6 months ago)

how do you splash the agar? it usually stiffens pretty damn fast once you've poured it IME.

and whats the cutting board with foil you talk of?


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Re: MEA and PDA agar questions [Re: spacechildo]
    #21920075 - 07/09/15 01:50 PM (8 years, 6 months ago)

I've had agar that stiffens really quick too, but I guess that depends on the temperature of the agar during pouring. I usually stack the petris in stacks of 3s, then slide them over when I'm done pouring them. This would cause the agar to swoosh around and touch the top of the petris. So using a flat surface (cutting board) helped me avoid this.


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Re: MEA and PDA agar questions [Re: mushpunx]
    #21920091 - 07/09/15 01:55 PM (8 years, 6 months ago)

Quote:

mushpunx said:
I found if you let the agar cool down alot.. longer than I usually would let it go the condensation is minimal. Almost non existent!




Yup, and stacking the dishes after pouring helps greatly too.


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Re: MEA and PDA agar questions [Re: mushpunx]
    #21920145 - 07/09/15 02:10 PM (8 years, 6 months ago)

Quote:

mushpunx said:
Quote:

bodhisatta said:
Pour your agar at 47C.





What do you use one of those thermometer guns? I found a sweet spot but its really hard to get it everytime. But when I pour at this temp I get no condensation, not even on the top plate. Its just hot enough still that it another few minutes itll start to thicken



Water bath. Or if you don't have a water bath put a tub of 55C water and your jars in it. When the water cools to 47c you can assume your agar jars are about that.


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Re: MEA and PDA agar questions [Re: shellzenone]
    #21920188 - 07/09/15 02:21 PM (8 years, 6 months ago)

Quote:

shellzenone said:
Some SAB's arent level at the botom, depending on how you're using the SAB. this causes uneven pooling on the petri. I have found that adding a cutting board wrapped in foil and sterilized with 91% iso al makes a better surface to work on. Also, be careful when you're moving or stacking your plates. You want to be as gentle as possible as to cause minimal turbulence so that the agar wont splash to the top of the plate and out the edges. Splashing the agar increases the likely of contamination.




That's why you set your SAB up so it's upside down when you work. Cut your holes so you can set the tote on a towel upside down. You'll be glad you did.


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

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21920521 - 07/09/15 03:35 PM (8 years, 6 months ago)

exactly, throw the lid away, and use some flat surface as the floor.


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21921628 - 07/09/15 08:02 PM (8 years, 6 months ago)

Quote:

NumeroEno said:
That's why you set your SAB up so it's upside down when you work. Cut your holes so you can set the tote on a towel upside down. You'll be glad you did.




I heavily spray the towel in my SAB with Lysol surface sanitizer.  This creates a damp surface which captures contams that get stirred up with movements inside the SAB.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21922231 - 07/09/15 09:47 PM (8 years, 6 months ago)

Yeah. That's good to do. I use a mix of water, bleach, dish soap, and jet dry for my SAB spray. I also put down fresh lysol wipes as my work surface.


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

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21923552 - 07/10/15 08:17 AM (8 years, 6 months ago)

Quote:

NumeroEno said:
I also put down fresh lysol wipes as my work surface.




Me too.  Also, instead of working directly on top of a towel, i created an elevated rack to work on. 
I used those rubber coated wire shelves.   


I took two wire shelf pieces, cut them to size and zip tied them together.  The shelf lip at each end creates an elevated rack of sorts.

Everything placed on the rack inside the SAB is not coming into contact with that much surface area at all.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21923704 - 07/10/15 09:10 AM (8 years, 6 months ago)

I'm curious to know, how long do you guys wait after pc-ing  to pour the agar?


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Re: MEA and PDA agar questions [Re: shellzenone]
    #21923943 - 07/10/15 10:31 AM (8 years, 6 months ago)

Put the media bottle in a dish of water at 60ish C. Once the water in the dish gets to 47, you can assume the agar media is around 47 too. Pour at that moment.


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Edited by Mad Season (07/10/15 10:32 AM)


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21926237 - 07/10/15 09:20 PM (8 years, 6 months ago)

Quote:

shroomyaxn said:
Quote:

NumeroEno said:
I also put down fresh lysol wipes as my work surface.




Me too.  Also, instead of working directly on top of a towel, i created an elevated rack to work on. 
I used those rubber coated wire shelves.   


I took two wire shelf pieces, cut them to size and zip tied them together.  The shelf lip at each end creates an elevated rack of sorts.

Everything placed on the rack inside the SAB is not coming into contact with that much surface area at all.






I put down a towel and place the open SAB top down on top of the towel(all sprayed of course), but I might replace it with this method, or a grid pattern created out of some sort of wire, nice engineering man


Also, I am going to start the agar tomorrow, the thing is, I still don't have a tek to follow, Im just going to use the pre mixed PDA that I have, and follow the LGM(RR) vid in terms of agar, then just kinda make up the rest from info on this post.  That is, unless someone has a good link to an agar tek for MEA...

also, using the pre made PDA powder is basically heat/mix with water, PC, then pour?


ALso, numero or anyone else, what temp should I heat the agar mixture too when heating in the saucepan?


wouldn't the plates being wrapped up interfere with the GE?


Edited by firstTIMER420 (07/10/15 09:34 PM)


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21930920 - 07/11/15 09:23 PM (8 years, 6 months ago)

Quote:

firstTIMER420 said:
I put down a towel and place the open SAB top down on top of the towel(all sprayed of course), but I might replace it with this method, or a grid pattern created out of some sort of wire, nice engineering man





Thanks man.

Quote:

firstTIMER420 said:
Also, I am going to start the agar tomorrow, the thing is, I still don't have a tek to follow, Im just going to use the pre mixed PDA that I have, and follow the LGM(RR) vid in terms of agar, then just kinda make up the rest from info on this post.  That is, unless someone has a good link to an agar tek for MEA...





I have pre-mixed LME from a sponsor that i mix with agar inside the flask. 
10 grams light malt extract
10 grams Agar
500 ml water

500ml can make 20 petri dishes. 

Quote:

firstTIMER420 said:
wouldn't the plates being wrapped up interfere with the GE?





I use parafilm.  It is gas permeable.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21930996 - 07/11/15 09:39 PM (8 years, 6 months ago)

Yup. 10 grams LME, 10 grams agar powder, and 500ml of water is a great agar recipe, and it will pour 20 100mm plates exactly :thumbup:


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

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21931030 - 07/11/15 09:49 PM (8 years, 6 months ago)

I mix those in a saucepan while heating right?  But, how much do I heat up the mixture, till its boiling or just a simmer?


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21931048 - 07/11/15 09:55 PM (8 years, 6 months ago)

Add your agar powder while the water is cold. Trust me.


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

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21931095 - 07/11/15 10:04 PM (8 years, 6 months ago)

It may not be exactly what you need but hope it helps.

http://www.shroomery.org/forums/showflat.php/Number/20111637/fpart/all/vc/1


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Re: MEA and PDA agar questions [Re: NumeroEno]
    #21931107 - 07/11/15 10:06 PM (8 years, 6 months ago)

so LME and water first, then add agar, then heat up while mixing.


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21931150 - 07/11/15 10:15 PM (8 years, 6 months ago)

Quote:

firstTIMER420 said:
so LME and water first, then add agar, then heat up while mixing.




No need to heat up.  I add dry ingredients to flask, add water then mix, wait, then mix again.


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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21931184 - 07/11/15 10:25 PM (8 years, 6 months ago)

gotcha, anyone else not heat? most teks ive seen say to heat I think to sterilize.


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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21931200 - 07/11/15 10:31 PM (8 years, 6 months ago)

Quote:

firstTIMER420 said:
gotcha, anyone else not heat? most teks ive seen say to heat I think to sterilize.




The sterilizing comes in the PC, after the mixing.


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OfflineICEMANOO9
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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21931229 - 07/11/15 10:40 PM (8 years, 6 months ago)

:whathesaid:


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OfflinefirstTIMER420
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Re: MEA and PDA agar questions [Re: shroomyaxn]
    #21931314 - 07/11/15 11:10 PM (8 years, 6 months ago)

im going to try giving it some light heat I guess, as I said most teks call for it.


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InvisibleNumeroEno
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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21931819 - 07/12/15 01:39 AM (8 years, 6 months ago)

It works best if you add the ingredients to cold water, and then turn the heat on and stir constantly until the agar is dissolved.


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:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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Offlinehamloaf
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Re: MEA and PDA agar questions [Re: firstTIMER420]
    #21932191 - 07/12/15 05:56 AM (8 years, 6 months ago)

Quote:

firstTIMER420 said:
im going to try giving it some light heat I guess, as I said most teks call for it.



The reason heat is generally applied during the mixing of the ingredients is to aid in the dissolving of the ingredients themselves.  The purpose of the pressure cooker is to sterilize the media.


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OfflinefirstTIMER420
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Re: MEA and PDA agar questions [Re: hamloaf]
    #21932477 - 07/12/15 08:25 AM (8 years, 6 months ago)

gotcha, thanks guys.  All I need now is a bottle to have all the ingredients in.

and  btw ham, I just looked at the cube monotub link in your sig...

  :youthemandawg:  :highfive: THEM PINS


have you written up the tek yet for the "hybrid" design of the tubs?

id also be inclined to see the Spawn to Sub ratios used in producing those tubs in that tek as well.. for information purposes, but also to rub it in peoples faces :lol:


Edited by firstTIMER420 (07/12/15 08:50 AM)


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