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Amazon Shop for: ½ Pint Jars, Agar, Autoclave, Laminar Flow Hood, Petri Dish, Potato Dextrose

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OfflineDeviant
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Agar disastar
    #2020926 - 10/18/03 06:16 PM (13 years, 1 month ago)

Today, for the first time I decided to try agar. I gathered my supplies that I recently bought and mixed 20g of potato dextrose agar with 500ml water. Pressure cooked for 45 min and let cool a bit. Then I put in my glove box and sprayed lysol a bunch. Let it sit for about 5 min and tried to pour the plates. I realize now that this is almost impossible to do in a glove box. After 5 plates I decided screw this and cranked up the oven and proceded to do the next 20 plates using the oven tek. Im almost positive that if I innoculate these plates nothing good is going to come of it. I also used glad wrap and sealed the edges. My question is: If I let the plates just sit somewhere for a week or so, if they are contimanted will they show signs of it. You know, other things growing on them without ever innoculating? I guess Im gonna have to save some money to build a flow hood. This was my origional plan, but once I started looking into it found out it was gonna be about $200-$400 bucks to build one, so decided to try it with a glove box. Never again will that happen. You should'ave seen the glove box, Agar all over the place, splattered on the walls and on the bottom. If I though of it sooner I would have taken a pic of it to show off my stupidity but I already cleaned it up. I guess the moral of this story is don't use agar in a glove box


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"How doth the little crocodile improve his shining tail, and pour the waters of the Nile on every golden scale! How cheerfully he seems to grin, how neatly spreads his claws, And welcomes little fishes in With gently smiling jaws!"


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InvisibleYidakiMan
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Re: Agar disastar [Re: Deviant]
    #2021012 - 10/18/03 06:52 PM (13 years, 1 month ago)

I'm glad another person has decided to start using agar.  When I started with agar I only had a glovebox and I figured out it would be pretty hard to pour it into petris right away.  Try using half pint canning jars or GLAD Mini-Rounds plastic containers instead.  They will be easier, but realize that a glovebox just plain sucks.  :crazy:

You mentioned leaving plates empty to check for contamination.  This is actually a common practice; however, it is used somewhat differently.  It is called leaving blanks.  A blank can be a petri dish, a grain jar, or substrate.  A blank is sterilized but not innoculated.  This allows the cultivator to go ahead and use the media he prepared as soon as it is ready.  If there is an outbreak of contamination, then the grower has a blank to check on.  If the blank has contaminated, then the grower must work on his sterilization technique.  If the blank has not contaminated, then the grower must work on his aseptic innoculation technique.  See how it is helpful?  The blank will help the grower target the vector from which the contaminant originated. 

You can build a flowhood for under $200.  If you are lucky you can build one for $100.  Mine cost $50 and I am a god damn lucky bastard.  :grin:
The key in building a cheap flowhood is the same of mushroom cultivation.  PATIENCE.  You must wait for deals to surface.  Deals are out there, just be patient and they will appear before you.   


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OfflineAnand
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Re: Agar disastar [Re: Deviant]
    #2021029 - 10/18/03 07:06 PM (13 years, 1 month ago)

i know the problem with agar in the glovebox...... :smile: so the second time i did it on a very clean kitchen counter and worked very fast (20 agar plates in under 2 minutes) then seal the whole stack at once with household plastic wrap.
Later once it has cooled i put the stack in the glovebox and sealed them individually for later use.

you could incubate the empty dishes to see if something grows but its not a fullproof testing method. 


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Offlinefalcon
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Re: Agar disastar [Re: Deviant]
    #2021054 - 10/18/03 07:27 PM (13 years, 1 month ago)

Use baby food jars , or half pint masons instead of petris. Cook up your agar mixture in pot and pour before you pressure cook. The agar won't dissolve completely so stir as you pour, this can be done on an open counter top. Pressure cook the jars for 20 minutes. Use a polyfill or tyvek filter on the mason jars, a big piece of foil on top of the lid of the baby food jars. Not as good as flow hood but it eliminates a step, pouring, that could let in contamination.


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InvisibleCow Shit Collector
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Re: Agar disastar [Re: falcon]
    #2021393 - 10/18/03 10:44 PM (13 years, 1 month ago)

Look in the marketplace, Bonsai is selling very nice flowhood units 2x2ft for less that what your planning on paying...


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Invisibledoktor_alternate
card-carryingAmateurMycologistsAnonymous member
Registered: 08/24/03
Posts: 119
Loc: vancouver
agar remedy, cheaply [Re: Cow Shit Collector]
    #2021571 - 10/19/03 12:10 AM (13 years, 1 month ago)

in my experience with agar:
make up some big fucking jars, as big as you can fit into you PC, add all your nutrients but dont stir in the agar, just leave it piled in the middle of your jar. you should pour it carefully so that no crystalline agar gets on the sides of the jars.

then autoclave for like an hour at 15psi min.

then take your jars out while theyre still hot (as soon as you can open the PC, do it) and put the jars into a hot water bath. this dosent have to be boiling, just 'hot' (80c is fine, 50c is fine).

then setup all your petri dishes to go using the stovetop variation on the oven tek. (i bought 2 cookie cooling racks for like $5 each that cover all 4 of my stovetop elements, and this works just fine)

then take the jars of molten agar out of the water bath and pour, pour, pour, pour.... until all the dishes are done.

then carefully slip all your liquid stacks of dishes into a clear garbage bag and stick em outside to cool quickly. if you put it all ontop of a slice of plywood before putting the plates into the bag its way easier to keep all the liquid plates upright!

once solid and cool, innoculate with myc or spores and ur good to go!

!adding some antibacterial drugs to the agar mix works wonders!
these you can order online, or from your local chem supplier.
your an amateur soap manufacturer, right? and you wanna make gourmet antibacterial soap, right? as a side note, this soap excuse works great for getting sassafras oil (for those of you who see the value in this...)!


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OfflineBaby_Hitler
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Re: agar remedy, cheaply [Re: doktor_alternate]
    #2024625 - 10/20/03 05:48 AM (13 years, 1 month ago)

If you're innoculating with mycelium just use peroxide. I've never contaminated a petri dish and I just do it on the kitchen table in open air.


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Invisibledoktor_alternate
card-carryingAmateurMycologistsAnonymous member
Registered: 08/24/03
Posts: 119
Loc: vancouver
Re: agar remedy, cheaply [Re: Baby_Hitler]
    #2025586 - 10/20/03 02:49 PM (13 years, 1 month ago)

well, when i worked for xenon genetics inc. we always used antibacterial drugs because we were cloning and expressing genes in resistant bacteria. i just adapted that procedure for home use.

of course if youre mega-anal about cleanliness you could probably innoculate inside your asshole...

but i do use the stovetop laminar flow hood tek for all my mycological operations these days and no problems with contams since i started.


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Invisiblemicro
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Re: agar remedy, cheaply [Re: doktor_alternate]
    #2025653 - 10/20/03 03:07 PM (13 years, 1 month ago)

TOPO sucks. We're using TOPO vectors right now, but most don't seem to want to clone because they're new strains and the primers we have don't really match. I'm lucky if I get a visible band at all (even lowering annealing temp and upping cycles) so most of the really light ones just won't fucking clone.

--
Micro


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Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


Edited by micro (10/20/03 03:10 PM)


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OfflineDeviant
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Re: Agar disastar [Re: Deviant]
    #2026056 - 10/20/03 05:27 PM (13 years, 1 month ago)

Thanks for all of the suggestions and comments. I definatly won't be using the glove box for agar again. I like the idea about babyfood jars, maybe ill give that a try if I don't get a flowhood soon. Thanks again everybody.


--------------------
"How doth the little crocodile improve his shining tail, and pour the waters of the Nile on every golden scale! How cheerfully he seems to grin, how neatly spreads his claws, And welcomes little fishes in With gently smiling jaws!"


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Amazon Shop for: ½ Pint Jars, Agar, Autoclave, Laminar Flow Hood, Petri Dish, Potato Dextrose

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