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Offlinenugjug
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Registered: 11/07/05
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A few agar questions
    #7415470 - 09/16/07 04:37 AM (16 years, 4 months ago)

Recently I got my feet wet with agar with poor results. The glove box I was using at the time was really small and I wasn't used to the motions and only had one of the 6 work out. Then all of the transfer plates from it failed.

My questions are these:
After you pour the plates, you wrap them with parafilm and let them cool. The only size parafilm I could find was 2''. This is kind of awkward to fold around the plates for me. Is this to thick? Do I just split the tray with it and fold the excess over the top and bottom? How hard do I push to make it seal? How good does the seal need to be? Because last time after they cooled the parafilm had kind of melted in place and was very difficult to remove which caused excessive handling of the plates. Also once I have inoculated the plates with spores or tissue, do I re-wrap the plates or is this only for cooling? If so do I use new parafilm or the original pieces used in cooling.

Lastly I used an 1000ml Erlenmeyer flask to prepare the medium in and the polyfill in the neck of the bottle kind of melted on the inside of the bottle. Is this unusual?

Thanks in advance for any help.


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Invisiblemusher_420
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Registered: 08/01/05
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Re: A few agar questions [Re: nugjug]
    #7415534 - 09/16/07 05:41 AM (16 years, 4 months ago)

Check this out if you haven't read it yet.
http://www.fungifun.org/English/Agar

Sounds like your not getting the agar cool enough before working with it. Let it cool down until the flask is cool enough to handle with bare hands without burning yourself.


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InvisibleautomanM
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Re: A few agar questions [Re: nugjug]
    #7415559 - 09/16/07 06:07 AM (16 years, 4 months ago)

Quote:

nugjug said:
After you pour the plates, you wrap them with parafilm and let them cool. The only size parafilm I could find was 2''. This is kind of awkward to fold around the plates for me. Is this to thick? Do I just split the tray with it and fold the excess over the top and bottom?




it isnt too thick. fold it over the top and bottom.

Quote:

nugjug said:
How hard do I push to make it seal? How good does the seal need to be?




you shouldnt have to push much. when you stretch it a bit over the plate, it should seal on it's own/ just be sure to overlap it a litle bit.

Quote:

nugjug said:
Because last time after they cooled the parafilm had kind of melted in place and was very difficult to remove which caused excessive handling of the plates.




i have never run into that. i dont pour my plates until just before they solidify, though, because i add hydrogen peroxide. if you add peroxide when the agar is too hot, it is destroyed by the heat. but if you do run into that problem again, how about cutting the seal with an exact-o knife?

Quote:

nugjug said:
Also once I have inoculated the plates with spores or tissue, do I re-wrap the plates or is this only for cooling? If so do I use new parafilm or the original pieces used in cooling.





yes. i use the same piece if possible making sure to put the same side in and same side out that i did before.

Quote:

nugjug said:
Lastly I used an 1000ml Erlenmeyer flask to prepare the medium in and the polyfill in the neck of the bottle kind of melted on the inside of the bottle.




why would you use polyfill on agar? just use a jar with the lid flipped and no holes.


--------------------
No, no, you're not thinking, you're just being logical. ~ Niels Bohr


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Invisibleorchidfanatic
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Re: A few agar questions [Re: automan]
    #7415580 - 09/16/07 06:24 AM (16 years, 4 months ago)

the best way to prepar agar is to get a short glass bottle at least 1/2 liter .. drill a hole in the cap and use a small piece of tyvek as a filter.. PC the agar in the bottle and let cool overnight ..
take it out and heat it up to be able to pour it ..
when the agar is cooling the tyvek keeps contams from being sucked into the bottle ..
good luck !


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InvisibleautomanM
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Re: A few agar questions [Re: orchidfanatic]
    #7415585 - 09/16/07 06:29 AM (16 years, 4 months ago)

i disagree. why go through the hassle of tyvek when you can just pour the plates as the agar is initially cooling? anad agar never reheats properly unless you spend a good bit of time heating it evenly. you end up with a lump of agar in the middle of the jar unless you leave it in hot water for a very long time. i'm not willing to spend that much time. pour as it reaches 105F.


--------------------
No, no, you're not thinking, you're just being logical. ~ Niels Bohr


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Invisibleorchidfanatic
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Re: A few agar questions [Re: automan]
    #7415771 - 09/16/07 07:48 AM (16 years, 4 months ago)

Quote:

automan said:
i disagree. why go through the hassle of tyvek when you can just pour the plates as the agar is initially cooling? anad agar never reheats properly unless you spend a good bit of time heating it evenly. you end up with a lump of agar in the middle of the jar unless you leave it in hot water for a very long time. i'm not willing to spend that much time. pour as it reaches 105F.




yes I agree that would be much easier and I do get lumps.
I prepared the agar hoping the petri dishes would come in the mail
now that I have them on hand I will do it the way you described much easier..
thanks for that


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Offlinemushyflushy
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Registered: 09/13/07
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Re: A few agar questions [Re: orchidfanatic]
    #7415963 - 09/16/07 08:58 AM (16 years, 4 months ago)

agree


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feen


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Offlinenugjug
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Re: A few agar questions [Re: mushyflushy]
    #7418333 - 09/16/07 10:47 PM (16 years, 4 months ago)

Thank you to everyone for your responses. One other question I have is how do you keep the disposable dishes sterile after you've opened the bag. I wrapped it up tight and put it back in a box. Will the plates be sterile or will I need to clean them before I pour my next batch of plates?


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InvisibleRoachMan
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Re: A few agar questions [Re: nugjug]
    #7418408 - 09/16/07 11:07 PM (16 years, 4 months ago)

You should only open the bag when you are ready to pour.

However, even if you opened the bag the inside of the closed dishes should still be sterile.

No further sterilization should be needed.


--------------------
"The mistake that I make is to try to come awake in a place you're just supposed to get shit faced or baked."


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Offlinenugjug
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Re: A few agar questions [Re: RoachMan]
    #7421361 - 09/17/07 08:47 PM (16 years, 4 months ago)

OK, that's what I figured. Just wanted to make sure. Thanks


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OfflineCyber
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Re: A few agar questions [Re: nugjug]
    #7421609 - 09/17/07 09:57 PM (16 years, 4 months ago)

Quote:

nugjug said:
My questions are these:
After you pour the plates, you wrap them with parafilm and let them cool.  The only size parafilm I could find was 2''.  This is kind of awkward to fold around the plates for me.  Is this to thick?





First off, ditch the parafilm. It is a waist of money! (I am a cheep bastard) Go get a roll of cellophane, take a gensu knife and cut it into 1" wide rolls. It works great and one of the rolls will do 3 hundred dishes. Oh, and you can get 10 to 12 rolls out of a single package of cellophane. It is also easy to roll around the edge of the dish.

Quote:


Also once I have inoculated the plates with spores or tissue, do I re-wrap the plates or is this only for cooling?  If so do I use new parafilm or the original pieces used in cooling. 





Yes you would re-wrap the plates.

Quote:


Lastly I used an 1000ml Erlenmeyer flask to prepare the medium in and the polyfill in the neck of the bottle kind of melted on the inside of the bottle.  Is this unusual?





No need to put the polyfill in the neck. Just cover the top with aluminum foil and Pressure cook for 20min @ 15PSI.

Quote:


agar never reheats properly unless you spend a good bit of time heating it evenly. you end up with a lump of agar in the middle of the jar unless you leave it in hot water for a very long time. I'm not willing to spend that much time. pour as it reaches 105F.





This is wrong, Just put it back into the Pressure cooker and run it for 20 min @ 15psi. I like to make mine in 1 pint jars. When it is cool the lids seal. once it is PC'ed and hot, the jars unseal and you can remove the lid with out getting the sucking sound. :wink:

Quote:


One other question I have is how do you keep the disposable dishes sterile after you've opened the bag. I wrapped it up tight and put it back in a box.





Ummmm, I tend to pour 20 dishes at a time, then wrap. They will keep for quite some time like that.

As to the contamination problem with your glove box. I gave up on trying to get agar to work in a glove box. The contamination ratio was just too high. There was no issue pouring them, but when I went to inoculate the dishes they would contaminate. Get a flow hood, it works much better. :wink:









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Offlinexhooliganx
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Re: A few agar questions [Re: Cyber]
    #7421767 - 09/17/07 10:33 PM (16 years, 4 months ago)

I personaly like parafilm more than cling wrap. the way you use parafilm you cut about 3 squares long piece hold one side against the plate and stretch it around the plate. you don't have to fold the edges over the top and bottom it pulls down on its own. glove boxes work great i've had 5 contamed plates in 100 dishes.


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Offlinenugjug
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Registered: 11/07/05
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Re: A few agar questions [Re: xhooliganx]
    #7425997 - 09/18/07 09:51 PM (16 years, 4 months ago)

Cyber and xhooliganx thank you both.


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