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Offlinemickle207
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Registered: 09/03/10
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A question about agar
    #14311020 - 04/18/11 09:05 AM (12 years, 10 months ago)

Ive been browsing through this forum on working with agar and I think I have it figured out now. Im going to try my first attempt on agar since I have to clean my only remaining sample of lc which is contaminated.
I do not have a flowhood but Im nearly done making a glove box for this and was just wondering if petri dishes are more suceptable to contaminates when it is outside the glovebox during the incubation stage since petri dishes are not airtight like jars (isnt it just two glass dishes put on top of each other to just close the other dish?) do you seal the two dishes with tape?
I know it might sound like a real noobish question but I just want to be sure before trying agar.

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OfflinePerun
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Re: A question about agar [Re: mickle207]
    #14311033 - 04/18/11 09:09 AM (12 years, 10 months ago)

I use parafilm to "seal" my petris! It keeps out the contaminants, but allows enough gas exchange for optimum growth of the mycelium.


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First:                                                        Then:

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Invisiblecyantific
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Re: A question about agar [Re: mickle207]
    #14311089 - 04/18/11 09:32 AM (12 years, 10 months ago)

just keep them in something with a lid like a tote to keep the lids from getting stuff landing on them ... the fact that they dont "seal" means very little as long as the cover is kept on ...

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InvisibleDoc_T
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Re: A question about agar [Re: Perun]
    #14311092 - 04/18/11 09:34 AM (12 years, 10 months ago)

I put them in ziploc bags.


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You make it all possible. Doesn't it feel good?

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Offlineesquaredx
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Re: A question about agar [Re: Doc_T]
    #14311278 - 04/18/11 10:31 AM (12 years, 10 months ago)

Quote:

Doc_T said:
I put them in ziploc bags.




You use parafilm right?


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InvisibleFungal growth
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Re: A question about agar [Re: esquaredx]
    #14311311 - 04/18/11 10:42 AM (12 years, 10 months ago)

i put mine in ziplocks too. do i still need parafilm?

and, if i can please jack this thread for 1 moment,
will the spores germinate at 65f or so the same as in
grains? i read recently they should be at 80-85, is
that right?

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Offlinemickle207
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Re: A question about agar [Re: Fungal growth]
    #14311317 - 04/18/11 10:45 AM (12 years, 10 months ago)

i dont know abt low temps but i incubate and fruit my shrooms just fine at 90-95

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InvisibleDoc_T
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Re: A question about agar [Re: esquaredx]
    #14311443 - 04/18/11 11:22 AM (12 years, 10 months ago)

Quote:

esquaredx said:
Quote:

Doc_T said:
I put them in ziploc bags.




You use parafilm right?




No, I put them in ziploc bags.


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You make it all possible. Doesn't it feel good?

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Offlineresptodd
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Re: A question about agar [Re: Doc_T]
    #14311482 - 04/18/11 11:32 AM (12 years, 10 months ago)

Agar was my first approach to cultivation. And I did it for a while just because I had sooo many supplies. But, IMHO, agar opens you up to more possibilities to contamination than working from a LC. If you are having problems colonizing a LC you may have to take a step back and reevaluate your clean/sterile procedures.
  Are you working from prints or syringes?


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Damn! I'm having fun! Just keep the GD monkeys away.

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InvisiblePrisoner#1
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Re: A question about agar [Re: Doc_T]
    #14311490 - 04/18/11 11:33 AM (12 years, 10 months ago)

Quote:

Doc_T said:
I put them in ziploc bags.





this and/or glad wrap. glad wrap allows for some gas exchange

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InvisibleDoc_T
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Re: A question about agar [Re: Prisoner#1]
    #14311523 - 04/18/11 11:41 AM (12 years, 10 months ago)

There's no particular need for gas exchange, there's plenty of air in a ziploc bag to allow the dish to colonize fully.


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InvisiblePrisoner#1
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Re: A question about agar [Re: Doc_T]
    #14311556 - 04/18/11 11:48 AM (12 years, 10 months ago)

conflicting statement, lol

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InvisibleDoc_T
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Re: A question about agar [Re: Prisoner#1]
    #14311582 - 04/18/11 11:55 AM (12 years, 10 months ago)

I've done it many, many times. I can tell you from experience there's plenty of air.
I squeeze out enough air that the dishes stack without any air cushioning, but not don't necessarily remove all the excess.
3 65mm dishes or one 100mm will colonize completely in a sandwich size ziploc.


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InvisiblePrisoner#1
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Re: A question about agar [Re: Doc_T]
    #14311613 - 04/18/11 12:02 PM (12 years, 10 months ago)

I'm not saying you havent or that there isnt, but what would be the need if gas exchange is unnecessary

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InvisibleDoc_T
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Re: A question about agar [Re: Prisoner#1]
    #14311628 - 04/18/11 12:05 PM (12 years, 10 months ago)

Of course a living mycelial culture needs oxygen.
There's oxygen in the dish. And the culture will excrete CO2, but not enough to inhibit growth.
So there's no need for gas exchange- in that you don't need to exchange the gases.
It's fine with the air it has.


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You make it all possible. Doesn't it feel good?

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Offlinemickle207
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Re: A question about agar [Re: resptodd]
    #14311718 - 04/18/11 12:27 PM (12 years, 10 months ago)

Quote:

resptodd said:
Agar was my first approach to cultivation. And I did it for a while just because I had sooo many supplies. But, IMHO, agar opens you up to more possibilities to contamination than working from a LC. If you are having problems colonizing a LC you may have to take a step back and reevaluate your clean/sterile procedures.
  Are you working from prints or syringes?




I doubt its my sterile technique because I havent had any contamination problems before this. The LC was made with honey water (which has proven successful to me before), but this time I knocked it up in an attempt to clone. The clone technique I used was the technique which involved piercing through the stem with a needle and shooting it into the honey water jar. I guess I wasnt sterile enough during this procedure since I did it in an open space with windows opened; thus leading to a contamed LC. However, this LC is the fastest colonizer i've used so far, but it stalls all of a sudden and produces green mold.

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InvisibleFungal growth
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Re: A question about agar [Re: mickle207]
    #14313112 - 04/18/11 04:14 PM (12 years, 10 months ago)

don't pierce it with a needle, there's contams
on the stem surface.
instead, tear it in half and with a sterile scalpel
remove a piece of the inner stipe. place it in a clean
ziplock and in a few days it will turn into a puffball
of myc.here's the whole tek

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Offlinemickle207
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Re: A question about agar [Re: Fungal growth]
    #14317386 - 04/19/11 01:16 PM (12 years, 10 months ago)

Ok, I'm in the process of making my first agar dishes and I was wondering why contamination is an issue with agar? Isnt agar used to 'clean' myc whenever it is contaminated by transferring the clean myc to other dishes? Did I get this wrong?

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Invisiblecyantific
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Re: A question about agar [Re: mickle207]
    #14317985 - 04/19/11 03:04 PM (12 years, 10 months ago)

ever seen fly paper catch flies before ? well thats what agar does with contaminate spores , thats the reason you got to be extra careful with it ... but yes it is used to clean up cultures and you cna have a perfectly good spot of myc inches away from some nasty stuff growing in the same dish ... its kind of a race against sporulation in your dish ... with all sterile teks being properly applied theres no reason a clean culture couldnt be obtained in 1 to 2 transfers off a dirty print or swab ... ive sometimes actually allowed some myc to "fight" with contamination's in dishes and grabbed the strongest section of growth still standing in the dish and cleaned it back up in a couple transfers and obtained some extremely virulent sub-strains that way ... agar is just fun man , its a whole new world , fun and exciting !

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Offlinemickle207
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Re: A question about agar [Re: cyantific]
    #14320047 - 04/19/11 09:51 PM (12 years, 10 months ago)

Wow man I cant wait till my agar plates start colonizing! I just knocked them up just now and will post the results of my first go. I didnt use a glove boxe though but i did it in a 1x1 meter room which used to be a closet. I nuked the whole room with lysol and hope it will be enough... If it doesnt work out then I'll try my second round in a glovebox and try to build a laminar. How many blank dishes do you reckon I should have a side to prepare for transfers?

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