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Offlinethisisfielding
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A couple general Agar questions for a newbie
    #21258635 - 02/11/15 01:34 PM (9 years, 3 months ago)

So I'm finally trying my hand at Agar and trying to wrap my head around it all. I've watched the RR video on it and done some searching through the forums but still have some questions.

Currently I have 4 ounces of "Agar Agar Powder by The Seaweed Solution (Excellent Gel Strength: 900 g/cm^2)" and a 20 pack of "SEOH Petri Dish 100 x 15Mm" petri dishes. They feel like they are plastic and are sealed in a bag.

So heres my questions : What precautions need to be taken to sterilize the petri dishes / keep them sterile / etc? Do the petri dishes need to be refrigerated after the Agar has been poured into it? If I'm going to clone, should I go head and fill ALL of the petri dishes I have, even though I'm going to be using one or two? After I have cut the flesh of the mushroom and put it on the agar, whats the general practices after that? Could I use a MS syringe to inoculate some of the other dishes?

I know this seems like a lot of questions and that I havent searched but I have tried to. A lot of the teks I'm finding has cheap solutions not using petri dishes or not using Agar / etc. I do have a SAB for the cloning portion of this all and the pouring of the agar. Thanks for any help!




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OfflinepsilocybeMAN
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Re: A couple general Agar questions for a newbie [Re: thisisfielding]
    #21258650 - 02/11/15 01:38 PM (9 years, 3 months ago)

The petris are already sterile.

1. Make a SAB (still air box)
2. after making your agar mix and sterilizing, place all items into SAB
3. open sealed bag and pour agar into each petri one by one
4. close petris and seal them with parafilm or w/e
5. let them cool and solidify wihtout refrigeration.
6. inoculate inside of your new SAB

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InvisibleDrRedBush
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Re: A couple general Agar questions for a newbie [Re: thisisfielding]
    #21258665 - 02/11/15 01:42 PM (9 years, 3 months ago)

They should come already sterilized

Yeah you can refrigerate turn them upside down so they don't condensate

I make mine as needed. Don't like em sitting around being possible vectors for contams

Yes you can use ms to inoculate them

General practices? Let it grow in ideal conditions

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OfflineX0cyris
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Re: A couple general Agar questions for a newbie [Re: psilocybeMAN]
    #21258667 - 02/11/15 01:43 PM (9 years, 3 months ago)



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Offlinethisisfielding
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Re: A couple general Agar questions for a newbie [Re: DrRedBush]
    #21258678 - 02/11/15 01:46 PM (9 years, 3 months ago)

Whats the purpose of the parafilm? Wouldn't the petri dishes have a chance of contamination after opening the bag and using only a few of them? Is there a safety practice for storing them so they have a less likely chance of contamination? Is there a TEK that incorporates SAB / the petri dishes I have and the type of Agar powder I have? Should I make the Petri dishes before I want to clone or when I have a mushroom ready to clone? Sorry for the rapid fire questions. Been reading on this for quite a bit and still have a ton of questions.

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InvisiblePirateSwazey
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Re: A couple general Agar questions for a newbie [Re: thisisfielding]
    #21258683 - 02/11/15 01:46 PM (9 years, 3 months ago)

Quote:

thisisfielding said:
So heres my questions :
1. What precautions need to be taken to sterilize the petri dishes / keep them sterile / etc?
2. Do the petri dishes need to be refrigerated after the Agar has been poured into it?
3. If I'm going to clone, should I go head and fill ALL of the petri dishes I have, even though I'm going to be using one or two?
4. After I have cut the flesh of the mushroom and put it on the agar, whats the general practices after that?
5. Could I use a MS syringe to inoculate some of the other dishes?




1. Only open them in your SAB / flowhood. Wrap them in parafilm (or saran wrap) after you pour them & they solidify. You can also place them into fresh ziplock bags.
2. No, they only need to be refrigerated if you are culturing on them and want to slow down the growth or store it for later use.
3. When I clone I put tissue to just a few plates, and then transfer it as soon as I see enough growth to make it happen.
4. See #3. Make a couple quick transfers to ensure you have a clean culture then inoculate a slant or jars.
5. Yes you can inoculate plates with a syringe. Just 1 drop should be enough to start growth.


Watch RR's "Let's Grow Mushrooms" DVD. I think it costs like $8 to download it. Sounds like it will
help you a lot, and is worth the investment. Have gone back to it over the years several times and
almost always have picked up a little extra nugget.


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Offlinethisisfielding
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Re: A couple general Agar questions for a newbie [Re: PirateSwazey]
    #21258700 - 02/11/15 01:51 PM (9 years, 3 months ago)

Thanks for all the quick replies! I do own the "Lets Grown Mushrooms" dvds and watched the bit on it. I guess where it ends is where a lot of my questions come up. The way I understand it is:
1. Mix Agar powder with distilled water
2. PC for certain amount of time
3. Pour into petri dishes
4. Let them solidify
5. Cut piece of mushroom flesh and place into petri dish
6. Mycelium growth/Colonization will happen in a few days
7. When need by, take sterile scapel and cut a small chunk off of growth and place in sterilized grain jar
8. Repeat as needed

Does this sound about right? Can petri dishes be stored for long term/repeated use or is it pretty much only good for the next round of grain jars ready to be colonized?

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InvisiblePirateSwazey
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Re: A couple general Agar questions for a newbie [Re: DrRedBush]
    #21258715 - 02/11/15 01:54 PM (9 years, 3 months ago)

Quote:

DrRedBush said:
Yeah you can refrigerate turn them upside down so they don't condensate

I make mine as needed. Don't like em sitting around being possible vectors for contams




If you stack your petris when you pour them the condensation will rise to the top plate. If you put a glass of hot water
on the very top it will keep condensation from happening at all. Not going to dig through to find it but there are a few
older threads where RR talked about storing plates upside down being bad practice.

As long as you wrap your plates they will be fine. I used to think that I had to use them all within a week or two after
I poured them when I first started working with agar, but now days I will pour 40-60 at a time and sometimes not
inoculate some for a couple months.

Quote:

thisisfielding said:
Does this sound about right? Can petri dishes be stored for long term/repeated use or is it pretty much only good for the next round of grain jars ready to be colonized?




Yes it sounds like you are on it. Petris can be stored for a few months if you need to.  It is best to keep them in an
insulated lunch box in your refrigerator. The lunchbox will help with the temperature swings as the refrigerator runs
its cycles, which will keep condensation down and your plates from drying out.


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InvisibleDrRedBush
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Re: A couple general Agar questions for a newbie [Re: PirateSwazey]
    #21258730 - 02/11/15 02:00 PM (9 years, 3 months ago)

Interesting thanks for the correction :thumbup:

I don't like to store just make them as needed

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Offlinemushpunx
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Re: A couple general Agar questions for a newbie [Re: DrRedBush]
    #21259247 - 02/11/15 03:38 PM (9 years, 3 months ago)

Is your agar powder just agar agar? You know you need to add malt extract or potatoe dextrose right?


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InvisibleFungus Mountain
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Re: A couple general Agar questions for a newbie [Re: PirateSwazey]
    #21259251 - 02/11/15 03:39 PM (9 years, 3 months ago)

I remember a few of those threads. This comment in favor of flipping the dish was pretty compelling. That said, I'm lazy and store mine normal side up. :tongue:

Quote:

mothy said:
So this is something I can contribute to, I did a masters in Molecular Biolgoy working in a bacterial culture lab :smile:

We always store agar plates upside down and in the fridge/incubators for some bacteria. It was quite common to get large amounts of condensation (a ml or two), which would easily contam plates if you weren't careful. Sometimes when someone flipped a plate back round the other way for use, if they weren't careful they would get some of the condensation on the agar which would then cause a contam. I beleive that this is the main reason why agar plates are stored upside down in a lab setting.

First post here, hi guys :cool:




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“Until they became conscious they will never rebel, and until after they have rebelled they cannot become conscious.”
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"If you can't explain it simply, you don't understand it well enough."
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Offlinespacechildo
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Re: A couple general Agar questions for a newbie [Re: mushpunx]
    #21259270 - 02/11/15 03:46 PM (9 years, 3 months ago)

Quote:

mushpunx said:
Is your agar powder just agar agar? You know you need to add malt extract or potatoe dextrose right?




just what I was about to say, you'll need some nutrients with the agar powder.

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Offlinethisisfielding
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Re: A couple general Agar questions for a newbie [Re: spacechildo]
    #21261910 - 02/12/15 12:03 AM (9 years, 3 months ago)

I did not know this about the nutrients. Thanks for the heads up! In the video RR talks about how he used to always mix the different ingredients for it but ended up finding that its less time consuming and not that much more expensive to buy the pre-packaged stuff (which is what I thought I purchased). So is what I purchased just the Agar and not the nutrients? Man I think I have a hold on this stuff and literally find out that I don't have a full hold on it.

So I find a fruit that has favorable genetics (grows fast, large,..etc),.. I harvest it,.. cut a piece of the flesh from the inside with a sterile scalpel and place it on a petri dish. Within a week or so it starts to grow... then I prepare grain jars,..inside SAB take a jar, cut a piece of the growth on petri dish,.. add it to the jar and continue to do this with the rest of the jars? Does a petri dish usually only function for colonizing one set of jars? Or is it - petri dish - cut a piece of growth off - add to one jar - then G2G for the rest? I guess I was under the impression that I could take a petri dish with cloned fruit on it and use it over and over (I guess I thought the growth in the petri dish would replace whatever was cut off).

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InvisiblePirateSwazey
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Re: A couple general Agar questions for a newbie [Re: thisisfielding]
    #21261960 - 02/12/15 12:19 AM (9 years, 3 months ago)

You have agar - agar powder. Needs to have nutrients added to it. I don't mix my own so I can't help you there but I'm sure someone else can. When you put tissue to a plate you want to transfer it to new plates once or twice before inoculating a jar, to make sure that it is clean. Yes you G2G your G1 jars (the ones inoculated with the agar wedges) and spawn from those (G2).


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