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Offlinesytar
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Some questions about inoculating with agar wedges
    #19508256 - 02/01/14 09:43 PM (10 years, 11 months ago)

1) should you wait for the agar to fully colonize the wedge before putting it with your grains?

2) why is it not a good idea to cut the agar into many pieces and then shake the jar after inoculation?
Presumably this would put a lot of stress on the mycelium and add quite a bit to recovery time, but is this not made up for with several additional inoculation points?

3) do you add recovery time to the mycelium by trying to shake the wedge into the middle of the grains?

4) would it be a terrible idea to inoculate with two agar wedges? One that has been broken up and shaken in, and another which is whole and relatively undisturbed?

5) is there any consensus on whether inoculating with the agar wedge face down vs face up has any benefits?

Edit

Bonus question: when transferring the leading edge of the mycelium, should I be cutting a small piece out with a scalpel or will a sterile inoculating loop work too?


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Edited by sytar (02/01/14 09:48 PM)

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Offlinenewera
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Re: Some questions about inoculating with agar wedges [Re: sytar]
    #19508373 - 02/01/14 10:22 PM (10 years, 11 months ago)

1)no
2)everytime you do another wedge you risk contams
3)yes, but i think the main issue would be not being able to see it, go for against the glass if anything
4)again it's the risk of contam, the less steps the better the chance of success
5)doesn't really matter, the wedge will colonize anyway
6)scalpel is much easier
7)i'm a noob so don't take my word as gold this is just my understanding

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OfflineCamtaro420
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Re: Some questions about inoculating with agar wedges [Re: sytar]
    #19508486 - 02/01/14 10:42 PM (10 years, 11 months ago)

1) No, take the healthiest, strongest, fastest growth. Make a couple transfers to ensure clean isolation

2) You want to keep the genetics pure to the piece of isolate and you want to make it quick and clean. Cuz it's fast and clean, no need for a bunch if you did you grains properly and use a SAB.

3) You let it sit on top for some colonization first, then shake later, like with spores. You wanna make sure everything is good to go, it;s already going through enough shock being cut from the agar.

4) Yes, See answer two.

5) I'd imagine face down would be better since the growth would hit the grains. Then see answer 3

Bonus question: Use a flame sterilized exacto knife if you are looking to go cheap. Otherwise scalpel.


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Edited by Camtaro420 (02/01/14 10:43 PM)

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Offlinesytar
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Re: Some questions about inoculating with agar wedges [Re: Camtaro420]
    #19509600 - 02/02/14 06:20 AM (10 years, 11 months ago)

Seems like a lot of conflicting answers so far.


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OfflineRogerRabbitM
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Re: Some questions about inoculating with agar wedges [Re: sytar]
    #19509671 - 02/02/14 06:46 AM (10 years, 11 months ago)

Don't fully colonize a petri dish.  You can't trust the edges to be clean and contaminant free, even when wrapped with parafilm. *unless you have a totally sterile lab and colonization area.

Shaking the grain jar after inoculation with agar knocks all the mycelium off the wedge, but it doesn't recover into the grains.  Instead, it has to grow back on the agar wedge, and then it can crawl into the grains.  Shaking slows down the process over simply dropping the wedge on top and letting the mycelium crawl off when it's ready.  Shake when a few dozen grains on top are covered.

Inoculate with the wedge face up if possible.  This causes the least disturbance to the mycelium, so once it recovers from being sliced, it can grow off the wedge and into the grains.  If it lands face down, put the lid on the jar and call it good.  Don't risk something nasty getting in there by trying to flip the wedge over.

When transferring, it's best to take a bit of the agar the mycelium is attached to.  You can shave with a loop or scalpel, but recovery will take a bit longer.
RR


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Offlinesytar
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Re: Some questions about inoculating with agar wedges [Re: RogerRabbit]
    #19509737 - 02/02/14 07:05 AM (10 years, 11 months ago)

Oh hell yeah, I got an RR reply!

Thanks RR. :peace:


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