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OfflineMushroomhead13
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Registered: 09/20/20
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Methods of inoculation
    #26993614 - 10/19/20 08:19 PM (3 years, 3 months ago)

I have a large live culture Creeper syringe. I was told that I could either put a drop on some agar and let it colonize or go straight to grain inoculation. I recently did some agar to agar transfers (8) a week ago. I've had to toss 3 plates already because of mold. So I'm thinking of skipping the agar and going straight to grain. What's a good grain tek when starting with LC? I have a pressure cooker.



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OnlineNichrome
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Registered: 12/17/18
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Re: Methods of inoculation [Re: Mushroomhead13]
    #26993672 - 10/19/20 09:06 PM (3 years, 3 months ago)

If you cant get anything but mold to grow on agar you will have loads of trouble with grain. I'd suggest keeping going with the agar till you have a clean culture for sure. Then just transfer a wedge to some grain.


--------------------
Better to be deprived of food for three days, than tea for one.


Freedom is not the right to do as you please, but the liberty to do as you should. ~Emerson


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OfflineMushroomhead13
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Re: Methods of inoculation [Re: Nichrome]
    #26993700 - 10/19/20 09:26 PM (3 years, 3 months ago)

The center of my agar transfers develop healthy mycelium its the outer edge that develops mold


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OnlineNichrome
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Re: Methods of inoculation [Re: Mushroomhead13]
    #26993720 - 10/19/20 09:39 PM (3 years, 3 months ago)

If your plates won't come clean your grain won't either. You need to hunt down where the mold is coming from. The only way to do that and clean up the culture is to work with agar. Just keep practicing until it works and try to keep track of what does and what doesn't. There are lots of good methods out there. Many many people use agar successfully and you can too.

It is nearly impossible to work with agar without at least an SAB. If you are using an SAB or a laminar flow HEPA filter properly, the most likely vectors for contamination are A) You and, B) your syringe. Are you wrapping your plates with Parafilm or plastic wrap? Did you flame sterilize the needle of the syringe before application each time? Lets try to narrow it down...


--------------------
Better to be deprived of food for three days, than tea for one.


Freedom is not the right to do as you please, but the liberty to do as you should. ~Emerson


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OfflineMushroomhead13
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Re: Methods of inoculation [Re: Nichrome]
    #26993746 - 10/19/20 10:00 PM (3 years, 3 months ago)

I have a SAB. I honestly don't know what could have caused the mold. There's so many different factors. I was told to wrap them in plastic wrap so I did... I'm getting some parafilm


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OfflineMushroomhead13
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Re: Methods of inoculation [Re: Mushroomhead13]
    #26993753 - 10/19/20 10:04 PM (3 years, 3 months ago)

I just feel better about working with grain because I feel like there's less chance for contam (opening and exposing agar dishes vs using an inoculation port). Also, a few years ago, I noc'd a few premade grain jars with a spore syringe and had no SAB at the time and had no contam issues.


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OnlineNichrome
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Re: Methods of inoculation [Re: Mushroomhead13]
    #26993838 - 10/19/20 11:35 PM (3 years, 3 months ago)

People win the lottery sometimes. I see so much failure putting iffy cultures or spores directly onto grain and very little but occasional success. I have done that successfully  a couple of times as well as failed. The amount of work that goes into all of this it is worth it to try do do it well. Spores directly on grain can and does work sometimes but more often it fails. Working with agar isn't hard ad can save you time and major headache in the long run.

To me it sounds like your syringe is the most suspect. If you are using an SAB and wrapping properly then the mold is either coming off of you or more likely is in the syringe. If there is mold in the syringe then putting that culture directly on grain won't help solve that problem. The only way to proceed is to transfer clean tissue to new plates till you don't see the mold anymore. Then you can use that culture to successfully inoculate grain in the method of your choice. Making a clean culture happen is not dependant on the medium such as agar or grain or wood or whatever. Once you learn how to handle the organism and keep it clean you can apply that to any venue universally. Agar is by far the most telling, cheapest, fastest, and easiest way to learn how to obtain a clean culture.

Plates are $0.18 usd if you buy a box full. Agar and malt are very cheap too. If you have a pc you can pour a hundred plates or more in just a few hours. Then you can transfer to your hearts content and not even worry about it.

The yields and quality you will get from using and maintaining clean cultures is worth the effort and there are lots of folks here who would be happy to help you figure it out, myself included. Just hoping to help you cut out some major headache in the long run, possible save some time and $, and I totally respect your guts, moxie, and you going for it.

Long story short, I think your syringe is fucked. Don't give up on agar. It's the way to go.


--------------------
Better to be deprived of food for three days, than tea for one.


Freedom is not the right to do as you please, but the liberty to do as you should. ~Emerson


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