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InvisibleCompilerV
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Agar Culture transfer to Liquid Culture Tek * 2
    #1644911 - 06/19/03 03:22 PM (13 years, 5 months ago)

You will need an agar plate with healthy mycelium growing on it. This example uses the Pearl Oyster.
You will need a sterile syringe and needle to make the transfer. Last but not least, you need a jar of liquid food. We are using a standard malt water mixture.



When working with agar plates you must work in a clean glovebox or in front of a HEPA hood. With this in mind we will continue.

To begin we will remove 2cc from the malt water mix.



Now carefully remove the lid on the dish. Locate the best looking mycelium on the plate, avoiding areas near the edge of the dish. Slowly squirt a small amount of the liquid from the syringe on this area. You want to be careful placing this solution on the plate. Your goal is to put a dime size ?drop? that will hold its shape from water tension. If you put too much on too fast it will run all over the dish, increasing the risk of contamination.



Now it is simply a matter of scraping the mycelium under the water ?drop? (with the tip of the needle) until some of it is suspended in the solution and then drawing this back in to the syringe. Working in front of a HEPA filter helps greatly here if you accidentally draw extra air in, but a good glove box will work fine also.



All that is left now to complete the transfer is to inject this syringe back in to the jar of malt water.



Your newly inoculated liquid culture jar should now be placed in an incubator at the correct temperature for the species. Once every 2 days you can swirl the jar around to re-mix the mycelium. Growth will be evident within hours with different mushroom?s mycelium. Growth will continue for some time but it will be optimal to use in 7 or 10 days.


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Invisiblemicro
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Compiler]
    #1645037 - 06/19/03 04:04 PM (13 years, 5 months ago)

It would probably be better if you didn't take the lid all the way off the petrie, and instead used the syringe like an innoculating loop and only cracked the petrie as much as you need to (be sure not to touch the syringe to the petrie, though.)

Just IME from working in a lab.

--
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InvisibleOscuro_lobo
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Re: Agar Culture transfer to Liquid Culture Tek [Re: micro]
    #1645073 - 06/19/03 04:15 PM (13 years, 5 months ago)

Where'd you get that lid for your jar? Hack an slash on an old mycobag?


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InvisibleCompilerV
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Oscuro_lobo]
    #1645330 - 06/19/03 05:32 PM (13 years, 5 months ago)

That particular lid was developed last year after using a cut up Mycobag. I have found the original cut up Mycobag version works just as well and is less expensive. I still have some of these around so I might as well use them up sense I have them.

btw, the cut up Mycobags work great for this. recycling is so kewl :smile:


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Re: Agar Culture transfer to Liquid Culture Tek [Re: Compiler]
    #1645989 - 06/19/03 09:50 PM (13 years, 5 months ago)

Nice post, thank you!
One thing could you either link or  list "standard malt water mixture" instructions. I assume its just malt and water but what ratio? Is it sterilized? For how long?
Forgive me Im still learning.. :wink: 


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Re: Agar Culture transfer to Liquid Culture Tek [Re: Effed]
    #1646053 - 06/19/03 10:07 PM (13 years, 5 months ago)

I had good results by just transferring a wedge to the liquid sugar solution... I think the act of sucking mycelium into the syringe adds an unneccessary step. With good technique a wedge of agar can be tranferred to liquid sugar solution (dextrose, malt whatever) with under two seconds exposure. If you add some peroxide to your liquid solution and transfer your agar culture to peroxidated agar (for storage) you should be fairly well protected against most contaminants...

Im not knocking the tek, nice pics and I have done similar things myself but I have found the above to be much more easy.

Anyone else agree?

Does anyone agree...


Edited by mesq (06/19/03 10:09 PM)


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Invisiblejojoba
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Re: Agar Culture transfer to Liquid Culture Tek [Re: mesq]
    #1646307 - 06/19/03 11:32 PM (13 years, 5 months ago)

yes mesq, i agree.. For me it is much easier to cut a wedge from the agar in a sterile glovebox or such, and just transfer to liquid medium ( i add peroxide ) :smile: seems to be workin like a charm.. nice fluffy growth on the oyster's. :smile:


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InvisibleCompilerV
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Re: Agar Culture transfer to Liquid Culture Tek [Re: jojoba]
    #1647739 - 06/20/03 11:33 AM (13 years, 5 months ago)

While a wedge transfer works quite it only provides one point of inoculation while this method will make dozens of inoculation points. It is another alternative to the more common methods, which I personally like best. Everyone has their favorites and should use what they are most comfortable with.

As far as the malt water formula it is very simple. 1/8 teaspoon malt-extract agar mixed with 200 ml water and mix well. I know there are tons of formulas for this but this is one of several I use. PDA works even better than the malt. By having agar in this mix the water turns in to a semi-gel which helps suspend the growing mycelium.

Edit - sterilized for 30 minutes at 15 pounds


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Edited by Compiler (06/20/03 11:35 AM)


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OfflineRichard_D_James
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Compiler]
    #1649438 - 06/20/03 09:21 PM (13 years, 5 months ago)

I just wanted to share the tek I use to change colonized Agar to Liquid Innoculum.

Tools Needed:
100% colonized agar plate(100mmx15mm should work fine)
Blender
Bleach
h202 (Hydrogen Peroxide)
Pint Jar with 1000ml of sterile water

I use a fully colonized agar plate. I take the best looking plate, one I know that is not likely to carry contaminate spores.

I then sterilize the blender with a strong Bleach/Water solution (20% bleach). I then blend on high for several minutes, shaking the blender vigorously to ensure total coverage of bleach. I then rinse with plain tap water (which is chlorinated itself), straight from the faucet. I do about 3 rinses to ensure all bleach residue is washed out.

Then, all in one big sweep, I throw a fully colonized agar plate, agar and all into the blender with my pre-sterilized 1000ml of water and around 75ml of Hydrogen Peroxide. I then blend on low until I have a liquid that looks something like milk. (this should be after about 5 secs of blend) I then immediately pour this milky liquid from my blender directly BACK into the pre-sterilized pint jar (which was holding your water) and put the lid on.

You can then use this innoculum anyway you wish. I do this in my dirty ass kitchen with great success. Doing this in front of a flow hood would be better, obviously. The hydrogen peroxide does its job at killing spore and bacteria. This is not the only way to get liquid innoculum obviously. Just another take on things.


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InvisibleCompilerV
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Richard_D_James]
    #1649505 - 06/20/03 09:52 PM (13 years, 5 months ago)

Sounds good, a nice way to make an "instant liquid culture"  :smile:
I rarely use my eberbach container I am ashamed to say  :blush:


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InvisibleAlkaloids
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Compiler]
    #1663605 - 06/26/03 05:03 AM (13 years, 5 months ago)

want to sell that Eberbach?  I've been looking for a new one.
  good thread.  I prefer wedge to Eberbach/blender work, but do what you like.  :smile: 


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InvisibleCompilerV
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Re: Agar Culture transfer to Liquid Culture Tek [Re: Alkaloids]
    #1667693 - 06/27/03 07:11 PM (13 years, 5 months ago)

Thanks for the offer but I will have to pass. I don't use it alot but do still use it and it took me a while to finally get one. They are a usefull tool to have when it is needed.


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