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Amazon Shop for: Agar, Paul Stamets, Petri Dish

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InvisibleATWAR
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Contam Sandwich Agar Technique... * 1
    #2823255 - 06/24/04 07:18 AM (12 years, 5 months ago)

This method is briefly outlined in Growing Gourmet and Medicinal Mushrooms by Paul Stamets, but it is not illustrated in the book. I decided I would demonstrate the process, as it has worked very well for me the few times I have resorted to it.

First you need the supplies:

10% peroxide solution (optional)
Sharp scalpel or knife
Inoculation loop
2 Petri?s of Gentamicin Agar. (I use 10mg per 200mg agar and I also used 2ml of 3% peroxide solution).




Rescue the healthiest looking mycelium from the wedge, as far away from the bacteria as possible. Try to get an area of 1cm square of mycelium, but do not get any agar. Scrape the mycelium free using your inoculating loop. Here you see me scraping mycelium that has grown onto the plastic of the bag the wedge was sent in.




Place the rescued mycelium in the center of one of the antibiotic agar plates after dipping into the peroxide solution. Be very careful not to smear the transfer around on the plate, or spread excess peroxide across the surface. You can omit the peroxide but I use it as an additional safety measure. Be sure that if you do use peroxide, you change it between each transfer.




Cut a 3cm x 3cm square from the other antibiotic agar plate you have. It is best to pour the agar thin for this plate, so you can see through it, and it is easy for the mycelium to grow through.




Place the square piece you removed earlier onto the rescued mycelium fragment. Making sure to cover it all, and making sure all edges of the agar sandwich are contacting the lower agar. Basically you want to seal the contaminated mycelium between the two pieces. Seal the petri and incubate as usual.




In a few days mycelium will poke up through the surface of the transferred piece of agar. This is an excellent sign that the culture has now been isolated pure.




And a few days after that be sure to watch that it colonizes down off the top piece and onto the bottom without delay. If it gets to that point and stops (or stalls), it may indicate that bacteria has crawled out from between the sandwich. Once it completely colonizes the dish, take a transfer 1-2cm away from the rim of the petri to ensure the cleanest tissue...

There you have it; it works very well to keep bacteria from crawling along with your mycelium, and reduces the chances of airborne cross-contamination when the nasties are sealed under the agar.


--------------------
To give is to live...



Edited by ATWAR (06/24/04 12:02 PM)


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Offlinedoc34
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Re: Contam Sandwich Agar Technique... [Re: ATWAR]
    #2823333 - 06/24/04 08:11 AM (12 years, 5 months ago)

I knew that!!! :rolleyes:




















































Ok,so I'm lying :grin:

Another "Technique"added to my list of FAV's!

Thanks

:cool: :thumbup:


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InvisibleATWAR
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Re: Contam Sandwich Agar Technique... [Re: doc34]
    #2823545 - 06/24/04 10:52 AM (12 years, 5 months ago)

I thought this might be interesting as well:

<- I transferred some of the mycelium to the dish with the missing piece (figured I wouldn't waste it). It recovered and has started to colonize the media, but once it hit the open non-nutritious space the growth became very agressive.



Then when it hit the media it thickens up and slows down. Interesting...


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InvisiblePinback
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Re: Contam Sandwich Agar Technique... [Re: ATWAR]
    #2823560 - 06/24/04 11:01 AM (12 years, 5 months ago)

Very nice! Have you tried this without (specific) antibiotics? Say standard PDYA, or peroxidated PDYA?


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InvisibleATWAR
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Re: Contam Sandwich Agar Technique... [Re: Pinback]
    #2823645 - 06/24/04 12:01 PM (12 years, 5 months ago)

No, I think it would spread just as usual (I would think regular agar would be destined for failure). Peroxide is not very effective againsed bacteria. It works fine for endospores, but some bacteria produce peroxidase and can live on the nutrified media. Their growth usually only becomes stunted. This method (in theory) forces the mycelium to grow through the agar which is antibiotic. When it grows through piercing it with tiny hyphae, the bacteria is left behind. Sometimes bacteria can live on the mycelium, traveling with it across the agar. This method prevents that from happening.

I have not tried it with peroxidated agar alone, although the actual mix I made with both peroxide and Gentamicin (MYA). I use 2-3ml of 3% peroxide to my 200ml agar mix (I forgot to add that part). I am still working on my contamination isolation techniques, especially with molds. Bacteria is quite simple in comparison. This method is basically fool-proof...


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InvisiblePinback
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Re: Contam Sandwich Agar Technique... [Re: ATWAR]
    #2823970 - 06/24/04 02:06 PM (12 years, 5 months ago)

I see... I was hoping for the bacteria to stay inside the agar sandwich while the mycelium grew out, because of their different mode of growth. Thanks for the explanation!


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Offlinehugo
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Re: Contam Sandwich Agar Technique... [Re: ATWAR]
    #2824139 - 06/24/04 02:56 PM (12 years, 5 months ago)

paul is such a smart guy, i met him once and he hand one of those cool hats made out of fungi


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OfflineMAIA
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Re: Contam Sandwich Agar Technique... [Re: ATWAR]
    #2838689 - 06/29/04 05:59 AM (12 years, 5 months ago)

Very, very good ATWAR.
Thanks for sharing :smile:

MAIA


--------------------
Spiritual being, living a human experience ... The Shroomery Mandala



Use, do not abuse; neither abstinence nor excess ever renders man happy.
Voltaire


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InvisibleATWAR
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Re: Contam Sandwich Agar Technique... [Re: MAIA]
    #2841198 - 06/29/04 09:52 PM (12 years, 5 months ago)

Thanks MAIA! I figured since Stamets left the illustrations for this technique out of his book, I would include them. IMO, you can't beat this method for contaminated wedges. Every time they have come out clean this way, and it seals the bad under the agar. Seriously limiting accidental cross-contamination. Here is the plate now, I almost forgot about it:



(this is Hypsizygus ulmarius)


--------------------
To give is to live...



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