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OfflineRohypnol
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Registered: 08/04/03
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Seperating mycellium from contam on agar?
    #2084914 - 11/08/03 09:05 PM (13 years, 10 months ago)

I have a SEVERLY contaminated peice of mycellim on agar. However, this mycellium is the fastest growing mycellium I have ever seen. It colonized a large portion of a karo syrup culture VERY quickly. Plus, it is all I have left of the strain - the syringe are gone. How would I go about ridding it of the contaminants? I assume it is contaminated with many different contams, but the only thing that shows up is black pin mold.


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Offlinewalk
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Registered: 06/22/03
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Re: Seperating mycellium from contam on agar? [Re: Rohypnol]
    #2085145 - 11/08/03 10:15 PM (13 years, 10 months ago)

by the time you see those black pins there will be spores everywhere so move to peroxidated agar. Cut a couple of pieces from the plate where there is no contam or where the contam is less dominant. Move slowly when you open the lid of the plate so as to minimize airborn spores. Put these new wedges on a H2O2 plate and then let colonisation begin...repeat as required.


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OfflineRohypnol
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Registered: 08/04/03
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Re: Seperating mycellium from contam on agar? [Re: walk]
    #2085166 - 11/08/03 10:21 PM (13 years, 10 months ago)

the problem is, its on peroxidated agar as of now and its not stopping contam at all


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OfflineRohypnol
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Re: Seperating mycellium from contam on agar? [Re: Rohypnol]
    #2085179 - 11/08/03 10:25 PM (13 years, 10 months ago)

do you recommend placing the wedge in pure (3%) peroxide for an amount of time to stifle contams?


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Invisiblemicro
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Registered: 05/09/03
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Re: Seperating mycellium from contam on agar? [Re: Rohypnol]
    #2086388 - 11/09/03 01:19 PM (13 years, 10 months ago)

You see, the problem is that that could easily kill it. If you have a chunk of mycelium you could always sterilize the outside of it, and then use the inside, but this is almost impossible to do from agar. You could try it, though -- just don't put all your eggs into one basket.

Your best bet would probably be to use an inoculating loop (sterilize each time) and simply try to isolate it from the contaminants on many dishes; hopefully you'll get an one that's o.k., and transfer this to like 20 dishes. Only take really small portions, though -- if you can't get an inoculating loop, use sterilized toothpicks.

Don't get your hopes up, but if you do it well and do like 20 dishes each time, you have a good chance of getting one -- just keep these dishes isolated (chances are you'll be growing contaminant) and right when you have one with mycelium that looks clean, isolate it from the rest really early, and transfer to like 20 dishes. Right when you see shit contaminate, throw it out.

--
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OfflineRohypnol
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Re: Seperating mycellium from contam on agar? [Re: micro]
    #2086899 - 11/09/03 06:55 PM (13 years, 10 months ago)

Thank you for the tip. I have another question, my black pin mold is growing only ON the growing mycellium. Could it be that all im growing is black pin mold and no mycellium at all?? Everytime there is a white slightly 3 dimensional growth, there is black pin in the middle of it.


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Invisiblemicro
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Re: Seperating mycellium from contam on agar? [Re: Rohypnol]
    #2088751 - 11/10/03 09:33 AM (13 years, 10 months ago)

Rhizopus, maybe? It's pretty likely -- those black dots are spores coming from the rhizopus mycelium.

Sorry, man.

http://complabs.nevada.edu/%7Ejbn/labpics/lab2/rhizopuszygospores.jpg

--
Micro


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Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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InvisibleSemilanceata
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Registered: 05/26/03
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Re: Seperating mycellium from contam on agar? [Re: micro]
    #2088996 - 11/10/03 12:04 PM (13 years, 10 months ago)

How do you sterilize the toothpicks ?


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OfflineRandolph_Carter
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Re: Seperating mycellium from contam on agar? [Re: Semilanceata]
    #2089003 - 11/10/03 12:10 PM (13 years, 10 months ago)

I'd guess an isopropyl bath would work just fine....but i'd let them dry out pretty well before use.
Stay Frosty.
R.C.


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Invisiblemicro
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Re: Seperating mycellium from contam on agar? [Re: Randolph_Carter]
    #2089010 - 11/10/03 12:12 PM (13 years, 10 months ago)

Just put them in a glass container, cover with tin foil and pressure cook.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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InvisibleSemilanceata
No god, no boss

Registered: 05/26/03
Posts: 841
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Re: Seperating mycellium from contam on agar? [Re: micro]
    #2089017 - 11/10/03 12:16 PM (13 years, 10 months ago)

But, you talk about wood toothpicks ?


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Invisiblemicro
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Re: Seperating mycellium from contam on agar? [Re: Semilanceata]
    #2089034 - 11/10/03 12:23 PM (13 years, 10 months ago)

Yeah. Just don't let them get soaked, but you can sterilize toothpicks.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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InvisibleSemilanceata
No god, no boss

Registered: 05/26/03
Posts: 841
Loc: República Federal Íbera
Re: Seperating mycellium from contam on agar? [Re: micro]
    #2089042 - 11/10/03 12:27 PM (13 years, 10 months ago)

OK man will do.


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Offlinefreebee
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Registered: 11/25/03
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Re: Seperating mycellium from contam on agar? [Re: Semilanceata]
    #2174241 - 12/13/03 06:59 AM (13 years, 9 months ago)


Snag a small piece of mycelium and transfer it to a new agar plate. Pour a second layer of agar media over it (let it cool as much as possible, to the point right before it starts to harden). When mycelium starts to show up on top of the second layer, snag a shalloow piece and transfer to a new plate. You may have to repeat this a couple times depending on how badly it's contaminated.

This method will clean out molds, bacteria, etc. The mycelium grows up through the agar but leaves everything else behind. Just make sure not to dig down to the first layer when cutting a piece out.

Using peroxide agar for both layers will also help. Hopefully the white mycelia in your dish is actually cubensis. If you clean it like this and it still grows black spores, the dish contains nothing but a contaminant, and no mushroom mycelia.

Freebee


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