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OfflineKingCloyster
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Registered: 05/27/13
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Contaminated syringe > WBS grain jar > agar (for cleanup and isolation)
    #19089198 - 11/05/13 09:29 AM (10 years, 2 months ago)

I have a couple syringes that are contaminated. Don't want to order more and I really like the strains (cambodian and Ksss).  I have been having problems with using the syringe directly on the agar so I nocd up a couple jars and planned on pulling out a few healthy grains of mycelium before the contaminant grows too large in the jars since they will most likely contaminate.  Is this method good? It's about all I can think of.... Also when I'm isolating the cultures should I do a few different ones or is one of each sufficient?


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Invisible8ow8
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: KingCloyster]
    #19089412 - 11/05/13 10:34 AM (10 years, 2 months ago)

Absolutely!

When I have contaminants that grow too quickly to be isolated on agar (sometimes the case with spore syringes), I find it very helpful to knock up a jar and to try to pluck out some healthy myc. Rye is naturally difficult for many contams to grow on, so you might get some myc growth away from the contams. If you get any aerial mycelium it is perfect for this. I would definitely take a few myc samples, drop them on agar, and grow it out a bit to make sure you've got something clean. You can do a few more transfers if need be. Using an antibacterial agar can also help. Also, once you have actual mycelium to work with, H2O2 enriched agar can beat back bacterial and yeast growth for a couple of days (don't use H2o2 with spores - only mycelium).

More generally, with isolates, there's always a possibility that you'll accidentally isolate a poor fruiting strain. So yes! Do a couple if you have the space! :smile:


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OfflineKizzle
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: 8ow8]
    #19090649 - 11/05/13 03:12 PM (10 years, 2 months ago)

You're probably adding too much spore solution to the agar. You just need a drop.


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OfflineKingCloyster
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: Kizzle]
    #19091184 - 11/05/13 05:07 PM (10 years, 2 months ago)

Honestly @kizzle I think its my shitty agar recipe becUse I had one germ on the dish a few days ago in a contaminanted dish. It's very cottony and spreading slow.


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OfflineKingCloyster
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Registered: 05/27/13
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: KingCloyster]
    #19091228 - 11/05/13 05:14 PM (10 years, 2 months ago)

@8ow8 thank you for confirming my hypothesis. H202 is hydrogen peroxide right? Do I just add a little bit to the bottle before sterilizing it?


Oh! One last thing.

How far should I let the mycelium and/or contaminate spread in the WBS jars before plucking some mycelium. Should I soak the piece and drain the water?


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Invisible8ow8
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: KingCloyster]
    #19094606 - 11/06/13 09:52 AM (10 years, 2 months ago)

You should let it grow until the instant you see some clean mycelium far enough away from the contams that you can harvest some without touching the contams. As soon as you think you can nab a little piece of unaffected mycelium, do so. If you do not see any contam, then I would wait a couple of days until either you have a little arial mycelium, or you can pluck from the center of a small colonized zone. 

And no, don't soak the mycelium first! It doesn't need it. After sterilizing everything, I follow the method below:

1: Put the myc source (dish, jar, or slant) and new petri dish next to each other in the (sterilized) glove box.
2: Remove any parafilm and loosen all lids.
3: Sterilize your scalpel or tweezers (flame or alcohol)
4: Using slow, sure movements, remove the lid from the source with your non-dominant hand, and pluck out a piece of myc with your dominant hand. Move carefully to avoid dropping the myc or bumping into anything else.
5: Replace the lid from the source with your non-dominant hand, and remove the lid from the new plate.
6: Gently place the mycelium on the center of the plate
7: Immidiately replace the lid of the destination plate, and seal with parafilm.

Once you get the hang of it, the whole transfer process only takes a couple of seconds, and is rarely (if ever) the source of contamination.

This whole thread is bringing back memories of a horribly contaminated syringe I picked up from an unnamed nonsponsor (coughcoughralphcough) that I managed to rescue by doing exactly this - knocking up a couple of jars, and relying mostly on luck to rescue some myc before the whole thing turned into a mush of black mold and mystery yellow goo.

Good luck!


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OfflineKingCloyster
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Registered: 05/27/13
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Re: Contaminated syringe > WBS grain jar > agar (for cleanup and isolation) [Re: 8ow8]
    #19094643 - 11/06/13 10:02 AM (10 years, 2 months ago)

Thanks man! I heard somehwhere that slimy bacteria doesn't really release airborne spores like fuzzy trich and penicillin do so I'm going to do this under a flow hood. Hope that's okay cuz I ditched my still air box a few months ago.


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