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OfflineThailandBound
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Stir bars causing contam
    #8822662 - 08/24/08 11:09 PM (15 years, 7 months ago)

Ok so lately I have been unable to get a clean LC. So I went back and reviewed my methods and the only thing that I can find different is the fact that I'm using magnetic stir bars and a stir plate. I have never had a succesfull LC since I bought these stir bars but all LC previous to this (4) were succesfull.

Is it possible that the stir bars I have (purchased on e-bay) are contaminated with something that PCing for 20-30 min would not kill?

If so would a long soak in alcohol followed by an hour or two in the PC fix this or should I pitch them and buy some more.


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OfflineRogerRabbitM
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Re: Stir bars causing contam [Re: ThailandBound]
    #8823820 - 08/25/08 09:17 AM (15 years, 7 months ago)

Quote:

Is it possible that the stir bars I have (purchased on e-bay) are contaminated with something that PCing for 20-30 min would not kill?




Nope.
RR


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Offlinegrod31
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Re: Stir bars causing contam [Re: RogerRabbit]
    #8823836 - 08/25/08 09:23 AM (15 years, 7 months ago)

Quote:

RogerRabbit said:
Quote:

Is it possible that the stir bars I have (purchased on e-bay) are contaminated with something that PCing for 20-30 min would not kill?




Nope.
RR



so RR you are saying that only pcin a lc for 20-30 mins is sufficient? is there no need for an hour at 15 psi ?


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OfflineHippieChick
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Re: Stir bars causing contam [Re: grod31]
    #8823919 - 08/25/08 09:53 AM (15 years, 7 months ago)

This is correct . 20 minutes is more than enough for me . I usually go 15-20 minutes .

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Offlinealkylbenzene23
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Re: Stir bars causing contam [Re: HippieChick]
    #8824029 - 08/25/08 10:18 AM (15 years, 7 months ago)

What about your coring needle?


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OfflineThailandBound
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Re: Stir bars causing contam [Re: alkylbenzene23]
    #8824254 - 08/25/08 11:08 AM (15 years, 7 months ago)

ok then so what could I possibly be doing wrong. Here are my methods:

-Scrape mycelium off of petri dish with flame sterilized scalpel and drop it into a LC. This is done in front of a flowhood following strict sterile technique.

-The LC is a Malt/Dextrose mix PC'ed 20min

-The contam is always the same, white stuff that floats on top and a cloud that looks similair to myc but its not.

My sterile technique and flowhood are good since im not getting contam anywere else. I haven't had a contamed dish since I built the flowhood. The LC is my only problem, very frustrating.


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OfflineMaverick
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Re: Stir bars causing contam [Re: ThailandBound]
    #8824275 - 08/25/08 11:11 AM (15 years, 7 months ago)

Maybe your petri dish is contamed.  Have you tried it from different dishes?  You're saying it's the same contam over and over and it started previously and you haven't had any luck since.  I'm guessing it's the petri dish.

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OfflineThailandBound
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Re: Stir bars causing contam [Re: Maverick]
    #8824870 - 08/25/08 01:11 PM (15 years, 7 months ago)

Yeah I have used 3 different dishes and the last 2 I kept to see if anything else grew and nothing did. I am pretty confident that the dishes are clean as they had no mold or bacteria visible and none ever presented itself.


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OfflineMaverick
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Re: Stir bars causing contam [Re: ThailandBound]
    #8824945 - 08/25/08 01:27 PM (15 years, 7 months ago)

Perhaps your flow-hood isn't working properly.  I don't see how you can get contams from a stir bar that's been PC'd.  Even if it was coated w/ spores of some kind, when you PC it, they'd be destroyed, as long as your PC is working properly.

You said the white cloud looked like myc in the LC, but isn't.  What did it turn into when you spawned it?  Have you tried dropping some agar off those plates into a jar of grain and letting it colonize?  Perhaps whatever the contam is looks like myc on agar too.

You could always make two LCs side by side with the same agar plate, one with a stir-bar and one without, and truly verify if it's somehow contaminating your LC.  Are you leaving the bar in the LC or pulling it out?

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OfflineNo_Life_G33k
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Re: Stir bars causing contam [Re: Maverick]
    #8825434 - 08/25/08 03:02 PM (15 years, 7 months ago)

Quote:

DRTMaverick said:
....
You could always make two LCs side by side with the same agar plate, one with a stir-bar and one without, and truly verify if it's somehow contaminating your LC.  Are you leaving the bar in the LC or pulling it out?




there ya go^^^^

20 minutes should be fine.


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OfflineRogerRabbitM
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Re: Stir bars causing contam [Re: No_Life_G33k]
    #8825522 - 08/25/08 03:21 PM (15 years, 7 months ago)

Quote:

-The LC is a Malt/Dextrose mix PC'ed 20min





Malt should be pressure cooked for 45 minutes.  Sometimes, it can harbor endospores.  What you describe sounds like bacteria contamination, so that could be the problem.  I use MEA agar and always PC for 45 minutes @ 15psi.  Ditto for malt/dextrose liquid culture.
RR


--------------------
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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Offlinenumonkei
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Re: Stir bars causing contam [Re: RogerRabbit]
    #8825537 - 08/25/08 03:26 PM (15 years, 7 months ago)

Is this open air?

The stir bar does move the surface of the liquid, so it WOULD be possible to introduce air-bourne contamination if it's in your medium, theoretically at least.

Practically, I don't know. It's almost definitely not coming from the stir bar, though.



~Monk

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