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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Where are my contams coming from? * NEW* I think I have my answer !!!!!
#20224070 - 07/04/14 01:10 PM (10 years, 6 months ago) |
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So I had a jar that had a contam, I emptied it and refilled it, pc'd it again (with the idea the next pc cycle would kill anything left in the jar) for AT LEAST 90mins, knocked it up with clean agar wedge, and now I'm seeing contams in a few jars, but only a few jars. And not even jars from the same petri dishes.
Is it possible cobweb among other molds can survive the pc cycle?
Is it possible for a pressure cooker to not be up to 15psi even though the weight rocks the whole cycle?
I use lids upside down with tight poly for ge, is it possible this is somehow harboring or letting in contams?
I highly doubt its my sterile technique as all my agar work is fine and my agar is visibly clean so I'm looking at other options.
Any obscure ideas?
Edited by Ghatti (08/05/14 07:55 PM)
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StygianKnight
A Mushroom

Registered: 03/12/12
Posts: 2,717
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Re: Where are my contams coming from? [Re: Ghatti]
#20224307 - 07/04/14 01:52 PM (10 years, 6 months ago) |
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Not likely, although if you are worried you can bump it up to 120min. If nozzle and weight are relatively clean and the weights rockin, it's up to pressure no matter what the gauge says. Yes, it could be possible your filter isn't tight enough or has contams. Don't dismiss your sterile technique just yet, while uncommon there are things that wont grow on low nutrient agar that will start to flourish with grains. My first guess is your transfer/inoc only because of all the steps this is always the highest risk.
My suggestion is to do a set of control jars, one gets PCed with the mix but not opened, another gets opened in your sterile environment, pretend you are inoccing it then close it, do the same to another but put a filter on it, etc.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: StygianKnight]
#20224337 - 07/04/14 01:58 PM (10 years, 6 months ago) |
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About the filter having contams. They are very tight so I'm relatively sure nothing is sneaking through them, and I didn't think poly could harbor contams if dry and pc'd?
And one would think if it was my technique that most of my jars would be compromised and not just a random splattering.
Maybe I got sloppy or slow on a jar or two but the percentage is too high to make that the likely culprit.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: Ghatti]
#20226004 - 07/04/14 09:54 PM (10 years, 6 months ago) |
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So upon further thought it might be from reusing a pair of tyvek sleeves I had likely laying out in a room where I had an outbreak.
Didn't think to sanitize them in any fashion. Is there anyway to reuse sleeves? I have always used new ones but I imagine loading a pair in a jar and pc'ing for a few mins should suffice to kill most everything off so I can take them out of the jar and get them dirty immediately again.
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cronicr



Registered: 08/07/11
Posts: 62,899
Loc: Van Isle
Last seen: 3 hours, 5 minutes
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Re: Where are my contams coming from? [Re: Ghatti]
#20226013 - 07/04/14 09:56 PM (10 years, 6 months ago) |
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try using your lids rubber side down
-------------------- It doesn't matter what i think of you...all that matters is clean spawn
I'm tired do me a favor
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: cronicr]
#20226031 - 07/04/14 10:01 PM (10 years, 6 months ago) |
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Have you ever seen an issue from using them upside down? I thought that was the recommended norm from RR among other old heads?
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MudaFuka
Poppin bottles



Registered: 12/14/13
Posts: 18,648
Loc: Canada
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Re: Where are my contams coming from? [Re: Ghatti]
#20226046 - 07/04/14 10:05 PM (10 years, 6 months ago) |
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It could be a few things. Do you let your PC blow off steam for a few minutes before you put the weight on? What type of PC do you have? Are you sure it go's up to 15 PSI. It is also possible your plates aren't as clean as you think they are. Yes you can PC your sleeves and reuse them.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: MudaFuka]
#20226065 - 07/04/14 10:10 PM (10 years, 6 months ago) |
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I let my pc vent for a few before I put the weight on, yes. Its a 16qt presto, with one of those tri weights, removable pieces for 5/10/15 psi. And yes I have all the weight on lol.
Well if the plates weren't clean, I would imagine that the majority of my jars would be bad and not a sampling.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: Ghatti]
#20226090 - 07/04/14 10:17 PM (10 years, 6 months ago) |
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The lid thing has me curious though. I've been having really shitty results lately and can't figure out why. It had been a while since I cultivated anything and always just remembered flipping lids, maybe my tops are warped enough to let some funk in?
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MudaFuka
Poppin bottles



Registered: 12/14/13
Posts: 18,648
Loc: Canada
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Re: Where are my contams coming from? [Re: Ghatti]
#20226098 - 07/04/14 10:18 PM (10 years, 6 months ago) |
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Then my guess would be something is getting in during inoculation.
I use plastic lids now but when I was using metal lids I used them upside down without issue.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: MudaFuka]
#20226205 - 07/04/14 10:43 PM (10 years, 6 months ago) |
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Some of my lids are quite old and getting rusty and pitting, could be it. I will clean my sleeves and for good measure right my jar lids.
Cronic may be onto something though because I had issues even before I reused these sleeves. I've just never heard of such.
Edited by Ghatti (07/04/14 10:48 PM)
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cronicr



Registered: 08/07/11
Posts: 62,899
Loc: Van Isle
Last seen: 3 hours, 5 minutes
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Re: Where are my contams coming from? [Re: Ghatti]
#20226310 - 07/04/14 11:15 PM (10 years, 6 months ago) |
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Quote:
Ghatti said: Have you ever seen an issue from using them upside down? I thought that was the recommended norm from RR among other old heads?
personnely no i have used them upside down and rubber side down but once they get bent up(which happens when your stabbing your hole for your poly sometimes) i dont' trust them at that point
-------------------- It doesn't matter what i think of you...all that matters is clean spawn
I'm tired do me a favor
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azur
God of Fuck



Registered: 04/21/12
Posts: 28,103
Loc: Daid
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Re: Where are my contams coming from? [Re: cronicr]
#20226373 - 07/04/14 11:39 PM (10 years, 6 months ago) |
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I wash all my jars, pc or not!! Are you soaking your grain?.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: azur]
#20226450 - 07/04/14 11:53 PM (10 years, 6 months ago) |
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Soaking my grain, using a sab, everything goes to agar first with multiple transfers, and never any contams to speak of.
There's really nothing else I can revamp as far as procedure or process, I've already reviewed everything I can think of aside from my lids and my pc. Which is where we stand now.
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azur
God of Fuck



Registered: 04/21/12
Posts: 28,103
Loc: Daid
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Re: Where are my contams coming from? [Re: Ghatti]
#20226454 - 07/04/14 11:54 PM (10 years, 6 months ago) |
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Poly may be the culprit
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: azur]
#20226477 - 07/04/14 11:59 PM (10 years, 6 months ago) |
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In what sense? It's likely tight enough I probably couldn't get a needle through it.
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cronicr



Registered: 08/07/11
Posts: 62,899
Loc: Van Isle
Last seen: 3 hours, 5 minutes
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Re: Where are my contams coming from? [Re: Ghatti]
#20226499 - 07/05/14 12:04 AM (10 years, 6 months ago) |
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until you know what type of comts your getting and where there starting it' s just a guessing game, things like penicillium have much smaller spores then things like trich where cobweb and trich are normally human error lol
-------------------- It doesn't matter what i think of you...all that matters is clean spawn
I'm tired do me a favor
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Kizzle
Misanthrope


Registered: 08/30/11
Posts: 9,870
Last seen: 2 months, 7 days
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Re: Where are my contams coming from? [Re: azur]
#20226500 - 07/05/14 12:04 AM (10 years, 6 months ago) |
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Quote:
I highly doubt its my sterile technique as all my agar work is fine and my agar is visibly clean so I'm looking at other options.
Did you check beneath the agar as well to make sure there were no bacteria colonies there?
--------------------
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azur
God of Fuck



Registered: 04/21/12
Posts: 28,103
Loc: Daid
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Re: Where are my contams coming from? [Re: Ghatti]
#20226503 - 07/05/14 12:04 AM (10 years, 6 months ago) |
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Well...lots of folks use it. I've never tried it or truly trusted it. Maybe it's too tight and you're not getting any gas exchange? What's the contam?
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Where are my contams coming from? [Re: azur]
#20226604 - 07/05/14 12:21 AM (10 years, 6 months ago) |
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That's the really interesting part. A few jars have cobweb and a few have an aggressive green mold, likely trich.
That's why I feel safe ruling out dirty agar. Even jars done from the same petri show diff contams so I'm leaning more towards the lid idea as it seems to fit best.
And even if my poly was too tight and there was 0% ge, I'd still likely only see bacteria.
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