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 Owl
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Registered: 03/20/04
Posts: 178
Loc: Netherlands
Last seen: 13 days, 7 hours
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 Bacterial contams - agar
#6081306 - 09/20/06 05:50 AM (1 year, 11 months ago) |
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I am getting a lot of bacterial contams in my agar lately and I am trying to find the source.
my PC time was 1 hour at 7.5 psi
glovebox is sanitized by spraying 70% alcohol
agar is poured and inoculated in a still air glovebox
I inoculated 7 plates and left three blank. All inoculated plates grew bacteria. Blank plates also grew bacteria but the colonies developed 10 days later.
I am sure that the inoculum is contaminated because of the difference between inoculated and blank plates. There must also be another source. What should be the first step to change (PC pressure and duration, glovebox sanitation, other)?
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RogerRabbit
Bans forPleasure
Registered: 03/26/03
Posts: 16,888
Loc: Pacific Northwest
Last seen: 29 minutes, 47 seconds
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Re: Bacterial contams - agar [Re: Owl]
#6081510 - 09/20/06 08:49 AM (1 year, 11 months ago) |
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It might be perfectly normal. We use agar to transfer the healthy mycelium away from the naturally occuring contaminants. As the agar cools after pouring into petri dishes, it shrinks, drawing in contaminants from the air, which is full of bacteria and mold spores.
It's important to wrap your petri dishes with parafilm as soon as they solidify after pouring.
When you see contamination developing in a petri dish, immediately transfer any good mycelium away from the contaminated dish into a fresh one.
It will help to use a filtered flask for PC'ing your agar to avoid contaminants being drawn into the flask as it cools. Allow the agar to cool until it's nearly solidified before pouring. This will cut down on the temperature differential between inside and outside the petri dishes, reducing the amount of contaminants drawn in as the dishes finish cooling.
RR
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Owl
Stranger
Registered: 03/20/04
Posts: 178
Loc: Netherlands
Last seen: 13 days, 7 hours
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Re: Bacterial contams - agar [Re: RogerRabbit]
#6085543 - 09/21/06 04:51 AM (1 year, 11 months ago) |
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Thank you for the tips. Yesterday I noticed that the potato broth (I use PDYA recipe) was extremely small for one litre. I probably forgot to turn down the heat when boiling the potato's, making the broth too concentrated and ending up with a media that favors bacteria instead of fungi.
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RogerRabbit
Bans forPleasure
Registered: 03/26/03
Posts: 16,888
Loc: Pacific Northwest
Last seen: 29 minutes, 47 seconds
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Re: Bacterial contams - agar [Re: Owl]
#6086826 - 09/21/06 02:16 PM (1 year, 11 months ago) |
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You still need to wrap the petri dishes with a breathable filter tape of some kind, even if left in a glovebox. Petri dishes don't seal by themselves so will definitely contaminate if not wrapped. I use Parafilm.
RR
--------------------
www.mushroomvideos.com
"I wouldn't want to belong to any club that would have me as a member".
Mark Twain, Woody Allen, Groucho Marx, and anyone else who wishes to claim credit for the quote.
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