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Scrape Bacteria from Agar plate

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#1 No-Idea



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Posted 28 June 2011 - 07:06 AM

How to scrape bacteria from agar plate?
I have 180 plates with hundreds thousand of single and each differentially unique colonies. I need to pool all the bacteria. The problem is some of the plates are stored in 4c for around one week and when I try to scrape them off, they are like mucus and very hard to resuspend them in broth. Even vortex doesn't help much. I try to pellet down the bacteria but there is no distinct pellet formed, but layers of insoluble "mucus". So I ended up only take the bottom layer and suspend it and discard all the upper layers. My question is: will it be possible the bacteria from the "old plates" (those that form mucus) is not present in the final bacteria suspension? As they are all in the mucus layers which I have already discarded. I just want to make sure my final bacteria suspension contains ALL the single unique bacteria from ALL the plates. Even one single bacteria will do. Any idea?

#2 pito



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Posted 28 June 2011 - 11:58 AM

the mucus is no problem... it contains the bacteria..

Just use an "eye" scraper (a needle with a little circle at the top) to get the mucus that contains the bacteria.

So just scrape the mucus and add that to your pool or to a tube with fresh medium...

What you did, is not that good..
(did you allready scrape all the mucus and centrifuged it or? I do not really understand what you tried to do..)
The mucus contains tha bacteria.. so adding the mucus in a tube, centrifuging it and then getting rid of the mucus.. I dont get it.. Normally you need to let the bacteria grow in the media before centrifuging it and discarding the suppernatants..

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.

#3 Phil Geis

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Posted 18 August 2011 - 05:15 PM

I think no-idea was having trouble separating bacteria from their mucus. He might try a little NaEDTA - say 0.05%. Alot of jucus is polyalginate and sequestering Calcium will break it up. You should run controls to msee if the cells are killed but thag level isn't usually bactericidal.

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