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Is it possible to know the live bacteria using PCR for a certain genus?


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#1 Abdelsalam Elawwad

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Posted 06 November 2010 - 01:28 PM

Hi all

I am doing starvation experiment for nitrifying bacteria and I want to show only the live bacteria for nitrobacter and nitrosospira for example. But the normal PCR will amplify the live and the dead bacteria DNA. Any suggestions?

Thanks in advance

Abdelsalam


#2 bob1

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Posted 06 November 2010 - 02:19 PM

PCR will amplify any DNA that is present, it will not matter if it is from live or dead cells.

#3 Abdelsalam Elawwad

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Posted 06 November 2010 - 03:38 PM

yes I know that . This because I need a method to see the only live cells for certain species. I am using T-RFLP based on PCR. So I need to know if it possible to modify maybe the method to get the DNA from the live cells only

#4 perneseblue

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Posted 06 November 2010 - 08:00 PM

Hmm...

Right now the only idea I have is to use a stain to differentiate life cells from dead. You can then use a cell sorter to separate the dead cells from the live ones.

Here is the title of a paper that is of interest
Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells.
May your PCR products be long, your protocols short and your boss on holiday

#5 Abdelsalam Elawwad

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Posted 07 November 2010 - 02:06 PM

Hmm...

Right now the only idea I have is to use a stain to differentiate life cells from dead. You can then use a cell sorter to separate the dead cells from the live ones.

Here is the title of a paper that is of interest
Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells.


Thank you. It seems a good idea. But It didn't be used it with nitrifying bacteria before and also there is some concerns about the staining for the live cells also!!!.

I am doing the sampling and preserve the bacteria at -40 for some months before analyzing. Is it ok to stain the bacteria after preservation or it should be done just after the sampling!!

Edited by Abdelsalam Elawwad, 07 November 2010 - 03:06 PM.


#6 perneseblue

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Posted 08 November 2010 - 06:21 PM

If you freeze the cells, many bacteria will die. I am not sure, how your experiment is structured. Is this an experiment to localise where live and dead bacteria were in a tissue sample? If that is the case, you have to stain the cells immediately. Then fix the stain and freeze the sample for later analysis. You probably can keep the tissue sample cool to better preserve the sample and give you more time to get from field/green house to the lab bench..

If this is an experiment to find out how well nitrification bacteria survive freezing.. then staining after freezing/thaw will be the way to go.

As for the idea of staining working in your bacteria species... the only thing I can say is give it a try. It is an experiment after all.

And read up the literature, surely somebody did an experiment similar enough to give you a few pointers on how to proceed.
May your PCR products be long, your protocols short and your boss on holiday




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