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Plating dilutions of lysates


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#1 repeatcell

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Posted 29 October 2009 - 08:05 AM

Hi!

I have real problems with dilutions of the bacteria. First, I make the lysates either in pure water (at 37 degrees for few minutes) or in 0.1% TX-100 in PBS. Then I store them in 96 well plates O/N. Next day, I make dilutions of these bacteria in PBS like 0.1, 0.01 and 0.001 and so on. However, when the plates are ready to count, I have problems like bacteria in 0.01 and 0.001 do not correspond to each other. Many times they are very close to each other and not exactly different by 10 times. Can anyone guide me how to get the perfect dilutions.

Thank you

#2 pito

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Posted 21 November 2009 - 10:01 AM

Hi!

I have real problems with dilutions of the bacteria. First, I make the lysates either in pure water (at 37 degrees for few minutes) or in 0.1% TX-100 in PBS. Then I store them in 96 well plates O/N. Next day, I make dilutions of these bacteria in PBS like 0.1, 0.01 and 0.001 and so on. However, when the plates are ready to count, I have problems like bacteria in 0.01 and 0.001 do not correspond to each other. Many times they are very close to each other and not exactly different by 10 times. Can anyone guide me how to get the perfect dilutions.

Thank you


How big are the differences?

its normal that its not "exactly" 10 times less or more. Its not an "exact" science, you cant be 100% accurate.

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


#3 HomeBrew

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Posted 21 November 2009 - 11:01 AM

Lysates of what? Are you freeing bacteria from eukaryotic cells? If so, releasing bacteria into just water or into PBS with a detergent is not going to be helpful to their viability, especially if the overnight incubation is in an incubator...

However, protocol aside, you must be sure that the bacteria are well suspended -- if they're aggregative, you're going to have sampling problems. Each aliquot must be well suspended and plated with a fresh tip. You should also be plating each dilution in duplicate (at least) to increase accuracy.

How far off are your dilution plates from on another? Are we talking orders of magnitude here?




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