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Issue with freezing and subculturing


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#1 Ryan Ho

Ryan Ho

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Posted 12 December 2011 - 01:44 PM

Hi there,

I have a question that pertains to culture storage. I'm working with Pseudomonas aeruginosa PA14 and a couple mutants. I was given the PA14 and the mutants and I stored them at -86 degrees Celsius. To do experiments I always take out the cryo tube on ice, put a pipette tip, swipe a bunch of frozen culture and streak on an LB plate. Then from the LB plate I inoculate into whatever broth is needed. I was using the frozen cultures a lot, and was actually running a little low at one point. So I subcultured by streaking on LB, inoculated into LB broth and froze some more with DMSO. So the original cultures of the PA14 + mutants which I shall call A, and the newly made cultures which I froze down which I shall call B have big differences between them which makes absolutely no sense to me.

They should be the same, however in experiments the behave differently. For example with congo red binding on plates, the culture A showed distinct congo red binding phenotypes. However when I redid the experiment for another biological repeat using culture B, there was absolutely no differences between them. This also holds true for other phenotypes such as swarming. With culture A the swarming is normal, however with culture B they just swarm so quickly they fill up the entire plate.

I have no idea how to account for this seeing as how culture A and B are pretty much the same, just that culture B has been subcultured and frozen once again. Furthermore where do I go from here? How do I know which is the "correct" phenotype? Can someone tell me the correct way to store strains? I was thinking of making a "master" stock of the mutants + PA14 and from this I would make a bunch of working stocks.




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