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Questions about overnight cultures


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#1 cspD

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Posted 30 November 2012 - 12:51 PM

Hi,

I have a few questions about overnight cultures. I have frozen stocks of the bacterial strians I work with in the -80. I've always been told to streak them on a plate first, then inoculate them into broth culture. I've also seen some people in my lab just inoculate broth culture straight from the frozen stock. Is there any disadvantage to doing this? I mean instead of spending 2 days setting up the culture, you have it ready the next day.

Also how long do you leave your plates/broth cultures in the incubator for? I've been told I'm leaving it in too long (~18 hours) and that this could cause problems.

Thanks

#2 bob1

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Posted 30 November 2012 - 01:57 PM

The way you have been doing it is the correct one, however the reasoning behind doing the culture straight from the glycerol stock is that it was made from a single clone in the first place, therefore it shouldn't need to be re-streaked.

For plasmids the optimium time is about 16 hours of overnight culture.

#3 cspD

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Posted 30 November 2012 - 03:05 PM

The way you have been doing it is the correct one, however the reasoning behind doing the culture straight from the glycerol stock is that it was made from a single clone in the first place, therefore it shouldn't need to be re-streaked.

For plasmids the optimium time is about 16 hours of overnight culture.


Yea that's what I was thinking. My freezer stocks were frozen down from liquid cultures from a single colony. So theoretically they should be from the same clone. So is it safe to just inoculate broth cultures from frozen stock? Also if I'm not doing plasmid work, is it alright to leave for 18 hrs? I ask because some people are really strict and say 16 hrs is the maximum

#4 John Forsberg

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Posted 30 November 2012 - 05:34 PM

Do you not have plasmids with antibiotic resistance markers in your bacteria? The main reason I've heard for keeping your cells from growing too long is to avoid cells without your plasmid from overtaking your cultures (as the antibiotics will be progressively degraded over time). I suppose even bacteria without plasmids will get further into stationary phase (with many more cells that are dead) the longer you incubate them as well.

#5 cspD

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Posted 01 December 2012 - 05:48 PM

Do you not have plasmids with antibiotic resistance markers in your bacteria? The main reason I've heard for keeping your cells from growing too long is to avoid cells without your plasmid from overtaking your cultures (as the antibiotics will be progressively degraded over time). I suppose even bacteria without plasmids will get further into stationary phase (with many more cells that are dead) the longer you incubate them as well.


No my mutants have a transposon inserted into the genome in the particular gene. So I wont run into this problem.

#6 John Forsberg

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Posted 01 December 2012 - 06:31 PM

No my mutants have a transposon inserted into the genome in the particular gene. So I wont run into this problem.


How do you select for your cells over contaminants? I'm not familiar with the techniques you're referring to. It looks like I'm out of my depth here, so I can't really say whether going directly from a glycerol stock would be okay with your system.

#7 cspD

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Posted 01 December 2012 - 08:03 PM


No my mutants have a transposon inserted into the genome in the particular gene. So I wont run into this problem.


How do you select for your cells over contaminants? I'm not familiar with the techniques you're referring to. It looks like I'm out of my depth here, so I can't really say whether going directly from a glycerol stock would be okay with your system.


When I first made my stocks I streaked my mutants on plates w/ gentamicin, then inoculated into broth which I froze down. I don't use antibiotics in the broth when I am sub culturing for an experiment. Contamination never really has been an issue, besides for the wild type, there is no resistance marker anyway.

#8 John Forsberg

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Posted 01 December 2012 - 08:57 PM

In that case, I'd probably restreak, make sure the colonies looked normal, then grow up from there. Even if you froze them down in media that originally had gentamicin (which will have degraded as the culture grew), I wouldn't rely on it being so clean that you won't get contamination when subculturing without antibiotics. That sounds like a solid case for picking a colony for an experiment. I realize that when using sterile technique people don't normally get contamination of their stocks, but you really (really) don't want that happening in an actual experiment. Sometimes waiting a day is worth it. If you were using antibiotics in your subcultures, then I'd say to not bother with the streaking.




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