9 replies to this topic

[9939]

One of my techs did some little mistakes while preparing ampicillin stock (50mg/ml). instead of aliquoting into small volume (we usually stored 0.5ml in -20), 5ml were aliquoted and all are now in -20oC.

However, we usually only need a couple microlitre of this ampicillin stock to make LB amp plates or broth. Thus there will be couple of ml of ampicillin stock left over =(
My question is - is repeat thawing and freezing of antibiotics affect its stability and its performance in agar plate or culture broth. i was wondering if i can freeze the remaining ampicillin and use them again next time, only that i'm not sure if this degrade or affect the antibiotic in any sense...

any suggestion?

[fishdoc]

wia84, on Jul 10 2009, 12:15 PM, said:

One of my techs did some little mistakes while preparing ampicillin stock (50mg/ml). instead of aliquoting into small volume (we usually stored 0.5ml in -20), 5ml were aliquoted and all are now in -20oC.

However, we usually only need a couple microlitre of this ampicillin stock to make LB amp plates or broth. Thus there will be couple of ml of ampicillin stock left over =(
My question is - is repeat thawing and freezing of antibiotics affect its stability and its performance in agar plate or culture broth. i was wondering if i can freeze the remaining ampicillin and use them again next time, only that i'm not sure if this degrade or affect the antibiotic in any sense...

any suggestion?

Thaw the 5 ml and re-aliquot at 500 ul. Basically that only adds one freeze/thaw cycle to that batch of antibiotics.

[phage434]

Or you can dissolve ampicillin in 50% ethanol and keep the solution liquid at -20C.

[GeorgeWolff]

Do you sufficiently control your work to know if there's signficant loss of activity?

[phage434]

We use 100 ug/ml final, so I probably would not notice a 2-4x reduction in activity, but we find it stable for at least a year.

[fishdoc]

GeorgeWolff, on Jul 11 2009, 07:00 AM, said:

Do you sufficiently control your work to know if there's signficant loss of activity?

We don't. We use 200 ug/ml for most cultures, though. At least for amp.

[GeorgeWolff]

So you run no "growth promotion" controls - to ensure bugs that should grow do and those you want to inhibit do not grow?

[phage434]

Yes, we run those negative growth controls, although not every time.

[fishdoc]

GeorgeWolff, on Jul 11 2009, 11:07 AM, said:

So you run no "growth promotion" controls - to ensure bugs that should grow do and those you want to inhibit do not grow?

Nope. At least I don't. Not sure if someone else in our lab does or not. To my knowledge, it's never been an issue.

But for the most part, we're not using this media in experimental designs, but rather for selection of clones or following conjugations. You can tell pretty quickly whether or not the antibiotic is working based on how much you have growing.

In the event selective media is used for an actual experiment needing statistical analysis, I'm quite sure we'd run the proper negatives on everything, but we don't do that sort of thing too often.

Edited by fishdoc, 11 July 2009 - 12:55 PM.

[molgen]

In the lab where I did my masters we used to rethaw the ampicilin at least 10 times without a problem.
We only used to change the vile when we'd start to get satellite colonies.
I don't do that now a days but it can be dun.