how to settle the FBS contamination - (Nov/24/2008 )
hello everyone ,
I am a fresher of this forum, and it's my first time to post a topic here. A little nerves...
May someone concern my topic, thanks very much.
I have a problem recently that I guess my fetal bovine serum may contaminate some bacterias.
It was because we aliquoted the thawed FBS to BD Falcon™ 50-mL polypropylene conical tubes,
then put them back to -80 centigrade refrigeratory. And a few days latter, we found the tubes cracked.
. It's dangerous, it must have to contaminate when thawing to use. I don't want to throw them for it's
waste of money, so i think i have to do something to them. After thawing, i filtered them through 0.22uM
filter. But soon i found it's hard to do so. The particles in FBS may block the filter. So i thought i must
get rid of the particles at first. The first thing i could think about is centrifuge. Then i spin one 50mL tube
to test. After 1000g 10min spin, i get the supernatant to the filter, but it made no difference----hard to get
through the filter. It makes me confused and so disappoint.
Could someone help me? Or some advise? Or maybe i should throw them?
I am a fresher of this forum, and it's my first time to post a topic here. A little nerves...
May someone concern my topic, thanks very much.
I have a problem recently that I guess my fetal bovine serum may contaminate some bacterias.
It was because we aliquoted the thawed FBS to BD Falcon™ 50-mL polypropylene conical tubes,
then put them back to -80 centigrade refrigeratory. And a few days latter, we found the tubes cracked.
. It's dangerous, it must have to contaminate when thawing to use. I don't want to throw them for it's waste of money, so i think i have to do something to them. After thawing, i filtered them through 0.22uM
filter. But soon i found it's hard to do so. The particles in FBS may block the filter. So i thought i must
get rid of the particles at first. The first thing i could think about is centrifuge. Then i spin one 50mL tube
to test. After 1000g 10min spin, i get the supernatant to the filter, but it made no difference----hard to get
through the filter. It makes me confused and so disappoint.
Could someone help me? Or some advise? Or maybe i should throw them?
Is this a serious question?
Ofcourse you could have added the contaminated serum to your medium and then pass it through a .22uM filter. That goes very well.
However, looking at the cost, throwing this serumtube away would have been far the cheapest. How much time, filters etc have you been wasting by now? And what about the cost of a whole experiment if you get an infection?
It was because we aliquoted the thawed FBS to BD Falcon™ 50-mL polypropylene conical tubes,
then put them back to -80 centigrade refrigeratory. And a few days latter, we found the tubes cracked.
Do you put 50ml of FBS in the 50ml tube?
When FBS freezes, it expands and cracks the tubes.
I think it is better to fill the 50ml tube with only 45ml of FBS, and allow room for the solution to expand.
I agree that you shouldn't fill 50mL conicals all the way. We do this for both FBS and trypsin. Personally, I just add the 45mL to the media and don't worry about my media being more of 9% rather than 10%. I just don't see how that little bit will make a difference. Especially since my cells are always cultured in the same percentage and all my results are repeatable. However, if you happen to be the type of person like one of my colleagues, you can thaw another tube and add the extra 5mL. A bit overboard if you ask me, but to each their own. Thaw all the FBS in the hood, re-aliquot into 45mL and put a big note to remind everyone that they must filter the media when they add this FBS.
You can use a 100ml sterile Scott bottle for freezing 100ml of FBS.
No, i think you misunderstand me. I just filled 45mL FBS in a 50mL conical, but it cracked at the bottom. I don't know why, and how to deel with it.
No, i think you misunderstand me. I just filled 45mL FBS in a 50mL conical, but it cracked at the bottom. I don't know why, and how to deel with it.
may be due to manufacture fault ie. there was already a small crack before you use it.
No, i think you misunderstand me. I just filled 45mL FBS in a 50mL conical, but it cracked at the bottom. I don't know why, and how to deel with it.
may be due to manufacture fault ie. there was already a small crack before you use it.
Thanks for so much concern.
But may you give some advise about settling the problem? To get rid of the precipitate in FBS, you may wish to use a filter paper.
Then you can filter it with a 0.22uM filter.
Then you can filter it with a 0.22uM filter.
Maybe you are right in a way.
However i don't agree to do that, i don't know how much nutrient will lose after doing that.
And if it can be used to culture cells still leave to be tested.