2 weeks ago I started an experiment with siRNAs to inhibit P21 expresion. The protocol needs the use of medium without Antibiotics. So, first I grew my cells (LnCap) in medium RPMI complete with antibiotics, everything was ok (Picture 1) (I was so happy)...the day of the experiment, I passed the cells to a 24 well plate and I changed the medium for a RPMI without antibiotic, I worked like always in a cabinet, with all the necessary safety. The next day... was the worse day... I found all my plates contaminated with bacteria (I wasn't able to take a picture, I was in shock).
Next day I incubated all the reactives and media that I used for the experiment, alone and in combination. The wells with RPMI, OPTIMEM, PBS 1X, RPMI with antibiotics, were clean! but the FBS alone and all the combinations with the others reactives were contaminated (Picture 2, FBS alone). So I discard the FBS, and I opened a new one... but in order to be more secure, I decided to incubate an aliquote (37°C)... what was my surprise... The sample was contaminated again! I don't understand what happend, I was really worry about that... So I tried with a new 3rd bottle, and It was the same.
The bottles are stored at -80°C, are from the same lot...but...they expired in 2020....this could be the problem? but they were at -80°C...so I really don't understand.
Some ideas??
Just for the sake of accuracy, how certain are you that this is bacterial contamination, and not (for example) precipitated serum proteins?
Does it progress over time? Does it turn the media cloudy, or is there just a visible precipitate? Do the particles move around rapidly (bacteria usually do, precipitated protein filaments really don't).
If you take the FBS and spin it (like, 1000g, 20mins) what collects at the bottom? If you take the uppermost fraction of the supernatant and incubate _that_ overnight, does it form near identical contamination, or not so much?
It's always formally possible that the entire batch of FBS is indeed contaminated with bacteria or fungus, but it's more likely (to my mind) that this specific batch is just more prone to protein precipitation.
Horse serum, for example, is far more prone to this sort of thing, and there you can sometimes see broad sheets of precipitated protein drifting by.
If it's microbial contamination, the particles very likely will move randomly, the phenol red indicator will turn yellow and the culture become really (visibly) turbid after some time and you even might note a strange smell.
As John Hildyard suggested, there might be just precipitates, maybe fibrin clots. To remove them, you may filter the serum through a 0.45um bottle top filter, if possible, use gravity flow over night (serum has a tendency to clog the membrane when using suction) and keep it in the fridge.
Regarding usability, the passed expiry date should not pose any problem, as you have stored it at -80. You just can't hold the manufacturer responsible anymore. If it should be contamination indeed, talk nicely to them in order to get a free replacement, as they might be interested in keeping their customers (happy).
Thanks for your reply Jhon Hildyard,
Yes, the media turned cloudy and the "particles" were moving rapidly. Actually, I have a vídeo, this particles are bacili.
I haven't tried to do the spin. But I could do it.
Lastnight I put another aliquotr from another bottle, but it is other batch and expires in 2024. I am going to tell you the results.
Thanks!!
@Wolfgang Schechinger thank You!
I agree with you about the expiry day, it wouldn't be any problem.
Regarding to the particles, all this particles were moving rapidly, and the media turned turbid and some yellow. The FBS alone, was turbid too. I don't know if it's possible ask for a replacement, because this lot was purchased in 2018 when I wasn't in this lab.
I am going to the lab in this moment...so I Will tell you what happens with the new batch.
Thanks
It does sound like an infection. And it also sounds like the FBS is the source: you've done pretty thorough detective work!
At this point, I would throw all of that batch away: you'll waste more time and money checking each bottle than you would just writing it off and buying more. It's annoying, but sometimes...that's science. You can, as Wolfgang Schechinger notes, contact the supplier for possible freebies.
I'd also clean all the hoods and incubators you've used, use fresh bottles of media etc (ideally use a separate TC room for this 'no-antibiotics' experiment).
Essentially you need to do the TC equivalent of "take off, nuke it from orbit".
It's the only way to be sure.
Hi dear John and Wolfgang!
yesterday I put two plates (from the new batch) in different incubators and guess what! both are clean, without bacteria, transparent, perfect. I think we can conclude that it was that batch of FBS. I feel better. Thank you both for the excellent comments and suggestions.
Hi Emily Toscano Guerra
As John suggested, Just discard that particular FBS bottle. Once we faced the same vibrating, long bacterial contamination. Before coming to the decision of throwing that FBS, we tried other options, such as filtering the media, cleaning the hood, sterilizing the incubators, etc. but this contamination was not going. We filtered the FBS. We tried cocktails of antibiotics to get rid of bacteria but no use. But, every day we lost cultured cells. The moment we changed the FBS, immediately, everything became fine. FBS is the only culprit here.
Good luck!
Thanks Saurabh! Yes I have discard all the bottles from that lot batch. I am working now with the new Lot.
Thanks!
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