Hello everyone,
Recently, I have encountered a problem in cell culture. I use Rpmi 1640. I have freezed SKBR-3 cells 8 months ago. I am defreezing them now. I defreezed two cryovials up to now and observed black sediments in both of them and approximately all of the cells died. Im wondering if they are contaminated or not (I uploaded its picture). Then, i put a flask containing complete meduim without cells in the incubator and 24 hrs later those sediments appeared and the meduim turned cloudy.
First of all, according to the picture I uploaded, I want to ask Are they debries or contamination?
And next, I'm going to use a new batch of meduim and Im going to defreeze another cryovial. What other precausions (except changing medium) do i have to take in order to stop probable contamination and loosing more cells?
Hello Sima Ravaei
If you are not sure that the culture is contaminated, just streak a few microlitres of the contaminated culture on the LB agar plate and incubate the plate at 37 degree C overnight. The next day if you see bacterial colonies, it confirms that your culture has been contaminated.
When you prepare a new batch of media, I hope you filter the media through 0.2-μm filter. Using 0.2-μm filtration which is referred to as 'sterile filtration', reflects the general belief that all living organisms are excluded from the filtrate.
The other precautions that you will have to take is to maintain sterility when you thaw a new cryovial. For instance, when you thaw the frozen vial, partially submerge it in a 37 °C water bath. A water bath is usually a shared device frequently accessed by many users to warm up different liquids like media, reagents, or frozen cells. The large water surface area of the water bath, it's warm and moist environment, as well as its frequent contact with unsterile vessels make it an ideal breeding ground for contaminants.
Another important step during thawing is to dry off the outside of the cryovial and wipe with 70% ethanol solution, carefully loosen and unseal the vial to prevent contamination.
Good Luck!
Hi, Sima Ravaei,
looking at the photo and to tell it is contamination is difficult. It looks like cell debris. Now that you have mentioned, the appearance of black particles and turbidity in the complete media alone, I suspect the media itself to be contaminated. You need to make a fresh batch of media and filter it using a 0.2-micron cellulose acetate or syringe filter.
I have two suggestions for your problem
1) Do not use a water bath for thawing the cryovials, I suggest hand-thawing them, once you are suited up.
2) It is ideal to clean the cell-culture incubator once in 15 days, as it would also be a source of contamination after repeated opening and closing during the work.
Hello Sima Ravaei,
I agree with both answers. Since the media turned cloudy the media is definitely contaminated. Other than the suggestions given I would also suggest to use new PBS and trypsin too, as they can also be contaminated from previous exposure.
All the best!
Thank you Kumudini Paliwal , Chriswin Saimon and Malcolm Nobre
Your suggestions helped me so much
Today, I opened a new batch of RPMI, serum and p/s and defreeze cells again in another incubator and hood. I hope I can solve the problem. Wish me luck!
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