For the past several months, I've been having trouble in getting contamination from thawed Sf9 cells. For the cells that's frozen in 2014 by other people, I have no problem thawing and growing them. However, any cells that I froze recently, when I thawed them, they first grow super slow. Then at the 3rd day, I saw the media turning really yellow and thick and my cells all died and from the microscope I saw black dots which I think is bacteria. However, when I split the cells for freezing, I also left out an aliquot (it's right before I put the resuspended cells into freezing vial) to directly seed in a flask and that grows fine. All my other Sf9 cells are fine; this weird thing only happened to my freeze-thawed cells.
My protocol to freeze Sf9: I grow the cells to a density of 8 million/mL in 100mL. The media I used to freeze Sf9 is 90% ESF+10% DMSO. I spinned down the cell at 200g for 5 min. I aspirate the media and resuspend the cells in ESF+DMSO media. The density of the cells resuspended is 40million/mL. I put the cells in a freeze controller box and put it in -80C overnight. The next day, I transferred the tubes into liquid nitrogen. When I thawed the cells, I added 1.25mL of the cells(50million) in 50mL of media in a 125mL flask.
Can anyone give me some advice on it? I also tried to add pen-strep and anti-micotic mixture, I don't see bacteria contamination this time, but I see the media turning slightly red and sticky... It's too gross that I don't want to look at them...
Hi Lan, I would toss out your freezing media . Do you thaw our the cell vials in a waterbath?, then toss out the water, clean the bath with 70% isopropanol, add new DI water with 0.05M EDTA.
Check the tubes you are using. They should be sterile Cryotubes with a screw cap (and sometimes an O-ring)
Spray them liberally with 70% isopropanol before putting them in the hood and spray the incubator thoroughly with isopropanol before putting any more cells in.
Hi Steingrimur, thanks for the advice. However, when I thawed my other mammalian cells in the same water bath, they are fine. When I thawed insect cells frozen by other guys 3 years ago, they are fine. And for my cells, the one that's not frozen but has the same freezing media, they are fine. The cryotubes are sterile, our entire lab use them.
Hi Lan,
It sounds like it is possible that you have introduced a microbial contaminant at some point. I would not assume that black dots that you see are bacteria unless you see directional motility (not Brownian motion) or the dots are forming chains, Staph-like clusters, tetrads, etc. and are uniform in size. As cells die, the lipid membrane breaks apart and forms little balls that appear black and are often confused with bacteria (some species of bacteria look similar). If you do have bacterial contamination, there are a couple of options, including getting rid of contaminated stocks and the source of the contamination as well as possibly treating the cells with a targeted antibiotic. My lab does a lot of sterility testing for cell cultures, and we often trace bacterial contamination to liquid reagents, water baths, and human skin microbiota. It is helpful to identify the contaminant, not only in the event that you want to treat the contaminated cells, but also in order to trace the likely source of contamination--to prevent recontamination later. My recommendation would be to try to culture the media on a wide array of permissive culture media. If you get bacterial or fungal growth, isolates can be identified by MALDI-TOF mass spectrometry or genetic sequence analysis. Considering the changes that you are seeing, if it is a contaminating organism, a direct inoculation protocol should be sufficient (see http://www.idexxbioresearch.com/sterility-testing-pricing). Good luck!
Marcus
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