I am beginner in mammalian cell culture, I am maintaining breast cancer cell line (t47d,mdamb231,mdamb468,mcf7).
Cells were going well but suddenly i observed so much cell debris and slightly early color change in media, I discarded flask in doubt of contamination but in newly revived flask also these dot like structure is visible, now I am confused that this is contamination or cells are in stress, i am attaching pics below.
Kavita Rana do you have Pen/Strep in your media?
If the media is getting exhausted before cells are confluent then it might be that you have contamination.
Also check for Mycoplasma contamination. Not all bacteria would turn the culture medium cloudy. I use Lonza's product for Myco detection but you can find more accurate PCR based assays easily.
Dear Dr Rana,
I am sorry to see your culture is not doing well.
I can see the problem you are facing. From your picture your cells do not look to be growing well.
I agree with Dr Gelincik, this is sound advice.
Use lots of 70% EtOH and UV light if you can, in your tissue culture hood.
Review your complete media, and expiration dates, like L-glutamine, and Pen/Strep.
Make new complete media with fresh pen/step and new cells from thaw.
Keep your data moving froward by not chasing the source of a contamination too much and focus on getting new ones up and running, by using good sterile methods.
This becomes more bother-sum if its pen strep resistant, or within the frozen source. I wouldn't not try and recover a contaminated culture.
If its cell stress..
Pre-warm all medium before use.
Try not to keep them out of the incubator too long.
I will take the flask I am using to place my new cells that are frozen, and put medium in it and place it into the incubator to let the CO2 equlibrate the media and warm too. Frozen cells into 37C bath and once cells are thaw just a little bit of ice left, I place them in the new flask, and wait about 4-8 hours for the live cells to attach, the replace the media with fresh media and let sit in the incubator overnight and check on them in the morning for out growth.
I know a lot of people like to spin them to remove DMSO in freezing medium once thaw... but even the i like to remove the media after some time to attach, just to remove any more trace amount of DMSO and also any left over dead cells.
Best Wishes
Thank you for advice..I have one more query that i have streptomycin and penicillin in powder form should i directly add powder in media or first i should make stock of antibiotic and then add 1% in media ??
I would make a stock.
something like... 10,000units/mL and 10,000ug/mL of pen/strep [100x], then aliquot and freeze in -20 for future use.
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