Hi Srijan Acharya.

     These are some of the reasons for your frequent contamination

Reuse your gloves and lab coat - not washed for long time,

Not wiping all the utilities with ethanol after thawing/ taken out from water bath -water bath contamination is major cause of cell culture contamination - these bacteria will grow slowly only visible if you continuously culturing the cells for longer time. Wipe the neck part of the bottle with ethanol where chance of contamination is more possible.

Reusing the plastic wares which may previously contaminated 

Not cleaning your bench/ hood and UV sterilization them before you start your work.

Touching the mouth part of your flask and media bottle.

Chance of your pipette touching external surface with your conscious.

Cell may be contaminated early when repeatedly using with antibiotic media which allows the microbes to raise tolerance.

Try to follow above things and sure you will get better results

wipe your hands before and after wearing gloves and touching the surface/ materials with ethanol 

All the best

Regards

Sundarajan K

Hello,

seems it is a microbial contamination. Do you have a possible for check this?

 Hi Srijan Acharya.

     These are some of the reasons for your frequent contamination

Reuse your gloves and lab coat - not washed for long time,

Not wiping all the utilities with ethanol after thawing/ taken out from water bath -water bath contamination is major cause of cell culture contamination - these bacteria will grow slowly only visible if you continuously culturing the cells for longer time. Wipe the neck part of the bottle with ethanol where chance of contamination is more possible.

Reusing the plastic wares which may previously contaminated 

Not cleaning your bench/ hood and UV sterilization them before you start your work.

Touching the mouth part of your flask and media bottle.

Chance of your pipette touching external surface with your conscious.

Cell may be contaminated early when repeatedly using with antibiotic media which allows the microbes to raise tolerance.

Try to follow above things and sure you will get better results

wipe your hands before and after wearing gloves and touching the surface/ materials with ethanol 

All the best

Regards

Sundarajan K

Sundarajan gives good advice - and you should also bare in mind that once you have a microbial infection, it can often persist for a couple of weeks before all traces have entirely cleared. One of our undergraduates contaminated a cell line when they started last summer, which spread quickly and was persistent for around 10 days,

It might be best (if you can afford the time) to wait a week before re-establishing your cultures. In this time, clean your incubator daily with ethanol, ensure aqua-resist is used in incubator water, and make fresh sterile reagents. Then pull up a new vial. This should eliminate the problem. 

Hope this helps!

Brittany

Dear Srijan Acharya 

I think it might be carryover contamination. because if we freeze contaminated cell line it will carryover whenever you revive.

so please check and revive new one.

prabu

It sounds to me like your frozen stock is contaminated.  When you thaw out a new vial have you tried a different freeze date?  If you don't have multiple stocks that have been frozen at different times, you may need to find someone to get a new stock from.  Also, test all your liquids (media, PBS, trypsin, etc.) for contamination by putting each in a flask for a week in the incubator to see if anything grows.

Kerry

Hello,

If you had a microbial contamination you could try to isolate these bacteria and evaluate of it drug sensitivity. After then you could try to use the antibiotics (to which bacteria was sensitive) for eliminate of contamination

hi!!

After all above answers, I just want to add one more thing that, change your medium bottle. I had this too last year and finally when I changed medium bottle, it was gone. Although, I just opened new one. 

Another thing is that now you have contamination, clean all the things in cell culture using uv's, ethanol, autoclaving etc. and then start fresh otherwise contamination will be continued.

Also it is better to have microbial contamination test from kits.

Hi, I´ll try too keep a sample of my medium outside the freezer 12 h at least, to see if it´s contaminated, as other answers is good to clean with ethanol the neck of the bottles and also the cap of your bottles of medium, personally I like to pass a lighter briefly on the neck of the bottles and caps

If you want to more systematically cross-check for & exclude any possibilities I´d recommend to (once again) read chapters 5 (Aseptic techniques) and 16 (Contamination) in R. Ian Freshney´s excellent manual of basic cell culture techniques. It´s called: culture of animal cells and I´m referring to the 3rd edition published "last century" i.e. 1994 by Wiley-Liss

I think it is better to use antibiotic anti mycotic  and before opening or closing any bottles especially freezing vials expose the openings to a flame

Hi, can you clearly describe the morphology of those black spots? I suspect those may cell debris because of mycoplasma or other parasite contamination which kill cells. If its bacteria, you could see those black spots moving when you observe under microscope and when they are not, then they may be cell debris. This occurs mainly due to handling. Since you are getting this again even after sterilizing all the required things for cell culture, as Prabhu and Kerry said, the thawed stock may be contaminated and those particles may get active when you revive back. I suggest you to obtain new culture because it is a carryover contamination and try doing fumigation. I had a similar problem and I restarted with a newly purchased running culture after fumigation. Hope this helps

It looks that your frozen stock is contaminated, in this case you don't have choice and you need to buy a new cell line. In case the stock is not contaminated, please check your media, PBS, trypsin and in addition it will be better to change the entire bottle. Good luck