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A question regarding contamination please help - (Mar/30/2012 )

Dear Guys.
I am culturing my cells without Antibiotics.
I have been doing since December 2011, I have some problems, but not true contamination, since I am new in cell culture and I have no previous experience or any one to help me.

Any way.
I have seen bacterial infection on my plate.
I have throw all things
clean the incubator.
Autoclave my electric pippetteing tips and 0.2 um filter.

And culture my cells from my stock for 9 day, and it was ok, no infection, again, very health and viability is very fine.

Yesterday, I replaced the medium in one of my flasks, Today it has the same bacterial contamination?????
I dont use aspirator, only disposable pippettes to added and aspirate the medium and saline.
I have blank plate on my incubator that contains medium only, and it was fine too no infection.
But Yesterday, one of my pippites has no plug (this white plug on the top of the disposable pipettes).
I didnt notice untill I was already using it
I am using this type

Any suggestion that my help me figure where is my problem????
if I added Antibiotics to my medium and culture my cells again, it will prevent this infection to reappear???

I want to restart another cell lines, but I cant as I have no stock from it,
we bought it recently, and I can`t risk the appearance of infection on this cell line.

finally, I am very grateful for those who is willing to share their experience



There are lots of things that can cause infection, and yes, pipetting without a 'plug' or filter can be one of them. As can working too close to the edge of the hood, keeping the dish open too long allowing bacteris to fall from your arm/labcoat etc into it.

In regards to you saying you have no stock and you cannot risk infection, you should make your own stocks right away, then if you do get another infection in the future you can start again.

Grow lots of cells and freeze some of them down in freezing media (10-20% DMSO in FCS) at around 10^6 cells/vial




Thanks in advance for sharing your thoughts with me.
I meant I have one vail of these cells, so I want to culture it in safe environment as if I got infection during the first plating I cant make stock.
So I need at least stable cell culture condition so I can make the required stock.

Another question, if I am getting my cells from stock cultured it in 6 mm plate, can I take 1 ml from this plate and make stock from these cells which are in stock from couple of days( I know it is not the same cells as they are constantly divide).
Thanks again