Seeking advice Regarding contamination - (Feb/10/2012 )

Dear Guys ,

At first I am very gratef ul for your comments and advices.
I have no one except you to seek for your priceless help.
I need your advice in two issues:

1) I am in a middle of experiment using keratinocyte from ATCC, I cultured it firstly with Antibiotic then without antibiotic.
it was fine and I was doing my experiment on 24 well plate, it takes around 4 days.
the first, second and thirds day was ok, on the third day I change the medium and add new medium, and on the fourth day I saw something like sever white turbidity in 17 wells out of 24, and I cant see my cells, they are gone, not attached to the surface of my plate, nothing only in 24 hrs.
I have added the new medium using the same pippte and the same tip for all wells, and the reagents for my experiment too.
The most strange things is in another cultured 98 well plate (3 out of 98) showed the same sever turbidity, this was cultured in another day.
the rest of my plate was fine too, no turbidity and cells looks fine under microscope.
So I have took some photo using 100 X (oil immersion) as these intruders are very tiny and cant be clearly seen under 40X.


2) I am using new FBS, on culturing another type of cells (No Antibiotics too), and what I noticed that there are a lot of things floating in my medium under microscope, but not turbidity which could be seen by naked eye, I dont know if they are cell debris or debris from my new FBS itself.
so my question, to identify contamination what shall I expect ( I am not using antibiotics),
Sever turbidity??? or slight one.
Contamination occupy all my plate or minor one??
death of my cells and the first issue ( but my cells are 97% viable using trypan blue.)
I have added a photo from American National Cancer institute which describes what you can expect in contamination, A is non contaminated cultured cells, so shall I expect something like that and my cells be normal.



Thank you guys in Advance,
Best Regards

It sounds like you have a bacterial contamination - yeast should be visible with a 20x lens and forms the classic budding structure. Bacteria are tiny and often appear as a cloudy looking mix under the microscope, without being easily able to distinguish cells. The contamination sounds like it is probably either in your cell stocks or in your medium.

Bacteria are very easy to spread - it will be best to make sure that you do not open the flasks/plates, just throw them in the biohazard waste. You should throw out any open solutions (medium, trypsin, glutamine, antibiotics (if you are using them), drug diutions etc (if they are very precious, you may be able to filter them)). You should clean down any thing that has potentially come into contact with the bacteria, such as pipettors, tips, etc. Change your lab coat and maybe clean out waterbaths too. You will also need to thaw a fresh tube of cells and examine them carefully for bacteria.

If you are new to cell culture, get someone experienced to observe you and help with/critique your sterile technique.

FBS often has debris floating in it, these usually look like small bits of fine cellophane and will not proliferate. There are quite a few threads on here about contamination and FBS debris comes up quite often - have a quick search of and you will see some pictures.

Dear
bob1

,
I am very grateful for your reply and priceless help
Best Regards