Contamination issue in cell culture - (Nov/16/2004 )
yes, U can disinfect the incubator with formaldehyde 10 - 15 % for 30 min.
the best method of effective fumigation method for incubators is......
set the temp of incubator at 80 *C and humidity to 95 % then place the 15% formaldehyde container in incubator for 30 min.
Humidified high temp with formaldehyde work better and best method of sterilizing the incubator.
good luck..
Hey everyone,
So I've been having the same problem and am desperately trying to figure it out. Not all cell lines in our incubator are affected. The ones on the top shelf (primary neurons and HEK 293A cells are fine), the ones on the middle/bottm shelf (HEK293FT, MES23.5, and PC12 cells) all look speckly at low magnification. The FT and PC12 cells keep dying when they start getting about 60 - 70% confluent. At high magnification I can see tiny dots swarming around like crazy and in some cases the dots are all linked together. When I thaw out new cells they are fine for a passage or two and then all of a sudden they are dead/or contaminated. I'm not sure if it's the water bath, the FBS, or what.... anyone have any suggestions? Does anyone know what this contamination is? I'm growing tired of these speckly little guys and would love for them to just leave!
Very Frustrated Ph.D. student 
when you open your individual package, do not tear the package with the pipette, but pull the two folders where it is written, unless you might touch the outside of the torn paper with the extremity of your pipette while pulling it outside (do you see what I mean?)
Do not lay down the bottles when you store them. no liquid should touch the cover (the cork).
when you work under the hood, wash your hand with detergent and then with alcool.
Keep in mind that gloves are not sterile, it's only to protect you.
always let a the lid on the bottle. Do not let an open bottle under the hood. you don't need to screw it each time, just let it on the top.
do not work too close to the opening. you must let around 10 cm to the opening.
if you need to pipet small volumes with a tip, never do it in a bottle, you have to transfer to a smaller volume so that the micropipette never enter the bottle.
clean all what enter under the hood with ethanol.
avoid to pipet too quickly because you would make some aerosols, that could contain contaminating particles.
Now throw away all your medium, antibiotic, serum aliquotes, take fresh cells and try again.
are you the only one that has contamination? maybe the incubator should be cleaned also.
there are some products to clean it (some are even autoclavable), you can autoclave the shelves.
Clean the hood also, especially under the surface where you work.
you should not keep contaminated cells in the incubator, but over confluent cells are not contaminated and this will not cause contamination.
do you work with petri dishes of flasks?
with petri dishes you will have more easily contaminations, especially if the incubator is over crowded, because persons caring less than you could move your plates to get their plates, and if you are not carefull (no hand wash...) you can split some medium to the opening, and contaminate the medium.
OK good luck
HI...
well, it's a great advice....
however, now, i'm facing a terrible "contamination" (i dont know it's a contamination or not)
i have working in cell culture technique for one year, and never facing situation like this before...
i worked on ROS 17/2.8 and gingival fibroblast cells, and i cultured them on flask
i always turn on the UV light and clean the bench using alcohol. i wore gloves cleaned by alcohol, and tried my best to keep my hands away from any bottles, or pipettes.
two days ago, i did routine subculture procedure. but, today, i found out that the flasks were turbid (all flasks), and the color didnt change (red). i use light microscopes to see the cells, but all the cells seems to detached from the surfaces, and just float in the medium. it happens to either ROS and GF cells. it's totally frustating. My medium in the bottle were fine
it happens only on my flasks, the other flasks inside the incubator were fine. When i did subculture, or change medium, there were some medium left in the flask opening, could it cause contamination? or, is it because my flaks is overconfluent ? is there any steps i need to give attention to (to prevent this to happen again) ?
Actually, in my lab, there are no other skilled technician, post doc, or ph D students working on cell culture.. so i dont know what to do, and what steps wrong in my procedures.....
need suggestion please.. any kind of advice would be usefull...
thanks in advance
delimartin
Hi, I'm another victim of this weird contamination, I don't work with primary cultures though 
.l Mine are MA-104 clone cells infected with the parasite Neospora, and since we obtain the isolate from mice to further adapt them to in vitro culture I guess some sort of contamination is coming out of this animal model. At the beginning we didn't figure we had this contamination because we usually work with medium containing Pen-Strept-Fungizone, but now we try to keep cultures without antibiotics and we have started to see at this tiny "bugs" with irregular shape, either alone or glued each other and sometimes adhered to the cells. 
At first, cultures look fine and medium has the appropriate pH but suddenly they raise their number and pH drops to 5.5 (amazing yellowish medium), so I must get rid of flasks before contamination affects the rest of what we keep on the incubator. We have run several tests (agar and brot culture, PCR for mycoplasma detection) and nothing shows up. If someone looks at the video and photo we are attaching, I would appreciate any advice you can give. Thanks!!! 
I too have the same issue as Delimartin!
I have been doing tissue culture for over a year and suddenly I am getting flasks with lots of cells floating in them 2-3 days after passaging (I am using LS 180 adherent cells). The floating cells look alive, which I confirmed using trypan blue exclusion. The media I used doesn't seem contaminated. The other flasks and plates in the same incubator at fine. In all of the flasks there are still a number of cells that have attached to the bottom, it's just that most are not. The media I'm using is the same as always DMEM with 10% FCS and pen/strep.
Any advice would be welcome as no one else in my lab has any idea!
Thanks
Have you changed supplyer of medium or any such things? Is the medium suitable for the amount of CO2 in your incubator? Some cell lines are also sensible to confluence, if you let them get too confluent at any time they will not feel well...
D
D
Or maybe you put too few cells in, they may not feel well, either. Or it is also possible that for that one day, you accidently did not loosen the flask cap?
Hello, I have a yeast contamination problem on my cell culture. I'm culturing hydridoma cells and I started working in an old cabinet, once they notice the problem they changed me to a new cabinet, where there is not contamination problems, I'm trying to get read of the yeast by washing the culture but it is almost impossible cause they proliferate very quickly. My hybridoma cells look fine, but the proliferation is limited because of the space the yeast are taking to grow. Any suggestions? I need to check on the antibody production on those hybridomas, but I guess I need to wait until I have more hybridoma cells proliferating per well in order to have enough antibody concentration in supernatant. Am I doing ok? What should I do?
Hi... like every suggestion's in this forum.. if any contamination happens, just draw out your flask, and start with the new one...
your problem is the same with mine, few months ago.. but it was too tiring wait until your cells proliferating, i think its kind of waste of time... and because i need to test the performance of biomaterials, the cell proliferation data seems not reliable if you use "sick" cells.. so, if you have any other vials, just start it with new cells... and dont forgot to clean your incubator too before start with these new cells... hope it works
D
Or maybe you put too few cells in, they may not feel well, either. Or it is also possible that for that one day, you accidently did not loosen the flask cap?
Hi... thanks all for the suggestions...
almasy = well ... i didnt think that i wasnt loosen the flask cap =P.... but, even i did.. two days seems to be too fast to cause this happens.. plus, it happens to all my flasks..
DLY = i didnt change the medium supplier... and about over-confluent things, i dont know...
.. my advisor just suggest me to draw out all the cells, start with the new one.. and that was what i did... it was a good lesson indeed... thx