Try preparing the media again and check for any such growth. I don't think excess alcohol usage would cause contamination.

Good Luck.

It seems that one of your routinely used items or ingredients is bacteria contaminated. Fungi vs bacteria infection should be readily obvious under the microscope (long filaments vs actual spherical bacteria). Based on the white density you describe, that is most likely bacteria. The contamination is potentially from the DMEM or the fetal bovine serum (if any). The cells seed itself may also be contaminated if they have been frozen from a contaminated batch. Suggest throwing everything out and re-start with everything new. If this happens again, then the "mother" cell line may be contaminated in which case you will need to either buy a new cell line or ask for a seed from a lab near by. Valuable (read custom-made) cell lines that get infected can potentially be "cleaned", but the process is a pain and would not be justified unless that is the only line in the world of its kind. It will however make a good story for the future. Best of luck!

Thank you for your suggestion. I already discarded all media, DPBS, and made new media. After I experience contamination, I discarded all dishes and took cells from my mentor. While mine is contaminated, his cells are perfectly fine ( same cell, same incubator). Last week, I thought it could be bacteria from my throat ( My throat have been sore for more than a month) so I wore face mask and rub my hand carefully with alcohol.  But it did not stop.  I seriously do not know what to do. 

Hi Taho, if you´re supplementing your DMEM media with serum, be sure your protocol of serum-inactivation is correct maybe the serum can form agregates or don´t be available for your cells. if you are suspecting for contamination I recomend to add copper sulfate this aid as fungicide

I didn't find any information about adding copper sulfate. Can you specify more about how can I add copper sulfate? Would it have any effect on cells? 

i agree Dr. Alice Ceacareanu .

I discarded everything including cells and media but it still happens. It is very strange that my mentor who conduct experiment with same mother cell, same incubator, same protocol did not meet this situation. He also checked my technique and I did not do anything wrong. It seems like contamination source is mine ??. But I wear mask during experiment ( I have had sore throat for one month) and always spray alcohol...

What else I can do? Help me please.  

you have to be methodical about this. I agree with the previous response that you need to throw all materials away and start again. As it still happens and others do not experience the same problem (I'm assuming that they use exactly the same media as you and cells? i.e if they made it up then you should use that or visa versa to check, you can aliquot a volume out for use by individuals), this does point towards your technique. Your supervisor needs to shadow you closely to make sure you are not doing something irregular when growing the cells. Your media can be easily checked: incubate (no cells) at 37C for a few days with shaking, if no growth then media is probably ok. You could also spike LB broth and grow for a few days. 

The main thing is, is it contaminating growth: sort this out first. As suggested easily done under the microscope. bacteria are black fast moving dots fungi are white filamentus. 

I would recommend to throw away every bottle that is open: antibiotic, PBS, serum, DMEM. The few times I had contamination issues the cause was traced back to the serum (incorrect inactivation prior to its use). If you cant afford to throw away your materials, then troubleshoot where is the source of contamination by plating combinations of your culture ingredients:

Plate 1 Serum

Plate 2 Serum and PBS

Plate 3 Serum and antibiotic

Plate 4 DMEM and Serum

Plate 5 DMEM and PBS

Plate 6 DMEM and antibiotic

Plate 7 DMEM only

Plate 8 PBS only

Etc

Incubate as per your protocol and check the combination thats giving you contamination. Think about cleaning your work area with a fungicide twice a week. If you can't find the source of contamination, and its not the plates, the incubator, the ingredients, have your mentor plate you a new batch of cells. 

Best of luck!

Add 1-2% of cooper sulfate to the water for the incubator before the sterilization. And the cleaning cicle we´re using is: clean the surfaces with et-OH 70%; benzal solution; finally et-OH. 

The cooper do not represent any risk for your cultures at this concentration, can actually increase viability throughout your culture. 

Added to the previous suggestions, you can also try filtering the complete media before seeding the cells using 0.45 um syringe filter. if the white cottony floats still appear, there must be a error with inactivation of serum or in the handling. We have faced such improper serum inactivation problem even in commercial inactivated EU certified serums. 

Another chance is fungal contamination, which you can identify either by prolonging the incubation (during which the spores or fruting bodies starts to appear on the surface) or you can plate it on PDA or SDA plates. if you find fungal contamination, discard all the components and start from day 0 and regularly fumigate the cabin and the hood with Potassium permanganate + Formalin mixture aided with regular bacterial and fungal screening.

Thanks and All the best.

Just a thought: If your cells are not adhering straight away after plating it may be a case of the media is not adequately equilibrated. Not sure what other agents you are adding to the DMEM but this media even though it contains Bicarbonate may for some cells lines require pre-incubation in the incubator to achieve a stable pH prior to plating. 

Hi,

we use DMEM/Ham's F12 media for culturing MDA-MB-231 cell lines.

Do NOT alcohol off your gloves. While it sounds like a good idea, to make them more sterile, it makes the glove more porous and increases your risk of getting something. One thing no one has mentioned it the hood, if you are using one. Take it apart and clean it well with alcohol or a detergent that is bactericidal and fungicidal. It can harbor contamination, especially under the work surface. Make sure you are working at least 6 inches in the hood (does not include grate). You can also put a piece of copper pipe or connector in the water bath of the incubator, it helps reduce contamination and keeps water bath from getting contaminated. Hope you figure it out.

I think is a contamination, sometimes the contamination is so big that it makes the flask cloudy and you would not be able to see nothing under the microscope.  You could check by staining with trypan blue and looking under the microscope, if it is a bacterial contamination you will see a lot of small, probably rounded deep blue dots. if it is fungi you will see a network of blue strains.

do mycoplasma test or ask experts in yeast just in case. If is not a contamination then my only suggestion would be to filter the  medium before using it   

 If it is a contamination discard everything, and I meant everything, not only the medium but the plates, the tips, pasteur, eppendorf, columns do a deep clean to the pipettes.

All the tips, pasteur pipettes, eppendorfs, should be autoclaved or individually wrapped for cell culture.Put the plates, reservoirs, flask and other things under UV light before it use if you consider it necessary.

Do not pour nothing, sometimes the last drop touch the outer face of the bottles that could be contaminated.

Clean the cabin  before and prior its use (that is the only  moment in which you should use paper) inside the cabin/hood (or if you had a spill). The paper could be contaminated with bacteria that will not die so easily even with alchohol.

try to avoid as much as possible to let the bottles open, and if you do it place the cap as far away as possible from your working area and never cross nothing over it. 

It is better to throw things away if  you suspect it could have touched a wall or your gloves or what ever than to face contamination

Check all the mediums by themself without cells in the incubator for one week

 I would change them and restart again in any case

Make your supervisor check not only your technique on the cabi/hood  but as well your technique of how you keep thing sterile, how you unpackage certain things, how you take out and in the cells from the incubator and how you clean the incubator and the cabin 

decontaminate again the incubator and the cabin if possible

and start again with some of the stocks of cells that your supervisor give to you

fingers cross 

Thank you for all your responses. I finally find the contamination source. It seems like the how batch of DPBS is contaminated so even after changing DPBS, the contamination still happen. Now it is ok with a new batch.