It is recommended in many laboratories to use antibacterial-antifungal antibiotics addition to culture medium, standard mixtures of pen-strep or ampicillin-fungicide are plenty and not expensive. I myself use antibiotics only for primary cultures, clean cell lines are Ok for months without treatment. And there is one more reason not to use antibiotics permanently - they induce resistancy development and alter protein expression profiles-phenotypes for in vitro cultures significantly. I do not let my students to use antibiotics in medium from the beginning but make them to watch their hands moving in laminar under my supervision within one-two months. It is worth doing until they can control their sterility themselves entirely and recognize immediately whether they touched internal part of the plate or flask with glove or outer part of bottle with steril pipette/tips end. Not everybosy is able to remove the cap from culture flask with left hand sterilly holding steril pipette in the right hand with closed eyes after many years, not two months. But in general it helps to prevent next years artefacts in post-graduate's data and saves stock cultures from new purchasing from Cell Banks. 

Besides, CO2 incubator internal surfaces regular treatment is also important. It should be dried inside and sprayed with ethanol once in a month, in case the instrument made of stainless steel, not copper - with antifungal solutions also.   

Greetings

P.S. Normally I wipe with ethanol-soacked cotton flask's and bottle's caps, plate's boaders - every edge brought from non-GMP room air into laminar - before openning it. Glass bottlenecks are even possible to treat with fire, but for plastic littlewiping is enough. 

It is recommended in many laboratories to use antibacterial-antifungal antibiotics addition to culture medium, standard mixtures of pen-strep or ampicillin-fungicide are plenty and not expensive. I myself use antibiotics only for primary cultures, clean cell lines are Ok for months without treatment. And there is one more reason not to use antibiotics permanently - they induce resistancy development and alter protein expression profiles-phenotypes for in vitro cultures significantly. I do not let my students to use antibiotics in medium from the beginning but make them to watch their hands moving in laminar under my supervision within one-two months. It is worth doing until they can control their sterility themselves entirely and recognize immediately whether they touched internal part of the plate or flask with glove or outer part of bottle with steril pipette/tips end. Not everybosy is able to remove the cap from culture flask with left hand sterilly holding steril pipette in the right hand with closed eyes after many years, not two months. But in general it helps to prevent next years artefacts in post-graduate's data and saves stock cultures from new purchasing from Cell Banks. 

Besides, CO2 incubator internal surfaces regular treatment is also important. It should be dried inside and sprayed with ethanol once in a month, in case the instrument made of stainless steel, not copper - with antifungal solutions also.   

Greetings

P.S. Normally I wipe with ethanol-soacked cotton flask's and bottle's caps, plate's boaders - every edge brought from non-GMP room air into laminar - before openning it. Glass bottlenecks are even possible to treat with fire, but for plastic littlewiping is enough. 

There are a number of things you should do all of which are small things in themselves but together will make a massive difference in getting clean cells WITHOUT ANTIBIOTICS. Antibiotics are dirty compound and WILL affect your cells:

Make sure your Class II cabinet is in a separate TC room.

Wear a clean lab coat which is specific for your TC room and get it cleaned on a regular basis.

Always wear gloves

Change your water in the TC incubator and the water bath EVERY week

Always wash your media bottles with 70% IMS from the waterbath

Always work in the middle of the laminar flow in the Class II cabinet and make sure that all the surfaces are clean and UNCLUTTERED.

Wherever possible use filtered (0.2uM) TC flasks for all your cell culture. When using dishes for experiments wipe them with 70%  IMS in and out of the Class II cabinet AND the CO2 Incubator. This will remove aspirates of media that are there but are not visible to the eye BUT will cause contamination if left.

Never share media or other TC components with other people

Regularly change your 0.2uM filter in your pipette aid. If you suck media into this then change immediately...but change them on a weekly basis. You can purchase 50 filters for about £35 so it is cheap to do this. Make sure also that the pipette aid is specifically used by YOU and that it stays in the TC room and is not used for other things i.e pipetting bacteria.

If you use Gilsons to add compounds for your experiments then remember that these are a major source of contamination....they have no 0.2uM filters and that people use them in all areas for all lab work. Again have specific Gilsons for the TC room and thoroughly clean them. Some commercial companies manufacture autoclavable Gilson type pipettes...beware of these as the autoclaving process can affect the precision of them, especially when still warm from the autoclave

Purchase CO2 Incubators that have a high temperature decontamination cycle on them. This means that if and when you contaminate your Incubator, it makes it a lot easier to deal with

Be trained by someone who has experience in TC techniques...this will save you time and money in the long run.

DO REGULAR MYCOPLASMA TESTING....these cannot be visualized under the microscope and will invalidate any results that you achieve

I hope this will help as a start there are many other things to consider

Hi there,

as you had contamination in some of your plates and not all, of course the media is ok. However, from my own experience even a simple and basic laminar flow can prevent contamination as long as the media, glassware, etc are sterilized. In fact, it might be normal, but annoying, to see contaminations sometimes. you should have a back up experiment and do sub cultivation at two different days for the same cell line in case one get contaminated.  solid plates as back up are extremely useful. it is because we can't control everything in 100% and all the time. everything's possible.

Regards, 

also keep and eye on your sleeves.  Try to make sure you are not passing them over and open plate/flask, or worse yet, touching them on the hood surface and then having them touch your open plate or flask.

Plates. dishes, Petris or flasks? Contamination happens. Do not worry, if you can still have normal cultures besides. Best is start over from stock unless it is a special one. And use permanently Ab-mix as you can read in papers (but i accept extra cases, of course). With everything fresh and new when your start over. One extra issue: slowly growing cultures are more likely contaminated if you forget to refresh medium and the Ab-mix in it regularly.

Am sure most of the above might cover this but I had similar issue couple of months back-- in petri plates!

First, make sure your incubator is devoid of contaminants..

For this, You can always place a plain bacterial media (if you think its bacterial contamination) and see if you are having any bacterial growth. 

Also If you have fresh vial of stock cells, try using media (or glassware)  from other lab   ---am saying this because I was trying to rule out each possible step where contamination could have happened...(may be glassware in your lab)-- this actually worked for me

Good luck!

I agree with all above answers . All answers are very useful. I also have problem of contamination but particularly when I have used 6 well plates, I have got contamination after 3 days. I have not get contamination in Culture flask, having same condition,same laboratory,same equipments. I can not found the problem. It was found on 2nd day,when I was performing Soft Agar Colony Assay with MCF-7 cells. Dirty white colored layer was formed above the agar in 6 well plate within 1 week. Media was changed on proper day(after every 2 days). However we got this type of Contamination . I cant find the reason.

Folks, thanks a lot for your replies. A few confessions - I am a rotation student in the current lab and don't have a lot of control over the maintenance and basic upkeep procedures in the lab. But, I will try to speak to my boss about it and see what he says. Also, the BSC seems to be a bit cluttered especially towards the corners.

As for an update on the situation - On 01 April,  I changed the media in the contaminated plates after 2 PBS washes. I added about 10 uL Ciprofloxacin to each plate. Please note that I used the same media as I had until now. Today (02 April), the previously contaminated plates still showed some floating particulate matter, but fewer in number than the previous episode. In addition, another set of plates (a different cell line) is now showing signs of contamination. Loads of floating particles that appear like small black dots, not perfectly circular, but kind of rounded. The media on visual inspection doesn't show any cloudy or particulate appearance. I am suspecting that the contamination is coming from the media. I don't remember cutting corners or being sloppy while perform any procedures. Anyway, that's not important.

My plan - all the plates will be given 3 PBS washes and the media will be replaced with a media from a new bottle. 10 uL of Cipro (per 10 mL of DMEM) will be added and I will check tomorrow. Each plate will be handled one at a time and the surfaces will be wiped clean after every plate. Also, I won't be taking the fresh bottle or PBS into the hood; instead I will be aliquoting the necessary amounts into tubes and taking them in as needed. Hands will be sprayed frequently with 70% alcohol. The plates will be placed on a different shelf in the incubator. As a test, I will be incubating a 10 mL sample of the old medium and checking tomorrow for any changes.

Keep the suggestions coming in as I hate contamination. It's extremely embarrassing and annoying. Thanks again, folks.

I suggest you take a look at this site with very detailed protocols and tips on the use of cell cultures http://cellculture.altervista.org/ hope this will be useful even if long time is passed now!

Best to use antibiotics if it is a primary cell line. In my experience, you will keep getting bacterial contamination regardless of aseptic techniques.