How To Prevent Cell Culture Contamination
Inside the body, cells are protected by the highly evolved and sophisticated immune system. Once isolated from tissues, cells in culture are “on their own”. We must therefore protect them with careful aseptic techniques to prevent their contamination from microorganisms that are present all around us, including inside the laboratory.
Various types of contamination exist: for example, cells can become contaminated with bacteria, mycoplasma, or mold. Some of them are harder to detect than others1 (such as mycoplasma) and can therefore have a massive impact on your results without you knowing it. Others will be noticeable and will force you to interrupt your experiments - which wastes time, energy and money. Contaminations in the laboratory can be a nightmare for scientists, especially as they can spread from one sample to all others present in the same incubator or hood. It is therefore an important responsibility for everyone in the laboratory to be as careful as possible, not only for the sake of their own experiments but for the sake of everyone else’s too.
Measures to prevent contamination are numerous and should be applied throughout handling. With experience, these measures become second nature, but it can still be easy to make involuntary mistakes. While the field of cell culture is moving towards higher levels of automation2 in part to prevent cell contamination, as of today we still rely mostly on manual labor, which presents many opportunities for mistakes in aseptic handling, especially for beginners. Here, we provide some essential tips to maintain an aseptic environment and prevent cell culture contamination.
1. Wear gloves, lab-coats and use hoods
This goes without saying, you must provide a barrier between your cells and the non-sterile environment that is your laboratory (or indeed yourself). Using gloves, lab-coats and hoods does just that. Use your lab-coats only inside your cell culture lab and have them cleaned often. Make sure your hood is serviced often to guarantee that it is properly working. Note that there are different types of hoods, depending on the level of protection you want for the cells and the users3.
2. Use your hood correctly
Having a hood is not enough to ensure a sterile environment: you must use it correctly. First, make sure that you are not blocking the flow of air inside the hood: do not place material over the air outlets or inlets that are often found at the front and back of your bench space inside the hood. In addition, make sure that you are working well inside the hood, and not too close to the edge to make sure that you are in fact operating under sterile conditions. You might want to empty your hood at the end of your day, and make sure to wipe it with 70% ethanol or 70% IMS before and after every use. Some groups choose to have a waste disposal bin inside the hood, if you do, empty it and clean it often. Others prefer not to and keep it outside the hood as having used material or reagents for a long time inside the hood can increase the risk of contamination. Finally, hoods often come with UV light: switch it on at the end of your day to sterilize your hood while it is not being used.
Some new incubators have self-cleaning capacities – others must be cleaned manually. Do so often; the protocol will vary depending on the incubator. If you use a tray of water inside the incubator, change the water often. You might sometimes need to add a chemical to the water to prevent contamination (always check that this chemical is compatible with the material your tray is made of). Similarly, the water in your water bath where you warm up your media should be changed regularly. Add a water bath treatment every time you change it.
4. Spray EVERYTHING with ethanol or IMS
Ethanol is typically used to kill bacteria. Make sure you use 70% ethanol mixed with water, and not a lower concentration. The water is known to increase the efficacy of ethanol in killing bacteria and some viruses. Spray your gloves and everything that you bring inside the hood with ethanol. It is important to realize that every single time you touch something outside of the hood you should spray your gloves again before placing them back inside the hood. This applies to automatisms that might go unnoticed such as moving your chair, throwing something outside of the hood or touching your glasses. Buy ethanol-proof markers to label your labware, otherwise spraying ethanol over it will remove your labeling.
5. Minimize exposure of cells to non-sterile environments
Since so much effort is dedicated to maintaining a sterile environment inside the incubator and the hood, make sure that you minimize the time cells spend outside of these environments. For example, when you check them under the microscope, or when you transfer them from the incubator to the hood. If you have to use your cells for extended periods of time in non-sterile conditions, for example during time-lapse imaging, and need to put the cells back in an incubator, designate a separate incubator for this purpose if possible. This way, the increased contamination risk will be confined to one incubator.
6. Buy sterile reagents and keep them sterile!
Be sure to buy reagents that are sterile. This encompasses media, PBS or any other product that will come into contact with the cells. If you use reagents for other work that is not sterile, such as antibodies, do not use them for your cell culture. Keep two separate stocks of sterile and non-sterile reagents. In addition, you can also choose to filter your media (or any other liquid reagent) through membranes that remove bacteria. Typically, 0.2 μm filters are enough to block bacteria. Another way to maintain the sterility of your reagents is to aliquot them. That way, if one aliquot gets contaminated the entire stock is not affected, and you can throw away the one aliquot instead of the entire stock. You can also buy pre-aliquoted reagents.
7. Use sterile labware
Apart from reagents, you must also sterilize the labware that cells will be in contact with. To sterilize these, you can use an autoclave machine. Make sure however that your labware can be autoclaved! Not all materials can sustain the temperatures of the autoclave. For example, scientists often autoclave pipettors, or pipette tips. If you can, buy consumables that are already sterile, such as flasks. To maintain sterility, open the bag of flasks only once you have sprayed it and placed it inside the hood. When finished, close the bag inside the hood before bringing it back outside. If you seal the bag with tape, and the tape is outside the hood, keep the bag closed with your hands until you tape it. You can also autoclave water, if you have a source of filtered water in your laboratory. Place a pressure-sensitive tape on the material before placing it in the autoclave: if the material has successfully been autoclaved and sterilized, the tape should change color.
8. Use filter tips and change them often
Pipette tips that contain filters prevent the solution inside the tip from touching the pipettor. Without filter tips, your pipettor can become contaminated, thus contaminating every other sample that it touches First, this means that if one sample is contaminated with microorganisms these can spread to other samples, but it can also mean that you can introduce cells involuntarily from one sample to the other. For the same reason, make sure you change your tips often during your experiments, especially between samples. When you operate with the pipettor be careful as to not bring the pipette tips in direct contact with anything else, and if you do change tip. Importantly, if you want to pipette something out of a bottle, avoid using a pipettor that you must insert inside the bottle, to prevent the pipettor from directly touching the inside of the bottle: instead, use a pipette gun that can be used with longer pipettes that can reach deeper inside the bottle.
9. Check your cells often
Even though some contamination, such as that associated with mycoplasma, is hard to detect visually, it is always wise to check your cells regularly under the microscope. If you do, try not to keep your cells away from the incubator for too long to minimize the risk of contamination. You can check for moving bacteria (do not confuse with cellular debris!), morphological changes or unusual proliferation rates. If you suspect your sample is contaminated with mycoplasma, there are commercial kits available to confirm this.
10. Bleach your contaminated samples
If you do think your sample is contaminated, fill it with 10% bleach, Trigene or Chloros in the sink or away from the incubator for a while. Empty it inside the sink and throw away the culture vessel. If you realize that your sample is contaminated, warn your labmates, especially those who have samples in the same incubator! This is really important, it’ll allow them to quickly react, check that their samples are fine and plan accordingly if they are not. If it’s possible to empty the incubator of all its samples, clean the incubator.
11. Use good labeling practice
Keeping good records of your stocks of cell supplements and media will help you identify sources of contamination more quickly. If you find that some cells are contaminated, you should be able to identify the stock to which these cells belong to and test whether the contamination is common to the entire stock. Similarly, if you know the batch of media or supplements you were using, you can check or throw away the affected stock to avoid repeated contamination. Good labeling can also prevent contamination of your experiments with other cell types: you want to make sure the cells you have inside your assay are the ones you think they are. This is a huge problem in the field, with up to 30,000 studies using the wrong cell lines4. Therefore, make sure you label all your flasks and vials properly, to minimize the possibility of cross-contamination between samples.
12. Common sense
Last but not least, common sense goes a long way to prevent contamination. For example, do NOT perform work with bacteria or molds in your cell culture lab. Needless to say, no sample containing bacteria should be placed in your cell culture incubator. If you are ill, try to refrain from doing cell culture work (we appreciate this may not always be possible). Minimize talking in front of open incubators or whilst working at the hood. Do not leave incubator doors open for too long. Wash your hands before and after cell culture handling!
1. Nikfarjam, L. & Farzaneh, P. Prevention and detection of Mycoplasma contamination in cell culture. Cell J. 13, 203–12 (2012).
2. Wendt, D., Riboldi, S. A., Cioffi, M. & Martin, I. Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing. Adv. Mater. 21, 3352–3367 (2009).
3. Cell culture contamination - Thermo Scientific.
4. Horbach, S. P. J. M. & Halffman, W. The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLoS One 12, 1–16 (2017).