Tips on Sterile Techniques for Cell Culture

There is nothing scary and disheartening than coming in Monday morning after a nice relaxing weekend, open your laboratory cell culture incubator and check your cells in your culture flasks to see them all contaminated.  There goes the week’s worth of experiments, time wasted in expanding the cells from the limited number, cost of wasted reagents.   I have done mammalian cell culture for more than 12 years and during this time I have picked up some wisdom regarding sterile techniques which may be useful.

Any reagent which may come in contact with the cells has to be sterile.  It is a good practice to write on the bottle if the reagent is sterile, especially if it is used by more than one person in the lab.  This will give the other colleague to use caution when using the reagent to use it sterilely.  If you are not sure if the reagent or culture medium is sterile, sterile filter the media. This is easier than using the media without filtering to contaminate the culture and waste time and cells.

Wear gloves when handling culture, this will protect both you and the cells from contamination. Since the gloves may not be sterile, rinse the gloves with 70% ethanol.

Use alcohol swab and wipe the mouth of the reagent bottles before using media to feed the cells.

When using Pasteur pipette always be aware of where the tips of the pipette is.  If you touch any surface by accident and put that pipette into the cell culture you can easily contaminate your cell culture. It is a good practice to keep the tip of the pipette far away, to avoid touching any surface.

When culturing multiple cell lines, handle one cell line at a time. There is a good chance of cross contamination if not careful by keeping different cell lines in the work space.

Check the cell culture everyday to make sure they are not contaminated.  Check frequently for mycoplasma contamination for purity and integrity of the culture. Mycoplasma contamination is difficult to see unlike bacterial or fungal contamination.

Make it a habit to get rid of the contaminated culture immediately by treating with chlorine. If you keep it around, the chances are the contamination will spread to other cultures.

Only use cell lines from reliable vendors or labs where they certify the cultures are tested negative for mycoplasma.  If cell lines are obtained from other local labs, confirm if that culture is tested negative for mycoplasma.

Keep the opening and closing of the incubators to a minimum. More often the incubator doors are open, the better the chance of the getting the cell culture contaminated.

If you are using a tray of water inside the incubator for humidity, make sure the water is always clean and changed frequently. Keeping dirty water is a great way to invite contamination.

Clean the warm water bath frequently and add fresh water for warming up medium, and other cell culture reagents.

If possible, reduce the foot traffic to the cell culture room. If the cell culture room is separated with a door, keep the door closed to avoid constant foot traffic to the cell culture lab. The more turbulent the air in the room, the more chances of bringing in contaminating organisms, especially during summer months fungal spores travel in the air.

If possible use filtered cell culture flasks instead of keeping the cap loose for aeration inside the incubator.

About James Maliakal

I am James Maliakal, writer of this blog and is with Pioneer Scientific. I have 12 plus years of experience working in the research lab in academic and leading life science companies. From my years of experience, I have designed and developed unique products which made my work a little easier and efficient in the lab. I have authored and published in peer reviewed journals. Pioneer Scientific is a leading manufacturer and distributor of innovative products, high quality laboratory instruments, and research tools. Contact me to know more.