Mammalian cell culture techniques are not something you learn from a book, per se. And because of this, it is important to be properly trained, especially in sterile techniques. It is important to keep your cell lines from contamination and just as important to keep yourself safe. Nevertheless, people tend to do things a little differently, even when they might tell you this is the only way. Therefore, there is some wiggle room in keeping your cell lines and techniques aseptic.

Leave the Cap Lids Face Up or Down?

Depending on whom you are trained by, your mentor either asks you to leave the lid of the cell culture flask face-up, face-down, or even on its side. I have personally never left the lid of my cap with the open side face down, but a former advisor asked that I leave the cap on its side. It was always difficult to prevent the cap from rolling away, but I managed. This did not seem to pose any more risk to contamination than putting the cap face up.

However, I believe it is easiest to set the cap open side face up and to move it far enough away so as not to accidentally wave my hand over the lid. In my experience, this works just fine as an aseptic method.

Opening Individually Wrapped Serological Pipets

Sometimes it is not so easy to remove the serological pipets from their individual packaging gently because it is difficult to peel the two pieces of paper back to remove the pipet, let alone quickly. So instead, you can actually tear or poke the pipets through the top, then pull the two pieces away from one another to remove the pipet from the packaging. I pretty much do this every time to remove the pipets from the packaging and rarely ever try peeling to open the pipet. I have watched others remove the pipet every time via peeling the two papers back even when it becomes a struggle to open the package.

How Often Do You Feed Your Cell Lines?

This is highly dependent on your cell line, and the way that you are growing the cells, but generally feed your cell lines as often as they need. I feed my healthy, growing cells 2-3 times a week with fresh medium. This may be overkill, but the cells are healthy and continue to grow at fairly decent rates. However, with cell lines that may be struggling to grow, I might feed the cells less and additionally feed them with a conditioned medium to help them along.

As a good rule, do not let the medium become too acidic, and as a result, turn yellow. This may also indicate that there are too many cells in the flask and they need to be passaged.

How Much Personal Protective Equipment (PPE) Do You Wear?

The amount of PPE to wear is highly dependent on the type of cell lines you are working with, the reagents you are working with, and the comfort level you have in working with all of these things together.

As long as you aren’t working with viruses or lentiviral plasmids, I think it is safe to say that nitrile gloves and a lab coat are sufficient to work in a biosafety cabinet with mammalian cell lines. There are also many people who do not wear lab coats each time they work with cell cultures, and I was one of them for a long time. For example, I was previously working with Chinese hamster ovary (CHO) cell lines, and now I strictly work with human-derived cell lines, which I feel pose a higher risk hence why I wear a labcoat now. However, I know people that work with human-derived cancer lines and still do not wear lab coats because they believe that it can lead to more contamination due to hanging sleeves and coats that aren’t washed often enough. Again, as long as you are safe and not contaminating the cell lines, then it is probably ok to keep doing what it is you are doing.

Keeping your cell lines from becoming contaminated is probably the most difficult part about cell culture techniques, but as long as you don’t contaminate your cells then it probably doesn’t matter which way you choose to leave your caps.

Just maintain a clean, clutter-free environment, don’t sneeze on your cells, and hopefully, these tips will help ease your mind while doing these difficult and sometimes tedious cell culture experiments.

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  1. I typically don’t have anything in the cell culture hood, and therefore have to bring everything I am going to use inside the hood. To do that I spray everything with 1 or 2 sprays of 70% ethanol- the tip boxes, medium, racks, pipets, bottles, etc. I do no spray the flasks carrying the cells as they should have just come from a clean/sterile environment (the incubator). I do disinfect my gloves with a spray of alcohol as well, especially if I am constantly going in and out of the hood. There is no hard or fast rule that i know of, but I tend to be more liberal about spraying with ethanol than not. I also do not spary up but my face, just down towards my hands or other objects so as not to inhale the alcohol. I have also used special fungicide like tergazyme in the water baths before, but do not use any currently. Clean your incubators often, as well as the water baths, changes filters and that should help cut down on contamination. Hope that helps a little! Thank you for the questions!

  2. There are even more things you take care of.

    How do you disinfect al materials that go under the hood. Do you disinfect the flask on the surface if there are already cells in it?

    Do you disinfect your gloves more often? you go often out of the hood and then back under the hood while working with cell lines.

    Do you use ethanol spray? that is harmful to your lungs!

    Do you use special anti-fungicides in your incubators?
    How do you handle our water baths?

    And there are more things I guess.

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