Advice for someone new to cell culture?
November 12, 2012 2:13 PM   Subscribe

I'm halfway through my first course in cell culture and I'm starting to get comfortable with the basic techniques of thawing, freezing, growing, splitting, and counting cells. Based on past experience, I know that any bad habits that I start forming now will be very hard to get rid of later. What kinds of bad habits do you see in cell culture labs? What advice do you have for someone new to aseptic technique, the laminar flow hood, and working with cell lines?
posted by sunnichka to Science & Nature (7 answers total) 5 users marked this as a favorite
Never trust the UV to work.
Never trust the person who used the hood before you.
Always make sure you have enough media before you start your days work.
Always make sure that there's ingredients to make media before you start your days work.
If you're doing higher level tissue culture, be sure to test each lot of FBS (or whatever kind of serum you're using) before starting to use it for experiments. We once had a lot of FBS that had no growth hormones and people didn't know that the lot number had changed until about two months later when we all started comparing our problems with cell lines.
Good pipetors are totally worth it - change your filter regularly.
To get comfortable manipulating plates, tubes and rollerbottles, practice outside of the hood with PBS and non-sterile stuff. It it completely worth it in terms of your confidence when you're doing the real thing.

A nice big carboy of 70% ethanol makes it easy to refill your ethanol spray bottle.
Get good ethanol proof sharpies (they call them industrial sharpies).
posted by sciencegeek at 3:01 PM on November 12, 2012

One more thing specifically: I still feel really awkward manipulating pipets inside the hood, I always feel like my arms go weird places. Sorry that is vague; hopefully it will make sense to someone.

posted by sunnichka at 3:41 PM on November 12, 2012

Never trust the undergrads/rotating students/total newbies in your lab to know how to do something obvious to assist you like realizing that making up cell culture media from the recipe they've just been handed involves filtered water, as opposed to straight from the tap.

Don't let your sleeves mess up the airflow vents keeping the TC hood sterile. Roll them up if your lab coat is way too big for you, like mine always was.
posted by deludingmyself at 5:34 PM on November 12, 2012

Is it ok to rest my forearms on the airflow vents when I'm pipetting?
posted by sunnichka at 5:47 PM on November 12, 2012

I'd call them ethanol resistant. They're not perfect, but they work more or less. Here's the VWR brand one. Learning how to label things well is important generally. If you've got bad handwriting, print deliberately clearly. Always label things so someone else can read them - you never know if someone else will need to split something for you.

It will just take time to get used to moving around in the hood. It takes practice. We always gave people new to tissue culture a couple of disposable plates of cells to try out for a few weeks before they started anything real.

I found that pipeting stacks of plates was easier to teach when I gave them to people to try outside of the hood - somehow working inside the hood stresses people out when they're first starting.

A lot of things also depend on the size of the hood you're working in. We had both huge and tiny hoods and it was much easier in the big ones.

Learn how to split your cells so you don't have to come in on weekends. This means learning the doubling times of your cells. Also learn how hard you can split them and have them still be happily dividing.
posted by sciencegeek at 6:27 PM on November 12, 2012

Is it ok to rest my forearms on the airflow vents when I'm pipetting?

It interferes with the air flow keeping the hood clean and theoretically increases your chances of contamination, but I still do it regularly without problems (ymmv, it is better technique not to do it).

Other things:

-airflow is the most important thing for maintaining a clean hood. Keep the hood as empty as possible and don't block the vents anywhere
-assume everything is contaminated and touch as few things as possible with your sterile pipette tip - ideally only the liquid that you're pipetting. Always aim for perfect technique, even though you'll bump things occasionally.
-depends on your lab, but I prefer to work without antibiotics since they can mask a low-level infection which can change cell behaviour, promote antibiotic resistance, and hide poor technique. Similarly, regularly test for mycoplasma infection, which is undetectable visually but can change cell behaviour
-never have two different cell lines open in the hood at the same time, and ideally not even in the hood at the same time. Contamination with other cell lines is extremely easy and often hard to detect, and of course makes experiments done with those cells worthless.
-know how your cells normally look and behave (growing times, etc) and pay attention if anything changes
-water baths are filthy cesspools of contamination. If you use them, be very careful that the water never gets near the neck of the bottle. Ethanol everything anyway, of course.
-don't pass anything over open containers ever if you can help it
-don't invert or tilt your media containers unless you know 100% that they're sterile - sometimes bacteria start to grow around the neck after a while. Also why you don't bump the neck with your pipette.

Hope that helps! 95% of it is just practice and paying attention to where you're likely to pick up contamination.
posted by randomnity at 9:33 AM on November 13, 2012

Thanks for these answers so far, they're very helpful. Is there an alternative to water baths?
posted by sunnichka at 1:52 PM on November 13, 2012

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