Virtually every research scientist has a use for sterile technique in the lab, whether you study infectious microorganisms, do tissue culture, or use E. coli for cloning. Good sterile technique is a basic lab skill required to avoid contamination of your materials and experiments; and fortunately, the principles are simple to learn and easy to do.
Glove up!
Wearing latex or nitrile gloves serves two purposes: to protect you from what you’re working with, and to protect what you’re working with from you. The majority of gloves sold for lab use are not sterile, so keep in mind that wearing gloves while working with sterile materials is not a guarantee of cleanliness. If you’re working with infectious materials, it’s important to always wear gloves to protect yourself. Keep in mind that gloves are designed as “single use” only, so make sure to discard them and put on a fresh pair after taking a break from the bench. Some people prefer to use bare hands when working with E. coli or tissue culture. So, if you’re one of them, it’s a good idea to spray your hands with ethanol to eliminate the majority of possible contaminants. Here’s a great tip though – if you do choose to use an ethanol spray, be sure to let your hands dry before lighting your bunsen burner!
Using your bunsen burner
If you do your sterile work at the bench, a bunsen burner is your best friend. The flame is used to directly sterilize glass bottles, spreaders, and other tools. The other major function of the bunsen burner is to create an updraft in the local area. Hot air rises so the heated air around a lit bunsen burner creates a slight current upwards. This means that any “hovering” contaminants in the air are wafted away from your work area, instead of falling into your work. While it’s important to work near the flame, don’t get so anxious about it that you burn yourself. Close is good enough.
Keep it clean: plasticware, media, and tips
All materials used for sterile applications should obviously be sterile, whether they’ve been purchased that way (i.e. plasticware), autoclaved (glassware, tips) or filter sterilized (buffers and media). Sterile materials should only be opened near a lit bunsen burner or in a biological safety cabinet (often called a “hood”, but not the same as a chemical fume hood). When removing tubes, petri dishes, or other sterile plasticware from plastic packaging, try to touch only the items you are removing, and reseal the packaging immediately to keep the remaining items from contamination. Keep your tip box closed when not in use, for the same reason. When using media in glass containers, run the mouth of the bottle through the flame of your bunsen burner before inserting a pipet and before replacing the lid. This helps to prevent contamination from the lip of the bottle. Of course, plastic bottles should not be run through a flame (at the risk of melting). Use them near a flame and try to avoid touching the mouth of the bottle with your pipet.
Basically, the rules for sterile technique are very simple. Use your common sense, and pay attention to what you’re doing. What are your tips and tricks for sterile technique? Let us know in the comments.
This is probably “common sense”, but I thought I would mention it anyway as I have found it was the most common source of cell culture contamination, at least in my hands.
When working in a biosafety cabinet (hood) with cells cultured in multiwell plates or 10cm dishes, be especially careful to never wave your hand over or across the plate when it is open for manipulation. Of course, you should only lift the lid of the plate/dish when you need to manipulate it, but in cases where the plate is exposed for more than a couple of seconds, always keep your hands to either side of the plate and never cross over it. The reason is that dead skin and bacteria are always being shed off your hands/wrists, and even a tiny fleck will contaminate your culture with bacteria. Even if you are wearing gloves, your wrists are most likely still exposed and you could still contaminate your cells.
To avoid this, always make sure that everything is arranged in the hood in such a way that you never need to cross your hands, for example by keeping all your tip boxes and your trash bin to the right if you are right-handed.
Another general rule is to never be stingy with tips or pipets. If you have even the slightest suspicion that your pipet tip might have touched your skin or anything at all, just eject it and get a new one. Never load a tip onto a pipet and then set it aside, always load the tip immediately before using it. Yes, tips and pipets are not free, but the cost of you having to repeat an entire experiment because of contamination would be much higher than the single tip or pipet you are replacing. Be overly cautious, and focus on getting good data rather than saving money.
Thanks for this simple and great article, the tip with “do not cross while working” will be kept in mind, been to many labs till now but never was able to do some cell culture, starting tomorrow i am doing cell culture for 1 month, we will see what happens :D