How clean are you? Top 10 tips for optimal sterile technique
By: Roche Life Science
Posted: September 25, 2015 | Everyday Essentials for Research
We know it's out there, lurking in the shadows. We try to pretend it only happens to other scientists. But eventually, when you least expect it, you open the incubator door and pick up your flask of cells, see the cloudiness and color change in the media of what can only be… contamination. You instantly hope that your remaining precious cell lines are free from this plague-like monstrosity and you have indeed identified patient zero of the tissue culture epidemic. And once the wave of death and destruction have passed, and your cells are blossoming once again, it is important to identify any potential future risk and work to optimize sterile technique to prevent future contamination.
Sterile (or aseptic) technique is the manner by which procedures or tasks are completed in an effort to minimize or prevent introduction of contaminating microorganisms from the environment. Indeed, many tissue culture mishaps and headaches are rooted in improper sterile technique methods. And while antibiotics are useful for reducing these complications, they do not prevent gross contamination from poor technique or even antibiotic-resistant organisms.
Therefore, learning and adhering to proper sterile techniques early on is essential for future success in tissue culture experiments, and will save you much-needed time and energy in the long run. Thus, here are our top 10 tips for optimizing your sterile technique:
1. Plan ahead. This might seem obvious, but it is often overlooked. Planning your experiments ahead of time allows you to minimize the number of times you leave the tissue culture hood and number of extra movements you need to make. Know how many tubes, flasks and plates you need. Set them up and label them ahead of time. Have reagents diluted and aliquotted as needed prior to using them.
2. Be clean from the start. Be sure the hood is clean (it should be cleaned with disinfectant before and after every experiment). Ensure everything you bring into the hood is clean and appropriate for a sterile experiment. Media and reagents should be purchased sterile (tissue culture grade) or applied through a sterile (0.22 micron) filter prior to use. If you wear a lab coat, it should be cleaned regularly, or even use a designated tissue culture lab coat. Keep a spray bottle of ethanol outside the hood and use liberally on the hood surface as well as any bottles, racks, or instruments going into the hood. You can wipe things down with a kimwipe.
3. Use aliquotted reagents. Media, serum, and antibiotics should be carefully aliquotted into appropriate volumes for individual use. This prevents potential contamination of stock reagents and disruption of multiple experiments should contamination occur. All aliquots should be dated, so any potential stock contamination can be traced to any other bottles.
4. Do the tilt. When pipetting liquid from a bottle, flask or tube, tilt the container toward the hand with the pipet. This will minimize exposure of the pipet tip to the neck/mouth of the bottle, which can potentially harbor organisms. Similarly, avoid rotating or turning flasks upside down or shaking. This can get media up to the neck of the flask, which can be a nidus for contamination. If media gets in the neck, carefully aspirate away with vacuum suction using a sterile tip.
5. Don't double dip. It's good advice for salsa at parties, but this etiquette rule also applies to your media stock bottle. Whenever in doubt, change pipet tips. It's not worth the risk of contaminating your stock bottle.
6. Caps on. Leave lids and caps on top of their respective plates and/or bottles as much as possible during your experiment. When you do need to remove a lid or cap, place it in the back of the hood with the underside up and be careful to replace them back on as soon as possible. It is easier to loosen caps ahead of time with two hands than trying to do it one-handed after your pipet is full.
7. One at a time. It's best to manage and handle only one cell line at a time in the hood. This eliminates the risk of cross contamination between cell lines when more than one cell line is in use at a given time. Once this happens, it can be difficult to trace back to when it occurred and can significantly impact experimental results.
8. Quarantine. All incoming cell lines should be quarantined and tested for mycoplasma prior to use. Alternatively, purchase your cell line from a vendor that certifies they are mycoplasma-free prior to arrival. This will prevent a lot of headaches if a new cell line turns out to be mycoplasma-positive.
9. Daily inspection. Cell lines should be evaluated daily for signs of contamination due to the risk of contamination to other cultures and experiments. If identified, cells should be promptly discarded. If an attempt is made to treat the cells instead, this should be done at the end of the day to minimize risk to other cells and experiments, and the hood should thoroughly cleaned afterward.
10. Sometimes, sharing is not caring. Cross-contamination can happen with the sharing of tissue culture reagents, and media and should be avoided between individuals. It may seem like the polite thing to do, but in the end, can result in significant issues should experiments become ruined due to contamination and other individuals are blamed.