Aseptic Technique: Keeping Cell Cultures Clean and Healthy
Aseptic technique refers to the set of practices used to prevent contamination from microorganisms such as bacteria, fungi, and yeast when working with cells. Because cell culture media is warm and nutrient-rich, even a tiny lapse in technique can allow contaminants to grow quickly and ruin an experiment.
Good aseptic technique protects your cells, your data, and your time.
Why Aseptic Technique Matters
Contamination can:
- Kill or stress cells
- Alter cell behavior and experimental results
- Spread to shared incubators and equipment
- Go unnoticed for days (especially mycoplasma)
Once contamination occurs, cultures often need to be discarded. Consistent aseptic technique dramatically reduces these risks and helps ensure reproducible, trustworthy results.
Core Principles of Aseptic Technique
At its heart, aseptic technique is based on a few simple ideas:
- Assume everything is contaminated unless proven otherwise
- Minimize exposure of sterile items to the environment
- Create a clean-to-dirty workflow
- Work deliberately, not quickly
Preparing to Work
Before starting any cell culture work:
- Tie back long hair and secure loose clothing
- Wash hands thoroughly
- Put on appropriate PPE (lab coat, gloves, eye protection as required)
- Disinfect gloves and the biosafety cabinet work surface
- Gather all materials before starting to minimize movement in and out of the cabinet
Preparation reduces interruptions, which in turn reduces contamination risk.
Working in the Biosafety Cabinet
Maintain Sterility
- Work 4–6 inches inside the cabinet
- Keep sterile items (media, pipettes) upstream of “dirty” items (waste)
- Never block the front or rear grilles
Handle Items Correctly
- Only open sterile containers when needed
- Keep caps facing down or held in your hand—never set them on the surface
- Avoid touching sterile tips or bottle openings
Move Thoughtfully
- Use slow, deliberate movements
- Avoid reaching over open containers
- Minimize talking, coughing, or sneezing toward the work area
Using Pipettes and Media
- Always use sterile, filtered pipette tips
- Avoid touching the pipette shaft to non-sterile surfaces
- Gently dispense liquids to avoid splashing or aerosol formation
When in doubt, change the tip—it’s faster than recovering from contamination.
Gloves: Your First Line of Defense
- Treat gloves as potentially contaminated
- Disinfect gloves frequently with 70% ethanol
- Change gloves immediately if you touch a non-sterile surface (e.g., phone, door handle)
- Never leave the lab wearing gloves
Clean gloves protect both you and your cultures.
Finishing Your Work
When you’re done:
- Close all containers securely
- Disinfect culture vessels before removing them from the cabinet
- Dispose of waste properly
- Wipe down all surfaces
- Allow the cabinet to run for a few minutes before shutting down or walking away
Good cleanup habits protect the next person who uses the cabinet.
Common Mistakes to Avoid
- Rushing through procedures
- Overcrowding the biosafety cabinet
- Storing items in the cabinet
- Reaching over open flasks or plates
- Assuming antibiotics will “fix” poor technique
Antibiotics can mask contamination but do not replace aseptic technique.
Final Thoughts
Aseptic technique is a skill developed through consistency and awareness, not speed. Everyone makes mistakes when learning, but careful habits and attention to detail will quickly become second nature.
Remember:
Clean work protects good science.
Reflection Questions
- Experienced scientists write their initials on their reagents and media bottles, and they expect other members of the laboratory not to use their supplies. Why might scientists be protective over their reagents and media?
- Fomites are objects that can carry pathogens, especially when someone handles them without aseptic technique or without disinfecting them. What are some fomites that might pass through a biosafety cabinet or clean room?
- Today, you plan to work with two cell lines. One of the cell lines came from a collaborator and has not been tested for mycoplasma. You plan to create a flask for mycoplasma testing and a flask for expanding the culture. In what order should you handle your cell lines today? (Work with your original cells and then the collaborator cells, or work with the collaborator cells first and then your original cells?) Why or why not?
- How do filtered pipet tips help prevent cross-contamination? What does the filter do?
- You have a meeting in twenty minutes. You think you can culture your cells in this time frame. You have cultured cells for months now.
Should you rush the procedure, or should you wait until after your meeting to perform cell culture? Why or why not?
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