Sterile techniques are critical in cell culture to prevent contamination from microorganisms (bacteria, fungi, and viruses) and cross-contamination between cell lines. Contaminants can compromise experimental results and harm the integrity of the cultures. Below is an overview of essential sterile techniques for cell culture:
1. Work Area Setup
- Use a Laminar Flow Hood or Biosafety Cabinet (BSC)
- Ensure the hood or cabinet is cleaned and decontaminated with 70% ethanol or a similar disinfectant before and after use.
- Maintain a steady airflow to create a sterile working environment.
- Avoid blocking air vents with equipment or arms during work.
- Minimize Traffic
- Limit movements and personnel in the cell culture room to reduce airborne contaminants.
2. Personal Hygiene and Protective Gear
- Hand Hygiene
- Wash hands thoroughly with soap and water before starting.
- Use gloves during cell culture work and replace gloves if they become contaminated.
- Protective Clothing
- Wear a lab coat, gloves, and in some cases, a face mask or goggles to reduce contamination risks.
3. Sterilization of Equipment and Materials
- Sterilize Consumables
- Use pre-sterilized disposable consumables like pipette tips, culture flasks, and petri dishes.
- Autoclave reusable items such as glass pipettes and metal instruments.
- Use Sterile Solutions
- Filter or autoclave all solutions (e.g., media, buffers) before use.
- Discard expired media or reagents.
- Handle Sterile Items Properly
- Open sterile packages only in the laminar flow hood.
- Avoid touching sterile pipette tips, bottle necks, or flask openings.
4. Handling Procedures
- Minimize Exposure
- Keep culture containers closed as much as possible.
- Work quickly and efficiently to reduce exposure to airborne contaminants.
- Proper Pipetting Technique
- Avoid touching the pipette tip to any non-sterile surface.
- Use aseptic technique to prevent contamination of pipettes or media bottles.
- Flaming or Sterilization
- Use a Bunsen burner or ethanol lamp to sterilize tools like forceps or scissors.
- Avoid excessive flaming that might damage materials or generate aerosols.
5. Cleaning and Maintenance
- Clean Work Areas
- Decontaminate surfaces with 70% ethanol or disinfectant before and after each session.
- Regular Equipment Maintenance
- Ensure incubators, water baths, and centrifuges are cleaned regularly and monitored for contamination.
- Use sterile water in incubator water pans and change it regularly.
6. Monitoring and Prevention
- Routine Checks
- Inspect cultures regularly for signs of contamination (e.g., cloudy media, abnormal cell morphology, or fungal growth).
- Use Antibiotics Sparingly
- Only use antibiotics when necessary, as over-reliance can mask low-level contamination.
- Mycoplasma Testing
- Periodically screen cultures for mycoplasma contamination.
By adhering to these sterile techniques, you can ensure high-quality, contamination-free cell cultures for reliable experimental results.