Maintaining Asepsis in Cell Culture: Essential Tips
Cell culture is a fundamental technique in biological research, but its success hinges on maintaining aseptic conditions. Contamination can lead to inaccurate results, wasted resources, and significant delays. This article provides essential tips and best practices to help you establish and maintain a sterile environment for your cell cultures, ensuring reliable and reproducible data. Remember that consistent adherence to these principles is key to successful cell culture. You can also learn more about Molecularbiology and our commitment to quality.
1. Proper Sterilisation Techniques
Sterilisation is the cornerstone of aseptic technique. It involves eliminating all viable microorganisms from surfaces, equipment, and media. Several methods are commonly employed, each with its specific applications.
Autoclaving
Autoclaving uses high-pressure steam to kill microorganisms. It's ideal for sterilising heat-stable items such as:
Glassware (flasks, beakers, pipettes)
Culture media
Aqueous solutions
Instruments (e.g., forceps, scissors)
Procedure: Ensure items are properly wrapped or contained in autoclave-safe bags or containers. Autoclave at 121°C (250°F) for at least 15-20 minutes, depending on the volume of liquid or the density of the load. Always allow items to cool before handling.
Common Mistakes to Avoid: Overloading the autoclave, failing to ensure proper steam penetration, and using incorrect cycle settings.
Filtration
Filtration is used to sterilise heat-sensitive liquids that cannot withstand autoclaving, such as:
Serum
Growth factors
Antibiotics
Procedure: Use sterile filters with a pore size of 0.22 μm to remove bacteria and fungi. Attach the filter to a syringe or vacuum system and pass the liquid through the filter into a sterile container. Ensure the filter is properly seated and pre-wet before use.
Common Mistakes to Avoid: Using filters with an inappropriate pore size, compromising the sterility of the filter during handling, and exceeding the filter's capacity.
Chemical Sterilisation
Chemical sterilisation is suitable for surfaces and equipment that cannot be autoclaved or filtered. Common chemical sterilants include:
70% Ethanol: Effective for disinfecting surfaces and equipment.
Bleach (Sodium Hypochlorite): Used for decontaminating spills and waste.
Vaporised Hydrogen Peroxide: Used to sterilise entire rooms or equipment.
Procedure: Apply the chemical sterilant to the surface and allow sufficient contact time as per the manufacturer's instructions. Wipe off any excess residue with sterile water or ethanol.
Common Mistakes to Avoid: Using expired or diluted sterilants, insufficient contact time, and failing to properly ventilate the area after chemical sterilisation.
2. Working in a Laminar Flow Hood
A laminar flow hood (also known as a cell culture hood or biosafety cabinet) provides a sterile workspace by filtering air and creating a unidirectional airflow. There are different types of laminar flow hoods, but those used for cell culture typically provide vertical laminar flow.
Preparing the Hood
Cleaning: Before each use, thoroughly clean the work surface with 70% ethanol. Allow the ethanol to evaporate completely.
Arrangement: Organise materials within the hood to minimise clutter and ensure smooth airflow. Place frequently used items within easy reach.
Operation: Turn on the hood at least 15 minutes before starting work to allow the air to purge the workspace.
Working Inside the Hood
Movement: Work deliberately and avoid rapid movements that can disrupt the airflow.
Placement: Keep all sterile materials at least 6 inches from the front edge of the hood.
Sterile Technique: Always work with sterile gloves and use sterile techniques when handling cell cultures and reagents.
Common Mistakes to Avoid: Blocking the airflow with equipment, working too close to the edge of the hood, and introducing non-sterile items into the workspace.
Proper Waste Disposal
Place a biohazard bag inside the hood for disposal of contaminated materials.
Seal the bag properly before removing it from the hood.
Dispose of sharps in a designated sharps container.
3. Regular Monitoring for Contamination
Even with strict adherence to aseptic techniques, contamination can still occur. Regular monitoring is essential to detect and address contamination early.
Visual Inspection
Daily Checks: Examine cell cultures daily for signs of contamination, such as turbidity, changes in pH (indicated by colour change in the media), or unusual cell morphology.
Microscopic Examination: Use a microscope to examine cells for the presence of bacteria, fungi, or other contaminants.
Antibiotic Use
While antibiotics can help prevent contamination, their routine use is discouraged as it can mask poor aseptic technique and promote the development of antibiotic-resistant microorganisms. Use antibiotics only when necessary and with caution. Consider our services for cell culture solutions.
Mycoplasma Testing
Mycoplasma are small, cell wall-less bacteria that are common contaminants of cell cultures. They can alter cell behaviour and affect experimental results. Regular testing for mycoplasma is crucial.
Frequency: Test cell cultures at least every 3-6 months, and whenever you suspect contamination.
Methods: PCR-based assays, ELISA, and fluorescent staining are commonly used methods for mycoplasma detection.
Common Mistakes to Avoid: Ignoring subtle signs of contamination, relying solely on antibiotics to prevent contamination, and neglecting to perform regular mycoplasma testing.
4. Handling Cell Culture Vessels
The way you handle cell culture vessels (flasks, dishes, multi-well plates) significantly impacts the risk of contamination.
Opening and Closing Vessels
Sterile Technique: When opening a cell culture vessel, avoid touching the opening with your fingers or any non-sterile surface.
Flaming: Briefly flame the neck of the vessel after opening and before closing to create a convection current that prevents airborne contaminants from entering.
Tight Sealing: Ensure the vessel is properly sealed after use to prevent evaporation and contamination.
Pipetting and Transferring Liquids
Sterile Pipettes: Use sterile, individually wrapped pipettes for all liquid transfers.
Aseptic Technique: Avoid touching the pipette tip to any non-sterile surface.
Slow and Steady: Dispense liquids slowly and carefully to avoid creating aerosols that can spread contaminants.
Common Mistakes to Avoid: Touching the opening of cell culture vessels, using non-sterile pipettes, and dispensing liquids too quickly.
Storing Cell Cultures
Proper Labelling: Label all cell culture vessels with the cell line, passage number, date, and any other relevant information.
Designated Incubator: Store cell cultures in a dedicated incubator that is regularly cleaned and maintained.
Temperature and Humidity: Maintain the incubator at the appropriate temperature and humidity for the cell line being cultured.
5. Personal Protective Equipment (PPE)
Personal protective equipment (PPE) is essential for protecting yourself and your cell cultures from contamination.
Gloves
Sterile Gloves: Wear sterile gloves when handling cell cultures and reagents.
Glove Changes: Change gloves frequently, especially if they become torn or contaminated.
Powder-Free Gloves: Use powder-free gloves to minimise the risk of allergic reactions and contamination.
Lab Coats
Clean Lab Coats: Wear a clean lab coat when working in the cell culture lab.
Dedicated Lab Coats: Use dedicated lab coats that are only worn in the cell culture lab.
Regular Cleaning: Launder lab coats regularly to remove contaminants.
Face Masks
Surgical Masks: Wear a surgical mask to prevent the spread of respiratory droplets.
Proper Fit: Ensure the mask fits snugly over your nose and mouth.
Common Mistakes to Avoid: Reusing gloves, wearing contaminated lab coats, and failing to wear a face mask when necessary. Consult the frequently asked questions for more information.
By consistently implementing these tips, you can significantly reduce the risk of contamination and improve the reliability of your cell culture experiments. Remember that aseptic technique is a skill that requires practice and attention to detail. If you have any doubts about your technique, seek guidance from experienced colleagues or consult relevant resources.