Aseptic Cell Culture Best Practices

Overview

These notes summarize key principles and practical steps for maintaining sterile (aseptic) technique in cell culture to prevent contamination and ensure reproducible experiments.

Importance of Sterile (Aseptic) Technique

• Sterile technique prevents contamination of cell lines and primary cells by bacteria, fungi, and mycoplasma.
• Contamination can ruin experiments and compromise data reliability and reproducibility.
• Microorganisms are present in air and on lab surfaces, so cell culture tasks must be done in a controlled sterile environment.
• Good technique is more crucial for sterility than the sterility of individual reagents or consumables alone.

Biosafety Cabinet (Cell Culture Hood)

• The biosafety cabinet (cell culture hood or tissue culture hood) provides a sterile environment for cell culture work.
• It uses laminar air flow as a barrier to reduce contamination by airborne particles in the room.
• Tasks involving open cell culture vessels should be done inside the cabinet to protect cells from airborne microbes.

Biosafety Cabinet Setup and Use

• Turn on cabinet lights and air filter before starting any work.
• Adjust the glass sash height to the manufacturer’s specified mark for proper airflow.
• Run the air filter for at least 15 minutes before beginning work.
• Avoid cluttering the cabinet; overcrowding can disturb the air curtain and reduce protection.
• Plan work so only necessary items for each step are inside the hood at one time.

Personal Protective Equipment (PPE) and Safety

• Before starting, wash hands thoroughly.
• Wear PPE: lab coat, long sleeves, gloves, and safety glasses.
• PPE reduces contamination risk to cultures and provides personal protection from biological materials.
• Follow local biosafety guidance, including procedures for waste disposal and handling specific materials.

Disinfection and Ethanol Use

• Use a spray bottle with 70% ethanol to disinfect gloved hands before work in the cabinet.
• Spray the interior surfaces of the biosafety cabinet and wipe with lint-free wipes or paper towels.
• Every item placed in the cabinet must be disinfected with ethanol and wiped before use.
• Pay special attention to disinfecting caps and necks of media bottles and reagent containers.
• If liquid spills or drops appear on the outside of vessels or in the cabinet, clean immediately with ethanol-sprayed wipes.

Water Bath Maintenance

• When pre-warming bottles in a water bath, add bactericidal and fungicidal agents to the bath water.
• Replace water in the bath regularly to minimize microbial growth and contamination risk.

Arrangement and Movement in the Cabinet

• To minimize disruption of the air curtain, avoid lateral arm movements whenever possible.
• Move arms straight in and straight out from the front of the cabinet.
• Keep pipettor and consumables on one side, depending on right- or left-handedness, for efficient workflow.
• Place other items toward the back, leaving the center as the primary working area.

Structured Summary of Cabinet Practices

Handling Bottles, Tubes, and Caps

• When removing caps from bottles or tubes, do not place them face up on surfaces.
• Place caps face down on the cabinet surface to reduce risk of contaminants entering.
• Optionally, hold the cap in the non-dominant hand while pipetting, which requires practice and dexterity.
• Replace caps on flasks, bottles, or tubes as quickly as possible to limit exposure to air.
• Ensure caps are threaded and closed properly to prevent leaks and contamination.

Pipetting Technique and Tip Use

• For serological pipettes, open the end of the wrapper and attach the sterile end firmly to the pipettor.
• Rotate the pipette so that graduations are clearly visible for accurate volume measurement.
• Avoid touching pipette tips to the exterior of any vessels to prevent contamination.
• Avoid contact between pipette tip and interior surfaces of flasks or bottles, especially necks and caps.
• If liquid gets onto the outside of a vessel, clean it promptly with ethanol-sprayed wipes.

Pipette and Tip Use Guidelines

Preventing Cross Contamination

• Use each pipette tip once; do not reuse tips between samples or reagents.
• Move through each step deliberately and efficiently to minimize open-time of cultures.
• Do not rush; haste increases risk of human error and contamination events.
• Experience and consistent practice are essential to become proficient in aseptic technique.

Labeling and Post-Work Cleanup

• Label all cell culture flasks, bottles, and tubes as needed before removing them from the hood.
• After finishing work and removing all items, wipe the interior of the cabinet again with ethanol.
• Maintain a tidy workspace to support both personal experiments and shared-use expectations.

Shared Cell Culture Room Etiquette

• The cell culture room and biosafety cabinets are usually shared resources.
• Schedule hood time considerately so others can access equipment as needed.
• Keep the area clean and organized for subsequent users.
• Communicate any issues (e.g., contamination concerns, equipment problems) so everyone can maintain successful experiments.

Key Terms & Definitions

• Sterile technique (Aseptic technique): Set of practices to prevent contamination of cultures by unwanted microorganisms.
• Biosafety cabinet (Cell culture hood/Tissue culture hood): Enclosed workspace using laminar airflow to reduce airborne contamination.
• Laminar air flow: Smooth, directional airflow that acts as a barrier against airborne particles and microorganisms.
• PPE (Personal Protective Equipment): Protective clothing and gear such as lab coat, gloves, long sleeves, and safety glasses.

Action Items / Next Steps

• Practice organized movement and pipetting inside the biosafety cabinet to build skill and consistency.
• Review and follow local biosafety and waste disposal guidelines specific to your laboratory.
• Regularly check cabinet function, water bath condition, and cleaning routines to maintain sterility.
• Continue to refine labeling and workspace organization to improve efficiency and reduce contamination risk.