Operating Procedures
When performing cell culture, it is essential to maintain precise and controlled movements. Avoid rushing, as this may increase the risk of contamination due to air turbulence. Always avoid touching sterilized tools with bare hands. If accidental contact occurs, immediately sterilize the tool using a flame or replace it with a new one. Keep your work area well-organized to improve efficiency. Generally, place your right-hand tools on the right side, left-hand items on the left, and position the alcohol lamp on the left. Ensure that all procedures are conducted in an orderly manner from start to finish, and never expose tissues or cells to the air before they are treated. Similarly, do not open culture bottles too early; if the bottle will not be used again, close it immediately to reduce contamination risks.
When handling liquids such as nutrient solutions, PBS, or cell suspensions, always use separate pipettes to prevent cross-contamination. Avoid talking or coughing within the sterile field, as this can introduce bacteria or mycoplasma. Before starting any procedure, wipe the clean bench and your hands with 75% alcohol. Never pass your hands or dirty objects over the mouth of an open container, as this is the most vulnerable point for contamination. If a pipette tip touches the bottle mouth, discard it immediately to ensure sterility.
Preparation Before Training
Before beginning any experiment, plan the procedure thoroughly. Calculate all necessary data in advance and prepare all required equipment and materials. Only after confirming that everything is ready should you proceed with disinfection. This helps minimize unnecessary movement in the lab and reduces the chances of contamination.
Disinfection of the Work Area
The sterile culture room should be mopped daily with 0.2% Xinjieer (a disinfectant), using a dedicated mop. Additionally, perform ultraviolet (UV) radiation for 30–50 minutes each day. Before each experiment, clean the ultra-clean workbench with 75% alcohol, followed by UV disinfection for 30 minutes. Be careful not to expose cultures or solutions directly to UV light. Avoid overcrowding the workbench during disinfection, as this can block UV rays and reduce effectiveness. Items like pipettes, waste containers, and test tube racks should be cleaned with 75% alcohol and placed on the table while UV is active.
In vitro cultured cells have no natural defense against infections, so preventing contamination is critical to the success of the culture. Even in a well-equipped lab, improper technique or carelessness can lead to failure. To ensure maximum sterility, every step must be carried out carefully and methodically, without shortcuts.
Washing and Attire
Follow strict hygiene protocols similar to those in surgery. If you are only observing, you may wear clean lab clothing for up to 30 minutes. When working inside the clean bench, insert your entire forearm into the box and wear long-sleeved lab coats. Disinfect your hands with 75% alcohol before starting. If your hands come into contact with potentially contaminated objects, wash them again with disinfectant before continuing. Before entering the primary culture room, wash your hands thoroughly, wear a mask, and put on a sterile lab coat.
Flame Sterilization
During aseptic procedures, always ignite an alcohol or gas lamp first. All subsequent actions—such as opening or closing a bottle, attaching a pipette tip—should be done near the flame to ensure sterilization. However, avoid leaving metal tools in the flame for too long, as this can cause annealing. A burned crucible should be cooled before use to prevent damage to the tissue. Once a pipette has absorbed nutrient solution, it should not be reused after being burned, as residual liquid can carbonize and contaminate the solution. When opening or closing a cell culture flask, keep the flame exposure short to avoid overheating the cells. Also, avoid holding rubber stoppers over the flame for extended periods, as this may produce harmful gases that could harm the cultures.
By following these guidelines, you can significantly reduce the risk of contamination and ensure successful cell culture outcomes.
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