Aseptic Techniques
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Aseptic techniques ensure that all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi, mycoplasma and cross contamination with other cell lines. All work is done under a tissue culture hood. A movable glass panel or sash covers the face area of the tissue culture hood and acts as a physical barrier that helps to maintain a particulate-free environment and laminar air flow.
In case the sash stop is overriden an alarm will sound.
Before you start your work:
- be sure the UV light is off;
- let the air flow for 2-3 minutes to filter and establish air flow patterns;
- spray interior surfaces with 70% ethanol and wipe surfaces dry;
- avoid rapid movements that will disrupt laminar air flow;
- all equipment that will be taken into the cabinet such as media bottles, pipette tip boxes, and your gloves should be sprayed with 70% ethanol.
Among the most common procedures performed at the clean bench are liquid transfers using a pipette or micropipette. Begin by loosening the cap of the conical tube inside the hood before you open the pipette cover. Open the cover from the bottom of the pipette. Make sure the tip of the pipette does not touch areas that are not sterile. Put the pipetter on the pipette. Aspirate the appropriate amount of liquid. Again, be very careful that the tip of your pipette does not touch the outside of the tube or any other area that is not sterile. Release the media. Do not reuse the pipette unless you are making multiple plates that are identical. Use a fresh pipette if you use different cells or different media.
When using the micropipette, spray it with 70% ethanol and wipe it dry. Inside the hood all work is done with sterile pipette tips. Once again, loosen the cap of the conical tube and your flask before you start. Insert the tip on the micro-pipette, taking good care that the tip does not touch any surface. Aspirate the appropriate amount and loosely set the cap back on the conical tube. Again, ensure that the tip does not touch any other surface, open the flask and release the liquid. Discard the tip.
When your work is done:
- dispose of all waste
- spray the work surface inside the hood with 70% ethanol
- dispose of tissue by autoclaving
- at night, turn the germicidal UV lamp back on
