1. Always label your flask/plates/dishes/any samples with your name, date of the experiment/seeding, and other information. Besides being standard cell culture practice, it helps other lab members identify your samples in the event you are out sick and need help or if they become contaminated.

2. No opened containers/medium bottles/cell culture flasks or dishes outside of the hood

3. No aspirations of medium or solutions outside of the hood The biosafety hood is meant to protect your samples as well as yourself, aspiration creates aerosolized droplets which can make contact with you if not properly shielded.

4. Certain supplies should always be kept in the hood (glass aspirators, pipettors, pipette tips, etc.) Removing supplies from the hood on an individual basis risks contamination regardless of how much tape/ethanol/preventative measures you use.

5. Ethanol and other cleaning solutions need to be used correctly Ethanol does not kill contaminants on contact, it takes 10-20 seconds to be effective and the heavier duty bio-disinfectant we have takes 5 minutes to sterilize a surface. → Briefly coating our materials in these solutions, wiping them down, and placing them in the hood does not prevent contamination

6. To avoid while working inside the hood:

• Talking/coughing/sneezing etc. during culture
• Frequently reaching in and out of the hood (every time your hands cross the grille plate, they are no longer sterile and need to be retreated with ethanol, therefor minimizing movements which cross that boundary are ideal)

7. Work flow during the day should be optimized from most sterile work to least sterile (e.g. if you need to do RT-PCR, nanodrop measurements, and cell culture medium change, start with medium change; bacteria and viruses are not typically airborne, but mycoplasma and fungal spores can and will jump from room to room)

8. Use of common equipment needs to be done as cleanly as possible

• Clean any microscope after you use it with appropriate solvents
• Hoods should be UV treated 15-20 mins before and after your session (should be done as a supplement for ethanol and bio-cide cleanings).
• Common bench space and tops of equipment should be cleaned on a regular basis (ideally every 2-4 weeks) → this will prevent build up of dust or other contaminants on working surfaces

• Everyone should self report any contaminated samples, or label your flasks so that others can identify. Accidents happen and everyone makes mistakes. This is not identification for punishment, but so that we can figure out who else you may have interacted with and which other samples may be at risk.
• Please ask Russ from Duarte-Campos lab for help.
• The flask should be treated with bleach or another ammonium based antiseptic for 20 minutes or until color change is complete.
• The flask will then be put in an S1 disposal bin, after removing the cleaning solution, then packed up and removed from the lab
• Everyone not affected will start testing their samples weekly for 3-4 weeks until everyone is clean.
• Incubators where contaminated cells were kept will be sterilized, along with the hoods where cell culture work was done
• Liquid waste bins and vacuum tubing will be changed as well.

General sterile work guidelines are very nicely described on the Thermo webpage: Thermo aseptic techniques