TC Best Practices

When handling mammalian cells (aka tissue culture) always work in the Biosafety cabinet (BSC). Best practices are outlined below.

General Guidelines

  • All TC room equipment (e.g., tube racks, timers, 70% ethanol bottles, gloves) should stay in the TC room. Also, outside equipment should not be brought into the TC room unless absolutely necessary – spray these with 70% Ethanol.

  • Do not wear your TC coat into the bacteria area. The white coats should be primarily limited to use in the TC, the exception would be getting media from the fridge (Sven).

  • Discard gloves used for bacteria work before entering the TC room.

Getting started in the Tissue Culture (TC) Room

  1. Wash your hands.

  2. Don a fresh pair of gloves before touching things in the tissue culture room.

  3. If you have long hair, it is recommended to pull it back before starting TC work.

  4. Find your lab coat which should be singly hung on a hook (not stacked).

  5. Spray your gloves with 70% ethanol before opening the incubators or hoods.

Getting into a biosafety cabinet (BSC)

  1. Pull up the sash to the height indicated on the hood (the hood will beep if the height is incorrect). Spray down the surface with 70% ethanol to ensure a clean space.

  2. Check the stocks of serological pipettes, pipette tips, and resupply anything that is low.

  3. Check the waste bottle. It should be clear or slightly yellow. If it is pink, add bleach.

    If the bleach is above the taped line (~70% full):

    • Replace the old bottle with a new bottle (there are extras under the sink). Add some (~100 mL) bleach to the new bottle.

    • Add bleach to the old bottle if necessary, and wait at least 20 min for the waste to be decontaminated. Then pour the liquid down the sink and rinse out the bottle.

  4. Optional: For ease of cleaning, place a clear trash bag or other disposable bag in the waste container. A trash bag is required for all BSL2+ work.

Note

  • If you need to bring any extra materials (clean pipettes, pipette aids, markers, tube racks, etc) into the BSC, always spray down and clean with 70% ethanol.

  • Ensure the blowers and vents are functional (laminar air flow run) by waiting for the BSC to say it is on. If the BSC alarm is going off the sash may be at the wrong height and must be adjusted.

  • Only bring items you need into the BSC. Having too many items in the BSC interrupts the laminar air flow and can cause contaminants to be introduced.

  • Do not place items on the BSC grill; this creates a gap in the air laminar flow.

  1. Check the media and cells before using the BSC—turbid media usually means that the culture is contaminated. If contamination is suspected, follow the guidelines below.

  2. Bring any cells and reagents you are working with into the BSC. Spray down outside of all bottles and tissue culture plates with 70% ethanol, but be careful that no ethanol gets into the filter cap of T75 and T185 flasks.

While in the BSC

  • The BSC is ordered from clean (left) to dirty (right) with clean tips and pipettes at far left and the dirty the waste container at the far right. Order things appropriately for the work you are doing.

  • Use ethanol and Kim wipes to clean up small spills as soon as they happen. Wipe the bottom of plates before putting back into the incubators to limit liquid-mediated contamination.

  • Always keep reagent bottles, tubes and culture dishes/flasks closed when not in use.

  • Do not work above or over open bottles/tubes/cultures/etc. Open containers should be put far away from you such that no work is done above or over them.

  • Be sure you are working INSIDE the BSC, a minimum 3 inches from the grill (not on or above the grill).

  • Do not use your phone or computer while handling cells—this leads to contamination!

  • Do not block the air in-take vents at the front and back of the BSC or the air flow will be disrupted. (For instance, do not make a wall of tip boxes at the back of the BSC.)

  • Do not discard your cells with the media in them. Aspirate all liquids before putting plastics in the biohazard waste.

Finishing up work in the BSC

  1. Put away all reagents, cultures, and other materials you brought into the BSC.

  2. Discard trash in the biohazard waste bin (NOT for BL2+ work, see step 5 below and refer to BL2+ guidelines); if not using a trash bag, spray the benchtop biowaste container with 70% ethanol and wipe clean before placing it back in the BSC.

  3. In order to minimize contamination and cross contamination, run ethanol or bleach through the aspirator tip after use.

Note

If you run concentrated (>10%) bleach through the aspirator, run sterile water through afterwards. Bleach residue can degrade the aspirator, especially the multichannel ones.

  1. Spray the surface inside the BSC with Pre-Empt, wait one minute for decontamination, then wipe down the surface with 70% ethanol. Pre-empt can be sticky so “rinsing” with ethanol can help remove residue that accumulates over time.

Important

Pre-Empt is required after BSL2+ work, and should otherwise be used at least once daily by the last BSC user of the day.

  1. Pull the sash down and allow the UV light to run for one cycle (minimum 15 min). Following the UV cycle, BSL2+ trash can be removed and placed in the biohazard waste bin.

  2. Again, wipe the surfaces with Pre-Empt and ethanol following the removal of trash.

  3. Check the liquid biowaste collection bottle. If contents looks pink, add more bleach.

  4. Remove lab coat.

  5. Discard gloves in biohazard waste containers (NOT the regular trash can).

  6. Wash hands.

Note

If working with BSL2 (NOT BSL2+), you can skip step 6, wiping down with Pre-Empt after the UV cycle.

If your cell culture is contaminated

Media that appears cloudy/turbid or the clear presence of fungal growth (e.g. fuzzy/fluffy white ball) indicates the culture is likely contaminated. If contamination is suspected:

  1. Immediately bleach the culture for at least 20 min. Pour the liquid down the sink.

  2. Discard the flask/dish/plate in a biohazard waste bin (step can) and close and change the bin. Change your gloves immediately.

  3. Decontaminate any surfaces the contaminated culture came into contact with using bleach or 70% ethanol.

  4. Clean any surfaces in the incubator where the contaminated culture was stored. (Do not directly spray into the incubator; instead wet a Kimwipe with 70% ethanol and clean the surface.)

  5. Check the media bottle you used for the contaminated culture—if suspicious, bleach and discard the media.

  6. Change gloves again and continue working with the rest of your cultures.