Cell Culture Rules

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Tissue Culture Room Guidelines

A. Safe Laboratory Practices

1. Always wear gloves (change gloves when contaminated, and dispose of used gloves with other contaminated laboratory waste).
2. Tie long hair; Wear mask in flu and allergy season or as needed (sick or sneezing).
3. Do not eat, drink, handle contact lenses, and apply cosmetics.
4. Decontaminate all work surfaces before and after your experiments with 70% Ethanol.
5. Antibiotics should not be used routinely in cell culture.
6. If you observe anything out of compliance or just odd, contact Assigned Caretakers (Nitha for Kirk Lab and Sara for Pak).

B. Rules

1. Mycoplasma prevention

• Treat any non-disposable materials brought into cell culture room with Mycoplasma Erase link
• Add Plasmocin link to media.

2. Incubators

• Quarantine incubator: Received cells will go into “Quarantine” incubator, and should be tested for mycoplasma immediately.

Once cells pass the test, they can move to “Primary cells incubator” or Kirk Lab incubator.

If the cells are positive for Mycoplasma, lines must be disposed and any other cells in same incubator.

• Test all remaining cells in the room and do not grow new cells until test results are completed
• Treat entire room with Mycoplasma Erase
• Throw out all media
• Sterilize incubators
• All newly grown cells need to be quarantined until verified clean

Note. Mycoplasma testing should be implemented as a regularly: Upon new cell line arrival and before transferring to a new location (i.e. incubator, culture room), also once a month as routine verification of contamination free cultures.

3. Hood

• Turn on hood by raising sash and turning on fan; spray entire hood workspace with 70% ethanol.
• Keep only necessary items in the hood.
• If you need to bring something into the cell culture hood that is not in a sterile wrapper (i.e.

markers, tube racks, Tupperware, ice bucket) it needs to be decontaminated with Mycoplasma Erase spray first!

• • SpraywithEtOH–>wipedown–>spraywithMycoplasmaErase–>wipedown–> spray with EtOH –> wipe down
• After usage of the hood, spray with 70% ethanol and turn to germicidal (at least 20 minutes).
• Suck bleach into the catch when you are finished in the hood every time.
• No waste containers in hood or in room (except for trash and glass box).
• Maintain an aseptic work area.

4.Water Bath

• Refill with water if the water level is low.
• Squirt some detergent into the water bath.
• If there are any spills into the water bath, empty, clean and refill with water.

5. Refrigerator and freezer

• Always label your stuffs before to store.
• Keep the refrigerator and freezer clean.
• Be organized.
• Discard old media.

6. Scopes

*Make sure to clean after use and turn off. 

C. If I have antibiotics, what to do?

Antibiotics should not be used routinely in cell culture, because their continuous use encourages the development of antibiotic resistant strains. Once the antibiotic is removed from media, and may hide mycoplasma infections.
Antibiotics should only be used as a last resort and only for short time, and they should be removed from the culture as soon as possible (pass the cells two times and remove antibiotics from the culture).

D. Mycoplasma positive, what to do?

• Throw away all cells that tested positive and any other cells in same incubator
• Test all remaining cells in the room – do not grow new cells until test results are completed
• Treat entire room with Mycoplasma Erase
• Throw out all media
• Sterilize incubators
• All newly grown cells need to be quarantined until verified clean

E. Duties

Every week

1. Spray down surfaces with 70% EtOH, including all “visible” parts inside the hood (pipettors, benchtop and walls, sash esp. the handles), benchtops in the room, outside of incubators (any part you touch i.e. handles and buttons), centrifuge buttons, microscope surfaces (avoid the lens), water bath weights, fridge handles, chairs, etc.
2. Empty vacuum catch and replace with clean, autoclaved catch (add a little 100% bleach to the bottom of the catch, also immediately suck 10% bleach through the tubing to purge that); wash old catch (add bleach and scrub if dirty-looking).
3. Check water bath levels.
4. Fill the dewars with liquid nitrogen (check 2x/week)... don’t let them get below ~2.5 inches of

nitrogen, but don’t fill above the top of the bottom rack of boxes.

1. Check the CO2 tank levels (do this whenever you walk through the hallway; when the dials start to decrease below ~500, that tank is almost empty; when ~500, switch the dial to the full tank (make sure the valves are open) and close the old tank valves so CO2 doesn’t flow back into the empty tank; mark with label tape that it is empty and place an order for a new tank using the order sheet located in the desk behind Joe, give the completed sheet to Joe, tanks are usually delivered Mondays and Thursdays).

Every month

1. Drain, clean, and refill water bath (make sure you add the water bath treatment solution).
2. When cleaning the catch, wash and autoclave tubing (after a few autoclave cycles, the plastic will start to break down, so you’ll eventually need to replace with fresh tubing, located under the culture room sink).
3. Do a more “thorough” EtOH spray of hoods (lift up the “benchtop” to clean underneath in addition to cleaning all the other surfaces. You might be able to do this without taking everything out, but if you do take things out, they’ll need to be thoroughly sprayed before putting back in).

Every 6 months OR after positive mycoplasma test:

1. Mycosensor PCR screen for mycoplasma of all cells and media currently in use.
2. Obsessively clean the hoods:take everything out, spray down entire inside of hoods (mycoplasma spray, then water, then bleach, then water, then EtOH; don’t forget to lift up the “benchtop” to get underneath), spray everything with mycoplasma spray (then water, then EtOH) before putting back in hood (open new tip boxes and glass pipette box), autoclave vacuum tubing and replace catch with clean autoclaved catch.
3. Sterilize incubators: for the “old” incubators, turn off incubators and shut off CO2 (using the nozzle in the room), remove shelves and shelf holders (spray with ethanol, when dry, wrap in foil and autoclave), spray insides of incubators with bleach, water, ethanol, when shelfs are cooled, replace and start the “steri-cycle” (consult manual, in culture room drawer under microscope), when complete ~24 hours later, start “auto-start” routine, when that is complete, check the temperature and CO2 calibration with a thermometer (it will have to be submerged, so equilibrate a beaker of water to the incubator temp, then read water temp with thermometer) and fyrite CO2 meter; recalibrate if needed (again, consult manual).

• • For the “new” incubators, consult .pdf manual ***there are parts (hypoxia chamber, etc.) that need to be removed from new incubators before running steri-cycle!