Central Cell Services | Protocols

The introduction of a cleaning routine into the tissue culture laboratory should result in lessening the risk of contamination, maintenance of researcher’s safety, and fewer problems with cell growth.

1. Check incubator temperatures and CO2 levels daily
2. Wipe down bench surfaces with 70% alcohol
3. Wipe the working surface of the laminar flow hood with 70% alcohol
4. If using a vacuum, check the pump is functioning and that the aspirator is clean and contains Clorox
5. Any media bottles, etc. should be wiped with 70% alcohol before placing in hood
6. Water baths should be cleaned regularly and replaced with fresh water
7. The use of Bunsen burners in hoods is not recommended as the flame disrupts the air flow
8. After use of hood, turn off vacuum pump, discard any waste into suitable bags for autoclaving, check there is sufficient Clorox in waste flask and the flask is not over-full, and wipe interior with 70% alcohol. Turn on UV for a short time
9. Cell cultures should be observed microscopically daily for one to become familiar with the morphological appearance of different cells. This will lead to an ability to see when irregularities and contamination occur which, in turn, may allow one to rescue or discard a culture at an early stage
10. At the end of the day remember to switch off microscopes, turn off water baths, etc. and check to make sure incubator doors are closed, and lids on LN2 tanks are replaced properly
11. Clean all hoods thoroughly, removing the working surface tray so that the base is accessible. Spillages can easily get through to the base. Clean with a bacdown solution and wipe with 70% alcohol
12. Clean out all incubators regularly and thoroughly, removing all shelving and paying particular attention to door seals. If a tray is been used to hold water for humidification, this should be removed, cleaned and refilled with fresh sterile water. Roccal (Benzalkonium chloride- from Sigma Aldrich) can be added to the tray at the specified concentration (1:64 from a 10% solution)
13. Check incubator water levels before the weekend and top-up if necessary
14. Check HEPA filter inside the incubator- replace every 6 months.
15. Cell stocks should not be in culture for more than three months. Recover a new vial and make sure cells are tested regularly for Mycoplasma. If cells are contaminated with mycoplasma it is wise to discard cultures. If that is not possible cells can be treated with antibiotics such as MRA (Mycoplasma Removal Agent) or Plasmocin. Double the treatment time to ensure mycoplasma is completely gone

Note: A thorough cleaning should be done every 6 months

1. Remove all items from the hood
2. Turn off alarm and lift front panel as high as possible
3. Turn blower and UV off
4. Remove bottom solid and grid trays by unscrewing both left and right
5. Keep screws in a safe place for later
6. The bottom tray is heavy so make sure there is help available
7. Bring the panels to sink area if possible
8. Wash well with bacdown and wipe with 70% alcohol solution
9. The base of the hood will be dusty and will need a thorough cleaning with bacdown and 70% ethanol
10. Clean interior walls of the hood as in step 8
11. Put grid panel back first, then put the large panel back and screw securely
12. Give a final wipe all over with 70% alcohol including glass
13. Put one opened nutrient agar plate in the hood for 1 hour
14. Remove agar plate, cover, wrap in parafilm and store inverted at 37°C
15. Close hood cover and turn on UV for a short time
16. Check plate next day for contamination growth. If there is any contamination the hood will need to be cleaned again

• General Cleaning and Maintenance of the Laboratory
• Preparation of Buffers for Dialyzed Serum
• Cleaning TC Hoods