Basic Sterile Technique

I found that spraying everything that goes into the hood with 70% EtOH, especially the gloves. helps cut down contamination rates to a minimum. Considering how cheap ethanol is, this is not the place to be economical. Do not cross hands while working to prevent cross contamination. Do not pour media out of a culture flask or plate; always pipette media in and out of the flask or plate so as not to establish a liquid bridge to the surroundings. Similarly, it is important to mop up any spills immediately and not to get media on the outside of the culture flasks. When adding media, pipette it on the side of the flask so that no bubbles are created.

It is convenient to aliquot out medium supplements (serum, pen/strep etc. ) so as not to subject them to repeated freeze-thawing. As a matter of routine, I add all supplements to the medium through a 0.2 micron filter. Serum frequently contains much particulate matter and requires more filtration. I denature fresh bottles of serum at 65 °C for 1hr, and then aliquot it into 50ml quantities through a filter. I filter the aliquots again when adding them to the medium.

For cell culture, the problem is not so much with bacteria contamination then with fungal contamination, as the growth media contains antibiotics. A common way to help minimize the chances of fungal contamination is to use 1% (w/v) copper sulphate solution in the incubator. Copper inhibits a specific fungal metabolism pathway. However, I find the effectiveness of this approach doubtful at best, since copper sulphate is not volatile. Better incubators are made with copper-lined internal surfaces, but they are very expensive. It is important to examine the cell culture visually before sub-culturing.

A more insidious problem is mycoplasma contamination, which is invisible even under the microscope. If abnormally slow growth is encountered, there is possible mycoplasma contamination or over-trypsinization. If slow growth is persistent over a few passages, contamination is probable. Throw away the culture flasks and start again, making sure that your stock medium is not contaminated as well. However, slow growth is usually encountered when starting a new culture, as the cells take time to adapt to the new environment. There are also mycoplasma detection kits available, although their use is more for peace of mind, as no effective remedial action is possible upon mycoplasma contamination (short of a few heroic measures, to be undertaken only if the cell lines are utterly irreplaceable).