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For those who do not wish this level of containment, the movement of people and equipment in and out of the room still should be minimized. People should have laboratory coats that are worn only in the culture room. The culture room should be maintained under positive air pressure and the air HEPA-filtered if possible. Disposable sticky mats just inside the culture room door are a reasonable precaution and minimize tracking dust and spores into the culture room.
The inside of the room or area used for cell culture should be well organized and not overcrowded. It is best to store cases of supplies elsewhere and have a cabinet in the room for the minimum of supplies necessary, for example, for a weeks' use. This can be restocked periodically. All tissue culture supplies should be used on a first in"Cfirst out basis, since sterility cannot be guaranteed after prolonged storage. The following tips will help maintain a clean culture room and minimize contamination:
1. Bench tops, hood surfaces, microscopes, and other work surfaces should be wiped down periodically with ethanol or another germicide.
2. The inside of refrigerators should be kept clean and uncluttered. Do not store tubes or bottles for long periods of time. Mold grows easily on labels, tube racks, and other containers left in the moist, cool environment of the refrigerator. Then, when the culturist removes reagents for use in cell culture, the contamination is spread to the working surfaces and the cultures.
3. Open water baths sometimes used in tissue culture laboratories to preheat medium are a major source of contamination. If these must be used, the water should contain a disinfectant and should be changed regularly.
4. The incubator is the optimal environment for the growth of many contaminants, as well as mammalian cells. The outside of plates can grow mold, as well as the water in the incubator or condensation on the doors, plates, seals, and surfaces. Water used for humidity inside incubators should contain a (nonlabile) germicide and surfaces should be wiped down with germicide frequently.
5. Trays that are removed from the incubator between experiments should be decon-
taminated or autoclaved and stored in a clean place until the next use.
6. Always clean up any spilled medium immediately since medium is the very best way to grow unwanted bugs, wigglies, balls of fluff, and any other not readily identifiable entities in the cultures.
7. Minimize movement of people in and out of the culture area. Needless to say (I hope), NO food or drink in the culture room.
What Can You Do to Get Rid of Contamination in Cultures?
As much as people do not want to hear it, the best way to get rid of contamination in cultures is to (carefully) discard all contaminated cultures. This can sometimes mean all the cultures in the laboratory. In such unfortunate circumstances, all plates should be discarded and new vials thawed from the frozen cell banks or obtained from the supplier of the original cells. Obviously, if cell lines are frozen down frequently, as suggested above, this will be feasible most of the time. The only time that one should even try to "cure" an infection is if the cell line at stake is irreplaceable. This can happen if one is developing a cell line or selecting a mutant cell line or a line with significantly altered properties. There is always a window of exposure during this process when the contamination of a single plate can mean having to start over and the loss of months of work, or worse. In this case, an attempt at getting rid of the contamination may be worthwhile. The process of curing a cell culture of adventitious agents is not easy and the gain must be weighed against the risk of spreading the infection to other cell lines in the laboratory. It is expensive and time consuming. There is often a high relapse rate and one should be prepared for failure.
The first step is to reduce the burden of infectious agent as much as possible. Thus, one can physically remove a fungal particle, or extensively wash cultures contaminated with yeast, bacteria, or mycoplasma. Remember that the density of contaminant per milliliter of culture medium may be several logs higher than the cell density. After this washing step, one can treat the cultures with bactericidal or fungicidal or fungistatic agents. All of these are toxic to cells at some level. One hopes to kill the unwanted contaminant before permanently damaging the culture. With bacteristatic or fungistatic agents, the goal is to cause a large enough differential in growth rate to let the cultures outstrip the contamination. At the end of this treatment, the cells should be recloned by limiting dilution. In this way, even if the culture is still contaminated, as long as the contaminant is at a much lower density than the culture, it should be possible to find wells containing only cells. This cloning and subsequent passages should be done in the absence of any antibiotics or other antimicrobials. After two to three passages in antibiotic-free medium, the clones picked should be rescreened for the contaminant. This is particularly important if the contaminant was one that is difficult to detect visually, such as mycoplasma. For a mycoplasma contamination, the cell lines should be rescreened a month later and at frequent intervals for a year before it is declared cured.