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If the microscopic examination of the cultures confirms a contaminant, tape the tissue culture dish closed or place it in a sealed bag to be autoclaved. Immediately wipe down the
Mold, yeast, and bacterial contamination in mammalian cell culture viewed through the phase contrast microscope. This is what you see when you look at the cultures in the microscope. Panels A, B, and C are viewed at 300x magnification and panel C at 60x. The culture in A contains bacteria and in B, yeast. The mold colony in C is growing over the grid of a 150-mm culture dish. The edge of the same colony is shown in panel D.
microscope stage, bench top, and your hands with a disinfectant such as alcohol. Mold can grow on the outside of the culture dishes and on the plastic, metal, or glass surfaces in the laboratory. Labeling tape and paper labels are particularly susceptible to growth of mold. If there are many contaminated dishes, they can be placed in a separate autoclave bag and immediately removed from the room for sterilization.
Yeast, mold, and bacterial spores are ubiquitous. They are found adhering to dust and water vapor in the air and to hair, skin, clothing, and shoes. While filtering the air entering the culture room through HEPA filters will help a great deal in keeping down contaminants, unless each person entering the room goes through a thorough shower and gowning to avoid contamination (something that is not done for a normal culture room), the bugs will catch a ride. Some of the worst instances of yeast contamination we have experienced have been traced to laboratory personnel who were baking bread or brewing beer at home. They were bringing their home to work rather than taking their work home. Therefore, the best safeguard is to avoid unnecessary traffic into the room where the culture work is done. A "sticky mat" placed inside the entrance door to the culture room will help remove dust and spores from the shoes of people entering the room. The paper must be changed at least daily to be effective. Laboratory coats that are to be worn in the culture room only and left, hanging near the door when the personnel leave should be available. Bacterial, mold, and fungal contaminants are the most common because they are designed by nature to be resistant to
Scanning electron micrographs of contamination in cell culture. SEM allows one to see more detail of these small organisms: (A) Bacterial contamination;
(B) sporulating mold; (C) pseudomonas on cells; and (D) budding yeast.
desiccation and to remain for long periods of time in a dormant state but to immediately replicate when they find a good food source, such as cell culture medium.
Food and drink should not be brought into the culture room. If it is possible to do so, locate the sink adjacent to the culture area entryway so that anyone working directly with the cultures or handling plates can wash their hands prior to entering the culture area.
Things You Cannot See Can Hurt You
Mycoplasma contaminants grow quite slowly, compared to bacteria and yeast, and do not immediately or directly destroy the cells. They are known to alter the function and metabolism of the cell culture, cause chromosomal aberrations, affect cell surface antigenicity, interfere with nucleic acid synthesis, and generally change cell behavior (Kotani et al., 1987; McGarrity, 1977; Van Diggelen et al., 1977a, b). Mycoplasms are too small to be seen directly except by fluorescence labeling and the use of very high power objectives or electron microscopy (Phillips, 1977, 1978), but any significant change in the growth and function of the cultures should alert the investigator to the possibility of a mycoplasma conta-
mination. In our laboratory, we mycoplasma test any culture coming into the laboratory and any stock to be frozen down. We also routinely test our cell lines every 6"C12 months. If contamination is detected, cells should be tested more frequently until it is clear that the contamination has been eliminated. In addition, we maintain our cultures in antibiotic-free medium. While this does not preclude the introduction of adventitious agents such as mycoplasma, it serves two purposes: (1) It requires exceptionally careful handling and manipulation of the cultures to avoid overt contamination; and (2) infections by other microorganisms will serve as a warning that there is an observable problem that needs to be dealt with, hopefully before the introduction of a mycoplasma.
There are fluorescent staining methods for detecting mycoplasma that exploit the ability of Hoechst 33258 dye to bind specifically to DNA. Mycoplasmas contain DNA and can be seen as bright, punctate staining in the cytoplasm (Fig. 7.3). This is a good routine test for screening purposes. Scanning and transmission electron micrographs of mycoplasma infected cells are shown in Fig. 7.4. These organisms are quite varied in morphology and much smaller than the host cells. Commercial kits are available for mycoplasma screening