The establishment of successful tissue cultures requires several important pieces of equipment: a flow hood, a temperature-, humidity-, and CO2-controlled incubator, an inverted microscope, a 4°C refrigerator, a −20°C freezer, and a liquid-nitrogen freezer for long-term cell storage. It is important that the flow hood, incubator, and microscope are located such that they are separate from the facilities used for the manipulation of bacterial or yeast cultures and that this area be kept absolutely clean. This is because sterility is the most important concern for the tissue culturist attempting to propagate and manipulate tissue culture cells for long periods of time. In fact, culture contamination is perhaps the greatest cause of wasted time and materials in gene-transfer experiments. Careful execution of the techniques described in this chapter will ensure a high degree of reproducibility and sterility and eliminate the possibility of cross-contamination during culture manipulations. A few minutes a day spent ensuring sterility will save the investigator weeks or months of grief regenerating precious but contaminated cultures.
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