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Introductionth to Cell and Tissue Culture - Jennie P.

Jennie P. Introductionth to Cell and Tissue Culture - Plenum Press, 2002.
ISBN 0-306-45859-4
Download (direct link): introductiontocellandtissueсulture2002.pdf
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Figure 2.1.
Suggested floor plan for a teaching laboratory uses bench top hoods and bench top or free-standing incubators.
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Figure 2.2.
Suggested floor plan for setting up a tissue culture area in a research laboratory.
The Teaching Laboratory
The teaching laboratory is a special situation, as it is often a place that is temporary at best and plagued by severe budgetary constraints. The goal here is usually to maintain as aseptic an area as possible for a relatively short period of time. The laboratory and equipment described below are the minimum necessary to teach students all the basic techniques necessary to do cell culture. While the equipment is fairly rudimentary, the concepts taught need not be. The pioneers of cell culture had even less to work with. Depending on the specific aims of the course, the teaching laboratory will minimally require a refrigerator and
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Figure 2.3.
Suggested floor plan for a separate standard tissue culture facility.
an incubator, although this need not be a CO2 incubator, as there are commercially available, appropriately buffered media that obviate the need for CO2. In this case, the incubator need only be able to accurately maintain the required temperature.
An inverted phase contrast microscope to be shared by all students equipped minimally with a 10x and preferably also a 20x objective should prove adequate for nearly all work done in a teaching laboratory. Most manufacturers offer an economical alternative to their high-end microscopes (Fig. 2.5).
Figure 2.1 illustrates how to set up a standard teaching laboratory for tissue culture using bench top hoods and CO2 cylinders. This setup requires a minimum of construction. A biosafety cabinet or tissue culture hood, while useful, may not be absolutely necessary in the teaching laboratory, especially if the facility is only temporary or being used for other laboratory functions. We have had success teaching students to work on open bench tops
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using flasks for cell culture. The medium can be equilibrated for 5% CO2 by blowing into the flask using a cotton-plugged pipette, or flushing with an air"C5% CO2 mixture from a commercially available gas supplier. Commercially available acrylic bench top hoods are satisfactory, but even this may be unnecessary if the immediate environment is relatively clean, the work area adequate (large enough and of a nonporous material so that it can be cleaned) and disinfected prior to use, and appropriate antibiotics used in a judicious manner. Minimally, the surface on which the culture work is to be done should be nonporous (e.g., stainless steel, Formica, or composite) and cleaned with a disinfectant before and after each use.
The Standard Tissue Culture Laboratory
This may be a core facility, working in conjunction with other laboratories, or it may be the primary facility for a cell biology laboratory. All basic cell culture studies can be done in this facility that would be adequate for cell culture use for most cell and molecular biology laboratories, as well as use by physiologists, biochemists, and others who might occasionally need access to a culture facility. If it is to be a shared core facility, the need for min-
Figure 2.4.
Suggested floor plan for an ideal high-use tissue culture facility or a core facility shared by a number of laboratories.
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Figure 2.5.
An inexpensive inverted phase contrast microscope suitable for a student laboratory, or as an extra microscope in a high-use laboratory.
imizing potential contamination vectors becomes paramount. This is critical not only with regard to bacterial or fungal contamination, but also with cross-contamination of cell cultures. Some facilities have an airlock that can serve as a buffer zone and somewhat of a deterrent to bacteria'Cfungal contaminants, largely because in its most minimal configuration, it discourages unnecessary traffic in and out of the primary laboratory (see Fig. 2.3). Optimally, the entryway might be large enough to include a sink for hand washing, storage for sterile tissue culture supplies, and even space for a freezer or cell counter. A high-use tissue culture core facility might be designed around a plan such as that shown in Fig. 2.4.
When this is not possible, or is impractical, the laboratory should be designed so that cell cultures can be "compartmentalized," that is, primary cell cultures can be handled in a specifically designated hood(s), and kept in an incubator chamber separate from other cell lines being maintained in long-term culture. Cell cultures coming into the laboratory, as frozen vials or as viable cultures, primaries, or established cell lines, should be quarantined in an incubator chamber and handled in a designated hood until they are tested for mycoplasma.
Incubators are available as two-gas (CO2"Cair) or three-gas models (CO2"CO2"CNO2), this being largely determined by cost and specific needs of the investigators. It is always possible to augment a two-gas incubator chamber when this has not been incorporated into the original design. Insulation is maintained by either a water jacket or an air jacket, with corresponding advantages and disadvantages. The water-jacketed incubator can maintain temperature over a longer period of time should there be a power outage, and this can be a critical feature for some installations. It is much heavier when filled, however, and the level must be maintained by periodic "topping off" and the jacket drained when the incubator has to be moved. The air-jacketed incubator is lighter, has more moving parts to fail, comes up to temperature faster, but will lose heat much faster when the fan goes off (e.g., in case of an electricity shutoff).
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