A variety of techniques are available to recognize the presence of viruses, identify them, and grow them in large quantities. The focus here is on methods for studying animal viruses, which are far more expensive and time-consuming than those used in studying phage.
Since viruses can multiply only inside living cells, such cells are needed to study virus growth. The study of bacterial viruses has advanced much more rapidly than investigations on animal and plant viruses, in large part because bacteria are much easier to grow in large quantities in short time periods. The primary difficulty in studying animal viruses is not so much in purifying the virions as it is in obtaining enough cells to infect. Some viruses can only be cultivated in living animals. Others may be grown in embryonated chicken eggs, those that contain developing chicks. Some animal viruses can be grown in cells taken from a human or another animal.
When animal viruses can be grown in isolated animal cells, the host cells are cultivated in the laboratory by a technique called cell culture or tissue culture. To prepare cells for growth outside the body of the animal (in vitro), a tissue is removed from an animal and minced into small pieces (figure 14.2). The cells are separated from one another by treating them with a protease enzyme, such as trypsin, that breaks down protein. The suspension of cells is then placed in a screw-capped flask in a medium containing a mixture of amino acids, minerals, vitamins, and sugars as a source of energy. The growth of animal cells also requires a number of additional growth factors that have yet to be identified but are present in blood serum. Tissue cultures prepared directly from the tissues of an animal are termed primary cultures.
The cells bathed in the proper nutrients attach to the bottom of the flask and divide every several days, eventually covering the surface of the dish with a single layer of cells, a monolayer. When cells become crowded, they stop dividing and enter a resting state. One can continue to propagate the cells by treating them with trypsin, removing them from the primary culture, diluting them, and putting the diluted suspension into another flask containing the required nutrients. This results in an established cell line.
Cells taken from normal vertebrate tissue die after a certain number of divisions in culture. For example, human skin cells divide 50 to 100 times and then die, even when they are diluted into fresh media. Accordingly, cells must once again be taken from the animal and a new primary culture started.
Cells taken from a tumor, however, can be cultivated in vitro indefinitely. Accordingly, they are much easier to use for growing viruses than normal tissue, and several tissue lines have been established from tumors.
Tissue culture is important for growing viruses in the laboratory. The virus is mixed with susceptible cells, and the mixture is incubated until the infected cells are lysed. Following lysis, the unlysed cells and the cell debris are removed by centrifugation. The cells and debris go to the bottom of the centrifuge tube while the light, small virions remain in the liquid, the supernatant. This liquid containing the virions is also termed a lysate.
Tissue culture cells can also be used in virus detection. When a virus is propagated in tissue culture cells, it often changes the cells' appearance. Often, these changes are characteristic for a
Chapter 14 Viruses, Prions, and Viroids: Infectious Agents of Animals and Plants
(1) Mince tissue into small fragments
(2) Incubate with a protease (trypsin) to disperse cells
(3) Place fragments into flask with growth media; allow cells to grow
(4) Cells settle on surface of glass and grow into a confluent single layer, termed a monolayer
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