ferred to as cell cultures (referred to by some as tissue cultures), originate as a few cells and grow into a monolayer on the sides of glass or plastic test tubes. Cells are kept moist and supplied with nutrients by keeping them continuously immersed in cell culture medium (Figure 51-21). Cell cultures are routinely incubated in a roller drum that holds cell culture test tubes tilted 5 to 7 degrees while they slowly revolve ('6 to 1 rpm) at 35° to 37° C (see Figure 51-5). Incubation of cell culture tubes in a stationary rack can be used in place of a roller drum. Rapidly growing viruses, such as HSV, appear to be detected equivalents by both methods. Comparative studies are not available for most viruses.
Metabolism of growing cells in a closed tube results in the production of carbon dioxide and acidification of the growth liquid. To counteract the pH decrease, a bicarbonate buffering system is used in the culture medium to keep the cells at physiologic pH (pH 7.2). Phenol red—a pH indicator that is red at physiologic, yellow at acidic, and purple at alkaline pH—is added to monitor adverse pH changes. Once inoculated with specimen, cell cultures are incubated for 1 to 4 weeks, depending on the viruses suspected. Periodically the cells are inspected microscopically for the presence of virus, indicated by areas of dead or dying cells called cytopathic effect (CPE).
Two kinds of media, growth medium and maintenance medium, are used for cell culture. Both are prepared with Eagle's minimum essential medium (EMEM) in Earle's balanced salt solution (EBSS) and include antimicrobials to prevent bacterial contamination. Usual antimicrobials added are vancomycin (10 pg/mL), gen-tamicin (20 ng/mL), and amphotericin (2.5 pg/mL). Growth medium is a serum-rich (10% fetal, newborn.
or agammaglobulinemic calf serum) nutrient medium designed to support rapid cell growth This medium is used for initiating growth of cells in a tube when cell cultures are being prepared in-house or for feeding tubes of purchased cell cultures that have incomplete cell monolayers. Feeding refers to the removal of old medium followed by the addition of fresh culture medium. Maintenance medium is similar to growth medium but contains less serum (0% to 2%) and is used to keep cells in a steady state of metabolism. Fetal newborn, or agammaglobulinemic calf serum is used to avoid inhibitors, such as specific antibody, and to be free of mycoplasmas present in serum from older animals.
Several kinds of cell cultures are routinely used for isolation of viruses. A cell culture becomes a cell line once it has been passed or subcultured in vitro. Cell lines are classified as primary, low passage, or continuous. Primary cell lines are those that have been passed only once or twice since harvesting, such as primary monkey kidney cells. Further passage of primary cells results in a decreased receptivity to viral infection. Low passage cell lines are those that remain virus-sensitive through 20 to 50 passages. Human diploid fibroblast cells, such as lung fibroblasts, are a commonly used low-passage cell line. Continuous cell lines such as HEp-2 cells (human epidermoid carcinoma cells), can be passed and remain sensitive to virus infections indefinitely. Unfortunately, most viruses do not grow well in continuous cell lines. The majority of dinically significant viruses can be recovered using one cell culture type from each group. A combination frequently used by clinical laboratories is Rhesus monkey kidney cells, MRC-5 lung fibroblast cells, and HEp-2 cells (Table 51-33).
Inoculated cell cultures should be incubated immediately at 35° C. After allowing virus to adsorb to the cell monolayer for 12 to 24 hours, it is common practice to remove the remaining inoculum and culture medium and replace it with fresh maintenance medium. This avoids most inoculum-induced cell culture toxicity and improves virus recovery. Incubation should be continued for 5 to 28 days depending on viruses suspected (see Table 51-33). Maintenance medium should be changed periodically (usually 1 to 2 times weekly) to provide fresh nutrients to the cells.
Blind passage refers to passing cells and fluid to a second cell culture tube. Blind passage is used to detect viruses that may not produce CPE in the initial culture tube but will when the "beefed-up" inoculum is passed to a second tube. Cell cultures that show nonspecific or ambiguous CPE are also passed to additional cell culture tubes. Toxicity, which causes ambiguous CPE, is diluted during passage and should not appear in the second cell culture tube. In both instances, passage is performed by scraping the monolayer off the sides of the tube with a pipette or disrupting the monolayer by vortexing with
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