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Learning objectives

  • Discuss why viruses were originally described as filterable agents
  • Describe the cultivation of viruses and specimen collection and handling
  • Compare in vivo and in vitro techniques used to cultivate viruses

At the beginning of this chapter, we described how porcelain Chamberland filters with pores small enough to allow viruses to pass through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to isolate and identify viruses.

Isolation of viruses

Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate (see [link] ).

Figure a is an electron micrograph showing pores and bacteria that are larger than the pores. Figure b is a drawing showing three particles in a container above a flask. A filter pore size of 5 µm blocks the largest of the particles and lets the smaller two through. The filter pore size of 200 nm (0.2 µm) only allows only the smallest particles through.
Membrane filters can be used to remove cells or viruses from a solution. (a) This scanning electron micrograph shows rod-shaped bacterial cells captured on the surface of a membrane filter. Note differences in the comparative size of the membrane pores and bacteria. Viruses will pass through this filter. (b) The size of the pores in the filter determines what is captured on the surface of the filter (animal [red] and bacteria [blue]) and removed from liquid passing through. Note the viruses (green) pass through the finer filter. (credit a: modification of work by U.S. Department of Energy)
  • What size filter pore is needed to collect a virus?

Cultivation of viruses

Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn ) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (see [link] ). The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected (see [link] ). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.

Figure a shows bottles laying on their side with red liquid; the bottles have screw-caps. Figure b shows 3 plates covered in bacterial growth (which is a smooth beige lawn). Each plate has small dots that are regions of no growth. Some plates have many of these plaques some have few.
(a) Flasks like this may be used to culture human or animal cells for viral culturing. (b) These plates contain bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells have been lysed. Viral titers increase on the plates to the left. (credit a: modification of work by National Institutes of Health; credit b: modification of work by American Society for Microbiology)

Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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