1. Cultivation of viruses
 Virus cultivation is also referred to as propagation or growth. To cultivate virus, it is 
 necessary to supply the virus with appropriate cells in which it can replicate. Phages 
 are supplied with bacterial cultures, plant viruses are cultivated in special plants or 
 in protoplasts (plant cells from which the cell wall was removed), while animal 
 viruses may be supplied with whole organisms, such as mice, egg containing chick 
 embryos or insect larvae. However, animal viruses are grown in cultured animal 
 cells.
  Tissue and cell culture consists of cells or tissues obtained from humans, animals, 
  or plants supplied with necessary nutrients grown in aseptic conditions (free 
  from bacteria and fungi). 
 Below are the kinds of cell culture flasks, plates and dishes
   2. Virus isolation
   •   Many viruses can be isolated as a result of their ability to form discrete visible 
       zones (plaques) in layers of host cells. Plaques may form where areas of cells are 
       killed or altered by the virus infection. Each plaque is formed when infection 
       spreads radially from an infected cell to surrounding cells.
   •   Plaques can be formed by many animal viruses in monolayers if the cells are 
       overlaid with agarose gel to maintain the progeny virus in a discrete zone. Plaques 
       can also be formed by phages in lawns of bacterial growth.
   •   It is generally assumed that a plaque is the result of the infection of a cell by a 
       single virion. If this is the case then all virus produced from virus in the plaque 
       should be a clone, in other words it should be genetically identical. This clone can 
       be referred to as an isolate, and if it is distinct from all other isolates it can be 
       referred to as a strain.
   •   There is a possibility that a plaque might be derived from two or more virions so, 
       to increase the probability that a genetically pure strain of virus has been obtained, 
       material from a plaque can be inoculated onto further monolayers and virus can be 
       derived from an individual plaque. The virus is said to have been plaque purified.
  • Production of plaques 
   by animal viruses (top).
  • Plaques formed by a 
   phage in a bacterial 
   lawn (bottom). 
    3. Virus purification
    •   After a virus has been propagated it is usually necessary to remove host cell 
        debris and other contaminants before the virus particles can be used for 
        laboratory studies, for incorporation into a vaccine, or for some other 
        purpose.
    •   Virus purification can be done by centrifugation which is the most common 
        procedure used for the purification of viruses. Partial purification can be 
        achieved be differential centrifugation and a higher degree of purity can be 
        achieved by density gradient centrifugation. 
    1.    Differential centrifugation involves alternating cycles of low-speed 
          centrifugation, after which most of the virus is still in the supernatant, and 
          high-speed centrifugation, after which the virus is in the pellet.
    2.    Density gradient centrifugation involves centrifuging particles (such as 
          virions) or molecules (such as nucleic acids) in a solution of increasing 
          concentration, and therefore density.
    •   Sucrose and caesium chloride are commonly used as a solute in different 
        concentrations to form density gradient.
    There are two major categories of density gradient centrifugation: rate zonal and 
    equilibrium (isopycnic) centrifugation.
    •  In rate zonal centrifugation the virus is layered over a preformed gradient 
       before centrifugation. Each kind of particle sediments as a zone or band 
       through the gradient, at a rate dependent on its size, shape and density. The 
       centrifugation is stopped while the particles are still sedimenting.
    •  Equilibrium centrifugation, in which the gradient is formed during 
       centrifugation, occurs when centrifugation continues until all the particles in 
       the gradient have reached a position where their density is equal to that of 
       the medium. This type of centrifugation separates different particles based 
       on their different densities.
    4. Structural investigations of cells and virions:
    I.   Light microscopy: light microscopy has useful applications in detecting 
         virus-infected cells, for example by observing cytopathic effects or by 
         detecting a fluorescent dye linked to antibody molecules that have bound 
         to a virus antigen (fluorescence microscopy).