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Cultivation of Viruses

Cultivation of Viruses - Microbiology

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Cultivation of Viruses

  1. 1. Cultivation of Viruses MICROBIOLOGY Islam Ghassan Sarakbi : 39
  2. 2. A virus is a biological agent that reproduces inside the cells of living hosts. When infected by a virus, a host cell is forced to produce many thousands of identical copies of the original virus, at an extraordinary rate. Unlike most living things, viruses do not have cells that divide; new viruses are assembled in the infected host cell.
  3. 3. Viruses can't be grown in culture media or on agar plates alone, they must have living cells to support their replication. The easiest viruses to grow are bacteriophages (because the easiest cells to grow in the lab are bacteria).
  4. 4. On the other hand, Viruses can be cultivated within suitable hosts, such as a living cell. To study bacteriophages, for example, bacteria are grown in a suitable growth medium, then bacteriophages are added. The bacteriophages multiply within the bacteria and increase their numbers substantially.
  5. 5. Purpose Of Virus Cultivation  The primary purposes of viral cultivation are: 1.To isolate and identify viruses in clinical specimens. 2. To prepare viruses for vaccines. 3. To do detailed research on viral structure, multiplication cycles, genetics and effects on host cells.
  6. 6. Virus Cultivation Systems  Tissue culture system.  Embryonated eggs system.  Whole animal systems.
  7. 7. Tissue culture system For Cultivation Of Viruses Animal and plant viruses are cultivated in cell cultures. A cell culture is prepared by encouraging cell growth outside the animal or plant source. The cells are kept alive in a suspension of growth factors within a Petri dish. A thin layer of cells, or monolayer, is then inoculated with viruses, and replication takes place.
  8. 8. Tissue culture system For Cultivation Of Viruses (Fertilized eggs and living animals can also be used to cultivate viruses.) For research study, viruses can be cultivated in large volumes by inoculations to tissue culture systems. After a time, the cells are degenerated, and viruses are harvested.
  9. 9. Growing Bacteriophages in the Laboratory: Bacteriophages can be grown either in suspension of bacteria in liquid media or in bacterial cultures on solid media. Solid media makes possible the plaque method for detecting and counting viruses. A sample of bacteriophage is mixed with host bacteria and melted agar. The agar containing the bacteriophages and host bacteria is then poured into a Petri plate containing a hardened layer of agar growth medium.
  10. 10. Growing Bacteriophages In The Laboratory (cont.) : The viral particles are concentrated by precipitation methods and purified by repeated centrifugations. Highly purified viruses can be obtained by crystallization and concentration under established conditions.
  11. 11. Flasks containing tissue culture growth medium which provides nourishment to growing cells.
  12. 12. The Cell Cultures • In modern usage, tissue culture generally refers to the growth of cells from a tissue from a multicellular organism in vitro. • These cells may be cells isolated from a donor organism, primary cells, or an immortalised cell line. • The cells are bathed in a culture medium, which contains essential nutrients and energy sources necessary for the cells' survival.
  13. 13. The Cell Cultures  The term tissue culture is often used interchangeably with Cell Culture.  Cell cultures have replaced embryonated eggs as the preferred type of growth medium for many viruses.  The literal meaning of tissue culture refers to the culturing of tissue pieces.
  14. 14. The Cell Cultures Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analysed.
  15. 15. The Cell Cultures  Contain cell lines, called diploid cell lines, developed from human embryos can be maintained for about 100 generations and are widely used for culturing viruses that require a human host.  When viruses are routinely grown in the laboratory, continuous cell lines are used. These are transformed (cancerous) cells that can be maintained through an indefinite number of generations, and they are sometimes called mortal cell lines.
  16. 16. The Cell Cultures  This cell deterioration is called cytopathic effect (CPE), can be detected and counted in much the same way as plaques caused by bacteriophages on a lawn of bacteria.  Primary cell lines, derived from tissue slices, tend to die out after only a few generations.
  17. 17. Cell cultures
  18. 18. CULTURE CONDITIONS • Culture conditions vary widely for each cell type. • The artificial media invariably consist of a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins,minerals), growth factors, hormones, and gases (O2, CO2). • It also regulates the physicochemicalenvironment (pH, osmotic pressure, temperature). • Most cells are anchoragedependent and must be cultured while attached to a solid or semi-solid substrate(adherent or monolayer culture), • Others can be grown floating in the culture (suspension culture).
  19. 19. Suspension Adherent
  20. 20. Differences between Adherent and Suspension Cultures ADHERENT CULTURES SUSPENSION CULTURES Most cells can be cultured this way Cells which are adapted to suspension cultures or non-adhesive cultures Passaging required at certain intervals Passaging is much easier, can dilute culture to stimulate growth Allows easy visualisation of cells Harder to view cells Cells dissociated enzymatically or mechanically Not require enzymatic or mechanical dissociation
  21. 21. DISADVANTAGES OF CELL CULTURES • Long period (up to 4 weeks) required for result. • Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen. • Susceptible to bacterial contamination. • Susceptible to toxic substances which may be present in the specimen. • Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.
  22. 22. Steps of cultivating animal viruses using tissue culture technique:  Cultivating animal viruses using tissue culture technique involves following three main steps: 1. Monolayer preparation. 2. Clonal cell line preparation. 3. Infection with virus.
  23. 23. 1,2) Monolayer & Clonal Cell Line Preparation: CELL CULTURE • Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment. • The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established. • Can be classified under the following cell lines. i) Primary culture. ii) Diploid cell lines. iii) Continuous cell lines.
  24. 24. i) PRIMARY CULTURE: Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence) (e.g. Primary monkey kidney and mice fibroblasts).
  25. 25. ii) Diploid Cel Lines: • After the first subculture, the primary culture becomes known as a Diploid cell line or subclone. (e.g. human fetal lung).
  26. 26. iii) Continuous Cell Lines: When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. Become immortal through a process called transformation. Can occur spontaneously or can be chemically or virally induced.
  27. 27. 3) Infection with virus • The clonal cell lines suspended in suitable media are infected with any desired virus which replicates inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus particles are released in the surrounding medium. • These newly produced virus particles (virions) infect the adjacent cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed.
  28. 28. Growing Bacteriophages In Living Animals: Animal inoculation may be used as a diagnostic procedure for identifying and isolating a virus from a clinical specimen. After the animal is inoculated with the specimen, the animal is observed for signs of disease, or is killed so that infected tissues can be examined for the virus.
  29. 29. Growing Bacteriophages In Embryonated Eggs: Growing viruses in an embryonated egg can be a fairly convenient and inexpensive form of host for many animal viruses. A hole is drilled in the shell of embryonated egg, and a viral suspension or suspected virus-containing tissue is injected into the fluid of the egg.
  30. 30. Growing Bacteriophages In Embryonated Eggs (cont.): There are several membranes in an egg, and the virus is injected near the one most appropriate for its growth. Viral growth is signaled by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes. This method was once the most widely used method of viral isolation and growth, and it is still used to grow viruses for some vaccines.
  31. 31. Detection Of Viral Growth In Embryonated Eggs: The signs of viral growth include: i) Death of the embryo, ii) Defects in embryonic development, and iii) Localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox). iv)The embryonic fluid and tissue can be prepared for examination with an electron microscope. v) Some can also be detected by their ability to agglutinate red blood cells or by their reaction with an antibody of known specificity that will affix to its corresponding virus, if it is present.
  32. 32. HARVESTED EMBRYOS
  33. 33. Viral Measurements Viruses are generally too small to be seen under the light microscope, and an electron microscope is usually necessary to make them visible. Once viruses have replicated and been harvested the concentration of viral particles (virions) in the viral stock solution must be determined.
  34. 34. Viral Measurements The most important method and the most commonly used to determine the concentration of a stock solution of bacteriophages is the Plaque Assay (plaque method).
  35. 35. The Plaque Essay (plaque method) : The plaque assay is performed by cultivating viruses on a “lawn” of host cells and noting the presence of clear areas where viruses have replicated and destroyed the cells (the virus lyses the bacteria). Each plaque is assumed to come from a single viral particle.
  36. 36. The concentration of the stock solution of the virus is given in plaque forming units.
  37. 37. Viral Measurements Another way of determining virus infectious units is by cultivating viruses in living animals and determining which dilution of virus is lethal to the animals. The end-point dilution can be calculated by this method.

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