Chapter 4 isolation identification-and-cultivation
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  • 1. CH T R 4 AP EISOL ION, QUANT ICAT AT IF IONAND identification OF VIRUSES
  • 2. REVISION VIRULENT VIRUS - Upon entering the host, the virus circular DNA will undergo multiplication and lyses the host • The Lytic Cycle to release the new virion. – Culminates in the death of the host cell • The Lysogenic Cycle – Virulent viruses – Replication of the viral reproduce only by lytic cycle. genome without destroying the host cell. – T4 virulent phages – A temperate virus may TEMPERATE VIRUS reproduce by either-Within the host, the virus’ circular DNAengages in either the lytic or lysogenic cycle. cycle. – Lambda virus (temperate- During a lytic cycle, the viral genesimmediately turn the host cell into a virus- phage): resembles T4producing factory, and the cell soon lyses and but only has a singlereleases its viral products. short tail fiber
  • 3. • Regardless of the type of virus, the parasite diverts the host cell’s resources for viral production.• The host cell provides:  Nucleotides for nucleic acid production  Enzymes  Ribosomes Machinery for protein  tRNA synthesis  Amino acids  ATP
  • 4. Phage GrowthGrowth curve for a bacteriophage: The eclipse phage represents the time afterpenetration through the biosynthesis of mature phages. The latent period representsthe time after penetration through release of mature phages. The number of virusesper infected cell is the viral yield, or burst size
  • 5. Lesson Outcome• Explain the cultivation and quantification techniques for bacteriophages
  • 6. Cultivation and identification of viruses 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. Bacteriophages – cultivation and identification is simple and easy, due to the simplicity of the host cells. Animal viruses – difficult, due to the properties of the animal host. Systems of cultivation with broader applications were developed, including in vitro* cell (or tissue) culture methods and in vivo* inoculation of laboratory-bred animals and embryonic bird tissues. - Such use of substitute host systems permits greater control, uniformity, and wide-scale harvesting of viruses.
  • 7. Cultivation and identification of phages1. Obtaining bacteriophage from sample2. Amplification/multiplication of phages Solution (sample) into liquid media (eg. NB, TSB) increase the numbers of phages Addition of host – sewage: enteric bacteria, faeces: the sewage sample) by (in E.coli allowing them to infect and Incubation: 37o C, 24 reproduce hrs within fresh host.1. Isolation of multiplied phages Separate the remaining host cell/cell debris Preparation of pure phage via centrifugation and filtration (0.2µm suspension filter)1. Plaque assay Detection, identification , phage isolation for storage and future research
  • 8. Isolation and identification of phages – Plaque assay technique Plaque assay technique Detection, isolation, identification, characterisation of STEPS: phages1. Serial dilutions –ten-fold dilution in preparation of phage suspension1. Add in host (log-phase growth) to phage dilution To allow infection of phage to host2. Incubation 37o C, 20 min3. Add in top agar4. Pour on solidified agar5. Incubate 37o C, 18-24 hrs.6. Observation of plaque formation
  • 9. Isolation and identification of phages – Plaque assay technique Plaque assay technique STEPS:1. Serial dilutions –ten-fold dilution in preparation of phage suspension2. Add in host (log-phase growth) to phage dilution3. Incubation 37o C, 20 min4. Add in top agar5. Pour on solidified agar6. Incubate 37o C, 18-24 hrs.7. Observation of plaque formation be collected for Plaque: can storage
  • 10. Identification of phages – Plaque assay technique Plaque ? Zone of cell death/ a clear area in a bacterial lawn culture where viruses have lysed host cells HOW TO IDENTIFY TEMPERATE- Cloudy PHAGE? plaque The basis is that one viral particle infects one cell, is replicated and the cell lyses. The nearby cells are infected and a ‘plaque’ of dead cells is formed over time.
  • 11. Identification of phages – Plaque assay technique Basis of plaque formation:  Plaque assay – also to calculate number of phages present.  The titer of a phage suspension, is determined by counting the number of plaques that form from a given volume of suspension. Phage titer is expressed as plaque forming units (PFU) per milliliter (ml). [pfu/ml] * measurement of the number of viable, infectious
  • 12. QUIZ1. List the replication steps for animal viruses. Adsorption, Penetration, Uncoating, Synthesis, Maturation, Release2. Name the point of entry and exit for animal viruses. Entry: endocytosis and fusion of virus envelope to host cell membrane Exit: budding/exocytosis and lysis3. Name the site for replication, protein synthesis and maturation step for DNA virus. Replication: nucleus Protein synthesis: cytoplasm Maturation: nucleus4. Define “plaque”. A clear area in a bacterial lawn culture where viruses have lyzed host cells5. How do you identify the present of lambda phage through plaque assay technique? Formation of cloudy/not clear plaque because lambda phage is temperate phage
  • 13. ISOL ION, QUANT ICAT AT IF IONAND identification OF VIRUSES
  • 14. Overview of Animal Viruses-Overview of animalvirus actions
  • 15. Lesson Outcome• Explain the cultivation and quantification techniques for animal viruses
  • 16. Isolation, Cultivation and Identification of animal viruses1. In living animals CULTIVATION/ ISOLATION - using live animal eg.mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey. - the animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood, muscle, body cavity, skin, or footpads. - use in example research to study the immune system’s response to viral infections. - HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin. IDENTIFICATION - The signs of viral growth include death of the animal and defects in animal development. The infected animal tissue can be prepared for examination with an electron microscope
  • 17. Isolation, Cultivation and Identification of animal viruses2. In Embryoted egg CULTIVATION/ ISOLATION - use embryonated chicken, duck or turkey for inoculation of viral suspension. IDENTIFICATION- The signs of viral growth include death of the embryo, defects in embryonic development, and localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox). The embryonic fluid and tissue can be prepared for examination with an electron microscope. - 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.
  • 18. Viral culture in eggs: Some viruses, such as influenza viruses,are grown in embryonated chicken eggs
  • 19. 3. Using cell culture CULTIVATION/ ISOLATION - preferred type of growth medium for virus, more convenient than the previous two methods - use isolated cell from animal that are cultured invitro. Normal cells will form monolayer.IDENTIFICATION - If viruses are present, the cells of monolayer will deteriorate as they multiply. Cell deterioration is called cytopathic effect (CPE). CPE can be detected and counted = plaques by phages (plaque assay). Microscopic observation via electron microscope (histopathology). A. Normal B. Transformed
  • 20. Culturing of using cell culture• Two discoveries greatly enhanced the usefulness of cell cultures for virologists and scientists1. The discovery and use of antibiotics made it possible to prevent bacterial contamination2. The discovery of proteolytic enzymes (e.g. trypsin) can free animal cells from surrounding tissues without injuring freed cells• Subculturing: the process by which cells from an existing culture are transferred to new containers with fresh nutrient media
  • 21. Identification of viruses1. PCR – polymerase chain reaction2. Restriction fragments polymorphisms (RFLP)3. Serological method – Western blot common method use4. Immunological test , ELISA, agglutination test – if specific antibody is A preparation of killed, inactivated available or attenuated microorganisms to induce artificially acquired active Vaccine development immunity1. Embryoted chicken egg – one the most used method of viral isolation and growth2. Still used to grow viruses for some vaccines – eg. Influenza vaccine3. Cell culture and animal tissue are also used in vaccine preparation for
  • 22. QUESTION• Briefly explain the culturing method used to identify, isolate and cultivate animal viruses. Embryonated eggs : use embryonated chicken, duck or turkey for inoculation of viral suspension. The signs of viral growth include death of the embryo, defects in embryonic development, and localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox). The embryonic fluid and tissue can be prepared for examination with an electron microscope. Tissue culture: use isolated cell from animal or plant that are cultured invitro. The cells will form monolayer. Thesign of viral growth detected through formation of plaque or looking at cytopathic effect. Animal : using live animal eg. mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey. The signs of viral growth include death of the animal and defects in animal development. The infected animal tissue can be prepared for examination with an electron microscope. - Identification: also by PCR, serology