Microbiology 2 PDF

 Laboratory diagnosis of viral infection A. Direct demonstration of virus in clinical specimen 1a. Detection of the virus particle By E.M. (Clinical specimen)  for research purposes. 1b. Detection of the virus particle By Immunoelectron microscopy (Ab) ?  We take the clinical specimen...

 Laboratory diagnosis of viral infection A. Direct demonstration of virus in clinical specimen 1a. Detection of the virus particle By E.M. (Clinical specimen)  for research purposes. 1b. Detection of the virus particle By Immunoelectron microscopy (Ab) ?  We take the clinical specimen  add the susceptible antibody to the specimen  the antibodies bind the viral antigens  aggregation. 2. Detection Inclusion bodies By L.M.  Inclusion bodies (IBs) are intracellular aggregates of viral components, such as proteins, nucleic acids that can be seen during the assembly stage.  Negri bodies are cytoplasmic inclusion bodies found humans infected with the Rabies virus. Negri bodies 3. Detection of Viral antigen e.g. HBsAg (ELISA) 4. Detection of viral nucleic acid of the virus (PCR) : very specific 5. Hybridization reaction (Tissue) ?  We bring radioactive complementary strand for the susceptible virus that is present in the tissue  it is called radioactive probe  we inject it inside the infected tissue  when it binds to the viral genome it will fluoresce.  Cultivation of the viruses / Virus Isolation  Virus isolation needs living cells (cannot live in artificial media)  Cultivation methods : 1. Laboratory animals : Interference with immune system ? We inject the virus in the animals to obtain it's characteristics/phathogenesis, used for research or for viruses that need animals in order to live (ex : rabies) BUT the animals immune system will attack the virus  interference. 2. Embryonated eggs/ Fertilized egg : its immune system hasn't been formed yet, we can inject the virus in 4 different places : 1)Chorioallantoic membrane 2)Aminiotic cavity 3) Allantoic inoculation 4) Yolk sac inoculation. 3. Cell (tissue) culture :  It consists of single layer (monolayer cells ? Single layer of cells).  The most widely used method.  Tissues (ex : kidney) + trypsin → separate cells due to trypsin  Separated Cells + growth media in flat-sided bottles  monolayer culture.  Law of contact inhibition : during cell growth, if the cell contacts other one  inhibition in growth  monolayer only.  Types of cell culture : 1. Primary cell lines : Obtained from organ fragments e.g. monkey kidney.  Only divide for several passages (10) and then degenerate.(we can do sub-culturing for 10 times only). 2. Human diploid cell line (2nd cell lines) :  Derived from human embryo tissues (Fibroblast)  high mitotic division.  Only divide about 50 -100 passages in culture e.g. human embryo lung tissues.  Rapid growth & increase subculture. 3. Continuous cell lines (3rd cell lines) :  HeLa cells are the first and most widely used immortal human cell line, derived from Henrietta Lacks, a cervical carcinoma patient, in 1951.  Hela cells Divide indefinitely until nowadays. B. Detection of viral growth 1. Cytopathogenic effect (CPE) : Changes in cells a) Cell death Detachment from glass surface e.g. poliovirus. b) Rounding e.g. Adenovirus c) Syncytium e.g. Measles & mumps a A syncytium is a multinucleated cell that forms when individual cells fuse together, creating a large cell mass with multiple nuclei. b d) Cell transformation Tumor viruses d c 2. Plaque formation  Virus infects cells, When using vital stain(neutral red), unstained areas for infected cells. 3. Inclusion bodies :  They are the site of virus assembly.  Intracytoplasmic (eg. rabies (Negri bodies)). 4. Haemadsorption :  Some viruses (eg. influenza virus) have hemagglutinin antigen  we add RBCs (O antigen) that have affinity to hemagglutinin  viruses bind the RBCs  Haemagglutination. 5. Fluorescent- antibody staining  We add certain antibodies that are labeled with fluorescence to the susceptible virus  if they bind the virus there will be fluorescence. 6. Neutralization test :  we add virus to tissue culture and there will be Cytopathogenic effect (CPE).  We add the same virus that is coated with antibodies to new tissue culture and there will be no CPE. 7. Detection of viral Ag : Such By using:- ELIZA- RIA (Radioimmunoassay)….etc. 8. Interference :  The replication of one virus in a cell usually inhibits the multiplication of another virus entering subsequently. i. Destroy the receptors (Prevent the attachment). ii. Prevent mRNA formation C. Detection of specific Antibodies Serological tests  Serum has Detection Antibodies that we put over antigens.  IgM  detected Early (Recent).  IgG  Past.  There is no point to detect the IgG because the patient may have been cured or got vaccination but it is useful if we do “Detection of rising titre of IgG by ELISA”. We detect this ratio from the After certain time (eg.week), if the titer patient specimen increased  the immunity is still fighting the virus