AGM 101 FUNDAMENTALS OF MICROBIOLOGY 11. Theory. Definition and scope of. Diagnostic Methods in Virology, Virological Methods, Virus Culture, Virus Isolation. Virological Methods Slideset. Individual Methods. In this section, some commonly used virological methods will. Virus Isolation. Electron Microscopy Complement Fixation Test Haemagglutination. Inhibition Test Enzyme Linked Immunoassay ELISA Single Radial Haemolysis Immunofluorescense Neutralization Ig. G antibody avidity Molecular Methods 1. Virus Isolation. Viruses are obligate intracellular parasites that require. Cultured cells, eggs and. Importance Of Serial Dilution In Serology' title='Importance Of Serial Dilution In Serology' />Although. The development of. To prepare cell cultures, tissue. The cell suspension is then placed in a. Eagles, and an animal serum. After a variable lag, the cells. Antistreptolysin O ASO or ASLO is the antibody made against streptolysin O, an immunogenic, oxygenlabile streptococcal hemolytic exotoxin produced by most strains. Original Article. MType Phospholipase A 2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy. Laurence H. Beck, Jr., M. D., Ph. D., Ramon G. B. Bonegio, M. Attachment to a. Primary and Secondary Cultures. Primary cultures are maintained by changing the fluid 2 or 3. When the cultures become too crowded, the cells are. EDTA, and. portions are used to initiate secondary cultures. In both primary. and secondary cultures, the cells retain some of the. Cell Strains and Cell Lines. Cells from primary cultures can often be transferred serially. The cells may then continue to multiply at a. Eventually, after a. For human diploid cell. During the multiplication of the cell strain, some cells become. These cells are immortalized and have an unlimited. However, they retain contact inhibition. Cell Cultures. Cell cultures are separated into 3 types Primary cells prepared directly from animal or. Semi continuous diploid cells which are derived. MRC 5 Continuous cells derived from tumours of human. Vero, Hep. 2 Cell cultures vary greatly in their susceptibility to. It is of utmost importance that the most. Specimens for cell culture should be transported to the. Swabs should be. put in a vial containing virus transport medium. Bodily fluids. and tissues should be placed in a sterile container. Upon receipt, the specimen is inoculated into several. The maintenance media. The inoculated tubes should be incubated at 3. C. in a rotating drum. Rotation is optimal for the isolation of. CPE for many viruses. If stationary tubes are used, it is. The inoculated tubes should be read at least every other day. Certain specimens, such as. CPE like effect. If toxic effects are extensive, it may be. Cell cultures that are. Cell cultures should be kept for at. CMV. Cell cultures. When CPE is seen, it may be advisable to. For cell associated viruses such as CMV and VZV, it is. Other. viruses such as adenovirus can be subcultured after freezing and. Cytopathic effects of enterovirus 7. HSV, and CMV in cell. Linda Stannard. University of Cape Town, Virology Laboratory, Yale New Haven. Hospital Cytopathic effects of mumps and measles viruses in cell. Courtesy of Linda. Stannard, University of Cape Town, S. A. Influenza and parainfluenza viruses do not ordinarily induce. CPE, however they possess haemagglutinins and thus the ability to. RBCs as they bud from the cell. This phenomenon. is known as haemadsorption. Commonly employed cell cultures. LLC MK2 and MDCK cells. The cell. cultures are incubated with a suspension of guinea pig RBCs at 4o. C. or RT for 3. 0 minutes. The unabsorbed RBCs are then removed and. Presumptive identification of virus isolates can. CPE, haemadsorption. For final. identification, immunofluorescence, neutralization. Advantages of cell culture for virus diagnosis include. It is limited by. Contamination by endogenous viral agents such as SV4. Another problem in isolating. Using fetal calf serum reduces. Rapid Culture Techniques e. DEAFF test. One of the most significant contributions to rapid diagnosis. For a number of years, it has been recognized that. The cell culture is stained by monoclonal antibodies for the. The best. known example of this technique is the DEAFF test used for the. CMV infection. In the DEAFF test, the specimen. After a period of 2. CMV early antigen. Therefore a rapid diagnosis of CMV infection can be made without. CPE to appear.       Left Haemadsorption of red blood cells onto the surface of a. Also note the presence of. Xp Professional Oem Crack Sp3 more. RSV Courtesy of. Linda Stannard, University of Cape Town. Right Positive CMV. DEAFF test. Virology Laboratory, Yale New Haven HospitalSusceptible Cell Lines. Herpes Simplex. Vero Hep 2, human diploid HEK and HEL,human amnion VZV. HEL, HEK CMV. human diploid fibroblasts Adenovirus. Hep. 2, HEK, Poliovirus. MK, BGM, LLC MK2, human diploid, Vero. Hep 2,Rhadomyosarcoma Coxsackie. B     MK, BGM, LLC MK2. Echo. MK, BGM, LLC MK2, human diploid, Rd Influenza A. MK, LLC MK2, MDCK Influenza B. MK, LLC MK2, MDCK Parainfluenza. MK, LLC MK2 Mumps. MK, LLC MK2, HEK, Vero RSV. Hep 2, Vero Rhinovirus. HEK, HEL Measles. MK, HEK Rubella. Vero, RK1. Electron Microscopy. Virus diagnosis by electron microscopy relies on the detection. A major advantage of virus diagnosis. EM is the ability to visualize the virus. By identifying the. Speed is. another advantage of EM as the specimen can be processed within. EM can be used as a rapid diagnostic. On the other hand, the main disadvantage of EM is its. Secondly. there must be a minimum number of virus particles present around. Some viruses. such as SRSV may give a non distinct morphological appearance. Finally, EM is a very. EM has found a particular niche in the detection of. Norwalk, and Caliciviruses. It is also used for the rapid. It is occasionally used for. In addition EM may be used to. There are two types of EM methods direct or immunoelectron. IEM. With direct methods, negative staining is. The specimens may be used. Several methods are available for. Fomvar. coated copper grids are used. Immunoelectron microscopy is a. EM and is. particularly useful in the following situations The number of virus particles present is small. Many different viruses have different morphology e. IEM may identify the. In an outbreak situation where the pathogens responsible. IEM. There are 2 types of IEM, simple IEM, where the specimen is. IEM. SPIEM, where the copy grid is coated with specific antibody. Electronmicrographs of viruses commonly found in stool. From. left to right rotavirus, adenovirus, astroviruses. Norwalk like viruses. Courtesy of Linda M. Stannard. University of Cape Town, http www. Complement Fixation. Test. The complement fixation test CFT was extensively used in. Wasserman in 1. 90. It. took a number of decades before the CFT was adapted for routine. CFT meet the following criteria it is. However, there is now a trend to replace the. CFT with more direct, sensitive and rapid techniques, such as. RIAs and EIAs. Although CFT is considered to be a relatively. In essence the test consists of two. The first reaction, between a known virus antigen and a specific. The complement is removed or fixed by the. The second antigen antibody reaction. When this. indicator system is added to the reactants, the sensitized rbcs. The antigens. used for CFT tend to be group antigens rather than type specific. In order for the CFT to be set up correctly, the. The following is a. Titration of haemolytic serum and complement. Dilutions of complement with 2. The following dilutions of. The following controls are required cell control unsensitized cells only complement control complement at different. The optimal sensitizing concentration OSC of haemolytic. One haemolytic dose of complement HD5. OSC of haemolytic. HD5. 0 of complement is used for the CFT b. Titration of antigen and antibody. Antigen at dilutions of 1 2 to 1 5.