Virology and Cell Culture Laboratory
Virology and Cell Culture Laboratory MIP 425
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This 2 page Class Notes was uploaded by Winona McClure on Tuesday September 22, 2015. The Class Notes belongs to MIP 425 at Colorado State University taught by Erica Suchman in Fall. Since its upload, it has received 52 views. For similar materials see /class/210243/mip-425-colorado-state-university in Microbiology at Colorado State University.
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Date Created: 09/22/15
CULTIVATION OF VIRUSES IN EMBRYONATED AVIAN EGGS Chorioallantoic Sac CAF paramyxovirus 1012 day old eggs less yolk sac Off center inoculation Hemagglutination assay Rapid 10RBC on a slide parvo only HAU titer lattice formation Yolk Sac Chlamydia 57 day old eggs more yolk Direct center inoculation Chorioallantoic Membrane poxvirus False air sac inoculation 1012 day old eggs Gimenez stain Chlamydiapink on green Heat fix fuchsin stain malachite green CELL CULTURE Primary cell line Subculturing MEM Serum 10 growth 2 maintenance Phenol Red redpurple redorange yellow Saline Trypsin EDTA Hemocytometry cellsx10quot4x1dilution cellsml 2x10quot5 8x10quot5 cellsml C02 when not sealed to maintain pH of cells as cells grow they Preservation of cell cultures 10 serum 10 DMSO freeze at 196 CPE stained and unstained Look at lowest MOI to determine rate of CPE 12 days is fast Hemadsorption paramyxoviridae blood cells stick to membranes INFECTIVITY ASSAYS Quantal Assay end point assay LD50 log10 of highest dilution dP05 Titer10quotnegLD50 LD50volume Quantitative Assay plaque formation plaques dilution x volume of innoculum PFUs Calculating titers CHEMICAL amp PHYSICAL CHARACTERIZATION OF VIRUSES 17 log10 difference aka 50 fold is significant use 2 serum after cells established Lipid solvents ether Virion size filters 022um and 01um lt50nm both filters 50100nm just the 022um gt100nm no filters Acid pH pH 30 High temperature 60C for 30mins non enveloped are more resistant Determination of nucleic acid type FudR fluorouracil deoxyriboside thymidine analog Inhibit DNA synthesis inhibition means DNA genome Giemsa staining and CPE bouin39s fix geimsa buffer gstain gbuffer acetone acetonexylene xylene SEROLOGY Immunofluorescence stain IFA look for presence of viral antigen green fluorescence wUV Direct Antigen Detection vs Indirect where only one antibody needs to be tagged permeablize with acetone and use fluorescein active FITC Immunoperoxidase stain IP brown residue on blue background from Harris39s hematoxylin ABC biotinylated enzymes avidinbiotinabhrp Antigen seral Ab biotinylated 2ndary ab ABC DAB substrate Block with normal horse serum Beta serum neutralization prescence of NAB prevents formation of plaques serum is diluted virus is constant 4 fold increase between acute and convalescent serum indicates recent infection fix with methanol stain wcrystal violet Hemagglutination inhibition where do you read the assay Heterophilic antibodies cause lattice formation even with inhibiting antibodies bind RBC Nonspecific inhibitors prevent hemagglutination regardless of antibodies cant detect in controls ELISA blue color after substrate positive phosphic acid fixed and turns it yellow Direct Indirect Ab capture Ag capture 17 log 10 increase in virus titre 4 fold increase in antibody titer MOLECULAR BIOLOGY OF VIRUSES Detection and Identification by RTPCR Gel electrophoresis Identification by BLAST ddNTPs chain terminating nucleotides PHYLOGENETICS Ebola and West Nile 70 bootstrapping