Lab4-2011

Lab4-2011 - BIOL 513 Lab 4: Virus characterization Part 2....

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Unformatted text preview: BIOL 513 Lab 4: Virus characterization Part 2. Sensitivity to lipid solvents 66 Lab 4: Virus characterization Part 2. Sensitivity to lipid solvents- lab designed for 12 groups Equipment for Lab 4: Day 1 Day 2 Day 3 Day 4 Day 5 CO 2 incubator + + Low-speed centrifuge + - 6-ft biosafety cabinet - 2 Light microscope - + Inverted phase contrast microscope + - Hemacytometer with coverslip - 12 Hand counter - 12 Vortex 6 - 2-liter vacuum flask with aspirator 2 2 Pipetman, 40-200 L 12 12 8-channel Pipetman, 50-300 L - 6 Safety bulb 12 12 Forceps - 12 250-mL beaker 12 - Rack for microcentrifuge tubes - 12 Rack for 12 75 mm tubes 12 - Rack for 15-mL tubes 12 - Rack for 50-mL tubes 12 12 Supplies & Consumables for Lab 4: Day 1 Day 2 Day 3 Day 4 Day 5 96-well tissue culture plate cluster - 24 Microcentrufuge tube - 12 Sterile 12 75-mm tube with snap cap 384 - Sterile dilution trays - 24 1-mL pipet 60 - 5-mL pipet 12 - 10-mL pipet - 26 Sterile Pasteur pipet 12 6 Sterile filtered tips in trays 12 12 Yellow pipet tips in trays - 12 0.4% Trypan blue stain - + Crystal violet stain in 50% methanol - + 70% ethanol in spray bottle 6 6 Chloroform + - Paper towels - + KimWipes + + BIOL 513 Lab 4: Virus characterization Part 2. Sensitivity to lipid solvents 67 Lab 4: Virus characterization Part 2. Sensitivity to lipid solvents- lab designed for 12 groups Materials for Lab 4 provided by the instructor: Day 1: Tuesday, February 22, 2011 FCV and FHV suspensions, 4 mL at 5x10 6 PFU/mL each in 15-mL polypropylene conical tubes 12 sets 50-mL tube with 50 mL CrFk cell culture medium (DMEM +10% FCS, 1 AA) 12 Extra 96-well plate cluster with seeded cells 6 Extra CrFk medium Day 2, Monday, February 28, 2011 24 mL suspension of freshly trypsinized CrFk cells @ 4 10 5 cell/mL 12 (from 5-6 150- mm plates @ ~2 10 7 cells/plate to get total 120 10 6 cells) Extra CrFk medium BIOL 513 Lab 4: Virus characterization Part 2. Sensitivity to lipid solvents 68 LAB 4 INTRODUCTION TO VIRUS CHARACTERIZATION Sensitivity to Lipid Solvents Enveloped viruses are usually susceptible to the action of lipid solvents such as ether or chloroform. Exposure to these solvents results in a marked decrease in viral infectivity (except for the poxviruses, which respond variably to ether). Non-enveloped viruses do not exhibit such a decrease in infectivity when treated similarly. Ether and chloroform dissolve the cell membrane that envelops the virus, removing viral proteins necessary for the attachment of virions to cells as well as possibly exposing the nucleic acid and protein core (for unencapsidated viruses) to the environment. Typically, non-enveloped viruses are resistant to lipid solvents due to the nature of the protein capsid, though this may not always be true. A given virus is considered sensitive if exposure to the lipid solvent results in a...
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Lab4-2011 - BIOL 513 Lab 4: Virus characterization Part 2....

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