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Lab2-2011 - BIOL 513 Lab 2 Introduction to virus...

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BIOL 513 – Lab 2: Introduction to virus quantification 27 Lab 2: Introduction to virus quantification - lab designed for 12 groups of 2 students Equipment for Lab 2: Day 1 Day 2 Day 3 Day 4 Day 5 CO 2 incubator + + + + Light microscope - + - + Inverted phase contrast microscope + + + - 6-ft biosafety cabinet 2 - - 2 Hemacytometer with coverslip - 12 - 12 Hand counter - 12 - 12 Vortex 6 - 6 - 2-liter vacuum flask with aspirator 2 2 2 2 Pipetman, 40-200- µ L 12 12 12 12 8-channel Pipetman, 50-300- µ L 6 6 - 6 Safety bulb 12 12 12 12 250-mL beaker 12 12 12 12 Rack for microcentrifuge tubes - 12 - 12 Rack for 12 × 75-mm tubes 12 - 12 - Rack for 15-mL tubes - - 12 - Rack for 50-mL tubes 12 12 - 12 Supplies & Consumables for Lab 2: Day 1 Day 2 Day 3 Day 4 Day 5 96-well tissue culture plate cluster - - - 24 12-well plate cluster - 12 - - Microcentrufuge tube - 12 - 12 Sterile 12 × 75-mm tube with snap cap 168 - 120 - 50mL sterile conical tube - - - - 1-mL pipet 24 - 12 - 5-mL pipet 24 - - - 10-mL pipet - 27 - 36 25-mL pipet - - 12 - Sterile Pasteur pipet 24 6 36 6 Sterile dilution trays 24 18 - 12 Sterile filtered pipet tips in trays 12 12 12 12 Yellow pipet tips in trays - 12 - - KimWipes + + - + Paper towels - - - + Crystal violet stain in 50% methanol - + - + 0.4% Trypan blue stain - + - + 70% ethanol in spray bottle 6 6 6 6 Ethanol - + - - 1% methylcellulose overlay - - 12 -
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BIOL 513 – Lab 2: Introduction to virus quantification 28 Lab 2: Introduction to virus quantification - lab designed for 12 groups of 2 students Materials for Lab 2 provided by the instructor: Day 1: Tuesday, February 1, 2011 FCV, 100 µ L at 5x10 5 PFU/mL in 12 × 75-mm snap cap tube – 12 FHV, 100 µ L at 5x10 5 PFU/mL in 12 × 75-mm snap cap tube – 12 CrFk growth medium (DMEM +10% FCS, 1 × AA), 30 mL in 50-mL tube – 12 Extra 96-well plate clusters with seeded cells – 6 Day 2: Monday, February 7, 2011 CrFk cell suspension, freshly trypsinized, 25 mL @ 2-2.5 × 10 5 cell/mL – 12 (from 3-4 150-mm plates @ 1.8 × 10 7 cell/plate to get total 60-120 × 10 6 cells) Extra CrFk medium Day 3: Tuesday, February 8, 2011 FCV, 100 µ L at 5x10 5 PFU/mL in 12 × 75-mm snap cap tube – 12 FHV, 100 µ L at 5x10 5 PFU/mL in 12 × 75-mm snap cap tube – 12 Viral dilution and inoculation medium (M199+2%FCS, 1 × AA), 14 mL in 15-mL tube – 12 Extra 12-well plate clusters with seeded cells – 6 Day 4: Monday, February 14, 2011 CrFk cell suspension, freshly trypsinized, 25 mL @ 4 × 10 5 cell/mL – 12 (from 5-6 150- mm plates @ ~2 × 10 7 cell/plate to get total 120 × 10 6 cells) Extra CrFk medium
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BIOL 513 – Lab 2: Introduction to virus quantification 29 LAB 2 INTRODUCTION TO VIRUS QUANTIFICATION Infectivity, or the ability to invade and parasitize cells in order to multiply, is the most important property of viruses. This fundamental biological property is used for both viral characterization and viral quantification. The presence of viral particles or virion components may be determined using biophysical or biochemical methods, including electron microscopy, spectrophotometry, immunologic detection (e. g. ELISA or western blots), or other methods and allow for an estimation of the nucleic acid or protein content.
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