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Unformatted text preview: Name_______ Key _______________________Perm______________Section day/time_________ Average = 21 points out of 28 points; Max score = 25 points. Quiz #1, VERSION1, 25 points maximum. April 30, 2009. You are working on a crime scene investigation involving the somewhat mysterious death of 21 polo ponies in Florida. The only information that you have regarding the death of these ponies is that they were attacked by an extremely fast- acting, and highly virulent animal virus, but it is safe for humans to work on in the lab. The name of the virus is PPV. 1. Your first task is to amplify the virus to high enough titers to enable characterization of the virus later on. Various cell types are at your disposal so you try infecting all of them with PPV. You use L, HeLa and CEF cells. a) (1.5pts) What is the animal of origin of each of the cell lines that you tested? L cells = Mouse, HeLa = Human, CEF = Chicken (Chick Embryo Fibroblasts) b) (1.5pts) The CEF cells are the only cells that can be infected with the virus. Why? They are the only cells that express a functional PPV receptor . c) (2pts) You titer your virus in triplicate at a dilution of 1x10 5 using 100uL of virus solution. On your plates you see 80, 78 and 82 plaques. What is the titer of your virus? Average # of plaques = (80+78+82)/3 = 80pfu and 100uL = 0.1mL (80pfu/0.1mL) x 1x10 5 = 8x10 7 pfu/mL d) (2pts) This is what some of your plaque assays look like (to the right) upon visual inspection after treatment with a stain for live cells (live cells take up the stain). What is wrong with each/any of them? Why? A. All cells took up stain, all cells appear to be live and there are no visible plaques. Your virus dilution must be too high (low concentration), or you are using a cell type that cannot be infected with PPV. B. An acceptable plaque assay (white spots = plaques, black area = live cells) with a countable number of plaques. C. No cells took up stain, all cells appear to be dead. You either used too low of a dilution of virus (high concentration) or you forgot to add your agar overlay. 2. You stumble upon a very disorganized collegue's lab notebook who is also working on PPV. The page that you are interested in has the following graph. It is thought-provoking because your collegue has labeled nothing, so you consider what the experiment could mean: a) (2pts) What is meant by relative plaque number? Relative plaque # = # plaques with a treatment (to the virus) # plaques without treatment (to the virus) b) (2pts) Which sample(s) would have a relative plaque number of ~1? Which sample(s) would have a relative plaque number of less than 1? I wanted you to recall figure # 3 from the Racaniello paper where they do a tissue binding assay and the samples with ~100% binding have low pfu's and therefore a RPE# less than 1 (Sample 3 definitely, Sample 1 would be close), and Sample 2 would have a RPE# close to 1 because the treatment was ineffective and they saw lots of plaques. Sample 4 would have a RPE# ofwas ineffective and they saw lots of plaques....
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- Spring '10