BIO XXXXXXXXXXDr. Hengartner Spring 2020 Assignment #3: Virus Titering Exercise (18 pts) Name: ________________________________________ Part 1. Preparing the plasma and diluting the virus sample. A...

This is a biology assignment only 16 questions


BIO 454-01 Dr. Hengartner Spring 2020 Assignment #3: Virus Titering Exercise (18 pts) Name: ________________________________________ Part 1. Preparing the plasma and diluting the virus sample. A sample of patient plasma was obtained and serially diluted in phosphate-buffered saline solution (PBS 1X) as described below: 1. Which of the following do you expect to remain in the blood plasma (circle one or more answers)? a) Blood cells (erythrocytes, leukocytes, platelets) b) Antibodies, non-blood clotting proteins c) Fibrinogen and other clotting factor d) Ionic salts and small compounds e) Virions 2. How would you change the protocol if you wanted to obtain serum, instead of plasma ? ___________________________________________________________________ ____________________________________________________________________ 3. What is the dilution in the last 3 tubes ? Insert the correct numbers in the figure above. 4. Circle the tube that is a million times more diluted than the original plasma. 5. The dilution process shown here is called a serial dilution. Please define what this term means: _____________________________________________________________________ ______________________________________________________________________ Part 2. Doing the plaque assay and calculating the titer. 300 μl of the last three tubes were used to infect a monolayer of mammalian cells. The experiment was done in duplicate (2 wells were each infected with 300 μl of the same tube). After a 3 day incubation, you stain the cells and look for the viral plaques, obtaining the results show in the figure on the next page. 6. Viral titers obtained from plaque assays are expressed in units of _________ 7. What does pfu mean ? ___________________________________________ 8. Pfu and number of virions may not necessarily be the same. Explain why ? __________________ _______________________________________________________________________________ _______________________________________________________________________________ 9. Which two tissue culture wells were infected by the last tube in the dilution series? ____________ 10. How many plaques do wells #b and #e have ? Well #b: _______ Well #e: _______ Mean of b and e: ________ 9.9 ml PBS 0.1 ml plasma 10-2 10-3 10-4 10-5 ____ ____ ____ Plasma Dilution BIO 454-01 Dr. Hengartner Spring 2020 11. (2 points) Use the mean value obtained in the previous question (average the pfu's in wells #b and #e), and calculate the virus titer in the second to last tube of the dilution series ? (Show your calculations and use appropriate units) ANSWER:____________ 12. (2 points) What was the virus titer in the original undiluted serum ? (Show your calculations and use appropriate units) ANSWER:____________ 14. If you had used the wells #c and #f to count the viral titer, you would have ____________. a) found the exact same viral titer b) found a grossly different viral titer c) found a similar, but less precise, viral titer 15. (2 points) You perform an infection by adding 300 μl of a 5.0 X 106 pfu/ml of virus sample to a 100 mm plate containing a monolayer of 3.0 X 106 cells. Calculate the MOI. 16. You decide to perform a one-step growth curve to examine the kinetics of viral replication. You would like to add enough virus so at least 95% of the cells are infected. What is the lowest MOI integer that would be sufficient ? Hint: Use the Poisson equation and calculate the proportion of cells that would receive zero viruses. Plug in an MOI of 1 into the equation, then 2, then 3, etc. into the Poisson equation until you find the MOI that gives an infection rate of 95% or higher. d f a b c e