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1 You have diluted a 1-ml sample of virus stock by taking 100 |ll from the stock solution and adding to it 0.9 ml of buffer. You then take 10 |ll of this dilution and dilute it into 1 ml. You then infect two plates that contain 105 cells each with 100 |ll. One plate had 25 plaques while the other had 29 plaques. What was the titer in the original stock?

2 One milliliter of bacterial culture at 5 X 10s cells/ml is infected with 109 phages. After sufficient time for more than 99% adsorption, phage antiserum is added to inactivate all unadsorbed phage. Cells from this culture are mixed with indicator cells in soft agar and plaques are allowed to form. If 200 cells from the culture are put in a Petri dish, how many plaques would you expect to find?

3 You have a series of culture dishes that contain "lawns" of HeLa cells (human cells). You plan to infect these cells with poliovirus type 1 under a variety of conditions. You will measure the ability of the virus to form plaques on these cells. In the table below, predict which of the conditions will result in plaque formation by poliovirus type 1 on HeLa cells. Indicate your answer with a "Yes" or a "No" in the table.

Experiment

Virus added

Treatment of cells

Plaques?

Negative control

None

-

No

Positive control

Poliovirus type 1

-

Yes

A

Poliovirus type 1

Cells treated with interferon

B

Poliovirus type 1

Antibody against rhinovirus added

C

Poliovirus type 1

Antibody against poliovirus type 2 added

D

Poliovirus type 1

Antibody against poliovirus type 1 added

4 You have performed a plaque assay on a stock of bacteriophage T4. Your results show an average of 400 plaques when you assay 0.1 ml of a dilution prepared by mixing 1 part of the original virus solution with 999,999 parts of buffer.

  • a) What is the titer of the original stock of bacteriophage?
  • b) What volume of this stock would you have to use to infect a 10-ml culture of E. coli, containing 4 X 106 cells/ml, such that the multiplicity of infection will be 10?

5 A stock of poliovirus is measured by plaque assay on a "lawn" of HeLa cells. When 0.1 ml of a 105 dilution of this stock is plated, an average of 200 plaques are observed.

  • a) What is the titer of this stock?
  • b) If 0.1 ml of this stock is used to infect 10.0 ml of HeLa cells containing 105 cells/ml, what is the multiplicity of infection (MOI) in this case?

6 Using the Poisson distribution, calculate the proportion (probability) of cells infected with the indicated number of plaque-forming units (PFUs), given the multiplicity of infection (MOI) shown in the table.

Proportion (probability) of cells infected with

MOI

0 PFU

1 PFU

>2 PFUs

0.01

0.1

1

10

7 You have three stocks of influenza virus that you have assayed by hemagglutination. The microtiter plate is shown below:

1/4 1/8 1/16 1/32 1/64 1/128

Graphic analysis of the data from Fig. 10.7. The percentage of infected wells as a function of dilution is shown on a semilogarithmic plot. The dilution at which 50% of the wells would be infected (the TCID50) can be estimated by graphic interpolation.

  • a) Which of the virus stocks has the highest hemagglutinin (HA) titer? Which has the lowest HA titer?
  • b) What would you report as the endpoint HA unit for stock 2?

8 Virus particles are very carefully isolated from an infected cell stock. You use this material to infect a culture of 106 cells with an MOI of 7 PFUs/cell. What is the maximum percentage of cells, which could be productively infected?

9 You apply a virus stock solution containing 3 X 106 virus particles to 3 X 105 cells. What is the MOI for this infection?

Physical and Chemical Manipulation of the Structural Components of Viruses

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