Questions (or scavenged from) the laboratory using cells of the living host. Viruses are cultured in or in broth seeded with the appropriate host. Animal viruses are grown in tissue culture, by the host cells. a monolayer or a are noncellular obligate intracellular parasites. They are composed of cells. Bacterial viruses are grown in soft agar on a host cell. They can only replicate using the machinery provided by a protective protein coat (and in some cases an outer membrane envelope). Viruses are inert outside of suspension of nucleic acid surrounded

formation. counting particles in the 3. Add 0.1 ml of the nutrient agar surface. the contents of each dilution to tubes by making dilutions of soft agar. Mix the appropriate dilution. Plaques Plaque count PROCEDURE the plate count of plaque-forming units (PFU), infectious phage particles, per ml of subsequent lytic cycles, the region are destroyed. The size of host cells to determine the

are clear areas on the lawn of both that plates and determine the point at which a single infectious virus (viable bacterium) in the host cells in the number of represent the virus and its host. the initial inoculum. A bacteriophage plaque count can be used to the replication times of bacteria in which each plaque (colony) represents a single infectious virus particle was deposited. As a result of the plaque is dependent on a 7. Examine the original suspension. Only those plates with 30-300 plaques are considered useful. Viruses harden and incubate the of phage particles, in the biological basis for plaque formation

OBJECTIVES: Students should be able to 1. Is the phage suspension by 10 to suspension provided. 2. Do of dilution series? 2. enumerate the virus titer in a control. 4. demonstrate the plates represent the soft agar overlay method

5. How would you determine the

basal layer

Sterile 1 ml pipettes

Sterile broth 4.5 ml per tube for in your environmental sample?

 

Phage susceptible bacterium in broth culture

Phage suspension

to 10

1. explain the phage of the concentration of the sterile broth.

At the beginning by adding 0.5 ml of the soft agar and maintain in the soft agar layer spread uniformly over the bottom agar layer?

4. Is the plate labeled correctly? ^plaq titer ^-1 6. Allow to find the agar to the plates for lytic animal viruses?

2. A virus suspension is enumerated by a higher count? Explain.

lyse the cell in order of to separate tubes of each tube of host bacterium culture to leave. Thus they can be enumerated by a plaque assay. By which method would you expect a seeded agar surface for

PFU/0.1 ml = No. plaques formed X dilution factor

MATERIALS

Since only 0.1 ml was plated you must multiply for 48 hours.

Plaque formation thus is analogous

1. Melt the PFU/ml.

PFU/ml = No. plaques formed X dilution factor X10 a suspension

CULTURES

3. calculate virus populations from PFUs

4. Add 1 ml on soft agar. Mark each tube with the electron microscope and by swirling and pour as an overlay onto the virus and assaying

3. Bacterial colonies may be observed growing in the plaque. Why would these develop?

4. How would you perform a water bath at 45°C. a plaque assay

total bacteriophage population Soft nutrient agar 2.5 ml per tube Nutrient agar

plaque