Laboratory Investigations in Microbiology

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Chapter 23: Viruses

Bacteriophages are viruses that can infect only bacteria. And just as animal viruses are specific to a particular host (human, dog, sheep), most bacteriophages are specific to one or a few closely related strains of bacteria. Some of the better-understood bacteriophages are lambda, T-2, and T-4 bacteriophages. Bacteriophages  that infect coliform bacteria are known as coliphages (a virus that infects coliform bacteria, primarily E. coli). Phages such as T-4 are complex, naked, dsDNA viruses that attach to the bacterial cell surface and inject their DNA through the cell wall and membrane into the bacterial cytoplasm. Once inside, phage DNA is replicated and transcribed/translated by the host bacterium, producing many viral proteins and DNA which self-assemble into viral particles (virions). Eventually, the phage lyses its host cell, resulting in cell death and release of thousands of phage particles into the medium.

When culturing bacteriophages in the lab, a lawn of E. coli sensitive to them must first be grown. Virions are mixed into the agar along with E. coli cells. As the E. coli cells start to divide and grow in the agar, bacteriophage particles attach to them, starting the lytic life cycle. As E. coli cells nearby get infected and lysed as well, a clear area soon begins to form around the point where the virion first infected an E. coli cell. These clear areas are called plaques. Each plaque was started by a single virion, and therefore the number of plaques on an agar plate give you an indication of the number of viral particles in the sample. (The number of plaques are used to determine the number of PFUs (Plaque Forming Units) in a sample. A PFU is a virion that actually caused formation of a plaque.) This method for determining number of viruses in a sample is very similar to the viable plate count method for determining bacterial numbers, as discussed in the previous chapter.

Common sources of bacteriophages include environments where such bacteria thrive, including water1 soil2, and sewage3. Bacteriophages can be isolated from these habitats by filtering or centrifuging samples to remove particulate matter (sewage, soil) and bacteria (using 0.2 micron filters). Clarified samples can then be analyzed for bacteriphages by culturing them with the appropriate host strain. Each bacteriophage has its own specific host.

In this lab exercise, we will be determining the phage titer (# phage particles/ml) of a stock preparation of T-2 Bacteriophage, as well as examining the host range of this bacteriophage.

 

Materials
Procedure
First period

        Step 1: preparation of plates

  1. Pour one melted bottom agar tube into each of your 4 sterile Petri plates; allow these to harden
  2. Label 3 bottom agar plates 10-1,10-2, and 10-3. These represent your diluted virus samples.
  3. Label 1 more bottom agar plate "host = (name of your second host culture)"
    Step 2: Serial dilution
  4. Pipette 0.1 ml of the virus sample into a 0.9 ml sterile saline tube. Label this tube "10-1". Vortex.
  5. Pipette 0.1 ml of the 10-1 tube into a 0.9 ml sterile saline tube. Label this tube "10-2". Vortex.
    Step 3: virus inoculation
  6. Pipette 0.1 ml from the virus sample into a tube of melted "TOP AGAR". Add 500 Ál of ECB bacterial broth, immediately vortex and pour into the Petri dish labeled 10-1
  7. Pipette 0.1 ml from the 10-1 tube into a tube of melted "TOP AGAR". Add 500 Ál of E. coli broth, immediately vortex and pour into the Petri dish labeled 10-2
  8. Pipette 0.1 ml from the 10-2 tube into a tube of melted "TOP AGAR". Add 500 Ál of E. coli broth, immediately vortex and pour into the Petri dish labeled 10-3
  9. Pipette 0.1 ml of the virus sample into a tube of melted "TOP AGAR". Add 500 Ál of your second bacterial broth (alternate host), immediately vortex and pour into the Petri dish labeled "host = (name of your second host culture)"
  10. Let plates harden before bringing up front for incubation.
Next period
  1. Obtain your Virus plates. 
  2. Count the number of plaques on each plate (if possible). Be sure to note overlapping plaques.
  3. Calculate the number of PFU for each plate counted.
  4. Report your data online to compare the number of phages in the virus sample

5. Evaluate your alternate host strain plate for plaques.

 

Data Sheet & Review Questions

 

References

  1. https://kb.osu.edu/dspace/bitstream/1811/23558/1/V093N3_048.pdf
  2. http://robles.callutheran.edu/~revie/virology/Soil-phage-isolation.pdf
  3. http://www.rivier.edu/journal/RCOAJ-Spring-2007/J91-Biology.pdf

 

 

ę 2003 - 2018 JosÚ de Ondarza, Ph.D.