Download Bacteriophage

MCB 3020L
Lab Experiment 10
Bacteriophage
A three lab session experiment
Bacteriophages are virus particles which infect bacteria. When a bacteriophage (commonly
called "phage") attaches itself to a bacterial cell, the genetic material of the phage (either DNA or RNA)
enters the cytoplasm of the host cell. The phage genetic material begins synthesis of more of the
genetic material and redirects the biosynthetic machinery of the host to make phage proteins. The
phage proteins and nucleic acids are then assembled within the host cell to complete virus particles that
are released to the environment usually by lysis of the host cell. This release occurs quickly and is
called a "burst". Burst sizes may vary, but for the most common phages are around 100 to 200 phages
released per host cell infected. When a burst occurs in a lawn of host bacterial cells, it creates a clear
zone called a plaque. This is due to the lysis of host cells by phages in this clearing.
Phages are usually very selective in their host range - that is the different strains of bacteria they
are able to infect. Generally, phages that can infect one species of bacteria are unable to infect other
species of bacteria. Generally where ever a species of bacteria exist, also in that niche are viruses
which can infect those bacteria. Since Escherichia coli is abundant in sewage, one would expect to find
coliphages there as well. We will attempt to isolate coliphages from FIU sewage be determining the
titer of coliphages in sewage that has been filtered to remove suspended solids and bacteria.
Materials
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Lab Session One
Fresh sewage that has been passed through 0.45 μm pore size filter.
Overnight Nutrient Broth culture of Escherichia coli B or E. coli C.
6 tubes 9 ml dilution blanks (0.1% peptone), sterile.
6 tubes Soft Agar (Nutrient Broth with 0.75% Agar), 4ml each in capped 13x100 mm tubes, molten
at 45oC.
6 Nutrient Agar plates.
Sterile 1000 µl pipette tips.
Sterile 200 µl pipette tips.
Lab Session Two
1. Broth cultures of E. coli B, E coli C, Staphylococcus aureus, Bacillus megaterium, Enterobacter
aerogenes, Pseudomonas aeruginosa.
2. 6 Nutrient Agar plates.
3. 6 tubes Soft Agar (see above), molten at 45oC.
4. Sterile 1000 µl pipette tips.
5. Sterile 200 µl pipette tips.
Procedure
Lab Session One
1. Prepare six serial 1:10 dilutions from the filtered sewage using aseptic 1 ml transfers. Refer
back to lab 3 if you do not remember how to do this.
2. Label 6 tubes of molten Soft Agar and 6 Nutrient Agar plates with each of these dilutions.
3. Inoculate 100 µl of the first into one tube of molten Soft Agar.
4. Add 50 µl of the E. coli B or C liquid cultures to the tube of Soft Agar containing an aliquot from
the serial dilution. Gently roll the tube to mix well, but do not create bubbles. Pour the tube
onto a Nutrient Agar plate. Gently swirl each plate so the Soft Agar is evenly distributed over
the surface. This is called an OVERLAY. You will have approximately 1 minute to complete
your overlay before the agar cools, so be sure you are efficient. Once you are done plating one
overlay, move on to the next until all 6 dilutions have been overlayed.
5. Allow the Soft Agar to solidify and incubate inverted at 30o or 37oC for 24 hours. If longer time
is required between lab sessions, remove the plates to a refrigerator after 24 hours.
Lab Session Two
1. Observe the plates and describe the plaques appearing on the plates. Are they all the same?
Calculate the phage titer in the enrichment tube. See lab 3 if you do not remember how to do
this. Are there surviving bacteria from the phage enrichment tube showing up on the plates?
Choose four different plaques, describe the phage plaque types (size, margin, clear or cloudy),
and label them 1-4.
2. Label 6 tubes of molten Soft Agar and 6 Nutrient Agar plates with each of the bacterial strains
you will be working with. Mark each plate on the outside bottom into quadrants.
3. Add 50 µl of the first bacterial liquid culture to the tube of Soft Agar. Gently roll the tube to
mix well, but do not create bubbles. Pour the tube onto a Nutrient Agar plate. Gently swirl
each plate so the Soft Agar is evenly distributed over the surface. Repeat this for all 5 cultures.
4. Stab with an inoculating needle (not loop) one marked phage plaque and stab each plate in one
quadrant those viruses on the needle. Remember to flame off the needle each time. Then
inoculate the other quadrants with the other 3 labeled plaques from step 1.
5. Incubate at 30⁰C for 24 hours and refrigerate.
1.
Examine your plates:
Lab Session Three
what is the host range of each plaque type (virus type)?
Questions
1. How could you devise an experiment to isolate marine Vibrio phages? (Hint: where are marine
Vibrios commonly found?)
2. Suppose you wanted to isolate viruses that infect cyanobacteria.
(Hint: where are cyanobacteria commonly found?)
Describe how you would do that.
3. Set up an experiment that would isolate bacterial mutants that are resistant to the phages isolated
in questions 1 and 2. What is the hypothesis you are testing?
4. Suppose you had isolated a new bacterial culture from nature and found that it could support
plaque formation by coliphage. (Hint: look up coliphage) What is the likely identity of this new
isolate? How would you prove that?