Bacteriophages are viruses that infect bacteria. They can be found wherever bacteria are found. Sewage is a rich source of bacteriophages that infect enteric bacteria such as Escherichia coli. In this experiment, a filtered sewage sample will be added to a rapidly growing culture of Escherichia coli. If phages are in the sewage sample they will go thru many cycles of infection and lyse the Escherichia coli.
Viruses are commonly characterized according to the type of cell they infect. The three major groups of viruses are animal viruses, plant viruses and bacterial viruses (bacteriophages or simply, phages). All viruses are obligate intracellular parasites and are incapable of functioning or reproducing outside living cells. Viral particles are composed of a DNA or RNA core surrounded by a protein coat (capsid). We will deal only with bacteriophages in this lab. Two cycles of infection (lytic and lysogenic) may be exhibited by bacteriophages. Virulent bacteriophages (e.g., T4) multiply rapidly after infecting a host cell and destroy the cell through lysis. Temperate bacteriophages (e.g., lambda) may lyse the host cell or lysogenize the host. If lysogeny occurs the phages produce a protein, called a repressor that prevents replication of the phage DNA. Instead the phage DNA integrates into the host genome where it is referred to as a prophage. When the bacterial host DNA replicates, the prophage DNA is replicated as well. Thus, all bacterial daughter cells carry a copy of the prophage DNA and are referred to as lysogenic bacteria. The lysogenic cells may exhibit new properties such as toxin production (e.g., scarlet fever, diphtheria or botulism).
The lytic cycle of virulent phage consists of 5 sequential stages:
1. Adsorption – A phage attaches to specific receptor sites on the surface of the host cell. This receptor conforms to molecules present on the phage tail. A phage cannot attack bacteria that do not have receptor molecules that conform to the phage tail proteins.
2. Penetration – The phage tail fibers contract and the baseplate settles down on the cell surface. The phage forms a hole in the cell wall using phage lysozyme, an enzyme, and drives the tail core through the cell wall and membrane. The phage DNA is then injected into the bacterial cytoplasm. The head (capsid) and tail stay outside and are referred to as the phage ghost.
3. Biosynthesis - Once inside the cell, phage DNA redirects host cell metabolism. It subverts the cell machinery to the exclusive manufacture of nucleic acid and protein molecules needed for the assembly of hundreds of new phage. For several minutes following infection, complete phage cannot be found; this is called the eclipse period.
4. Maturation - Phage DNA, head protein and tail proteins are synthesized separately and then assembled during this step. The head protein is packaged with phage DNA and then the tail is attached. The eclipse period is over when the first complete phage appears in the cell.
5. Lysis - Lysozyme coded for by the phage DNA causes the host cell to burst releasing intact phages to infect neighboring cells. For some phage, the cycle may be completed in as quickly as 15 minutes.
Bacteriophages can be found wherever their host cells reside. If one wishes to isolate phages that infect Escherichia coli, which is found associated with fecal material, then sewage would be a good source. If one, instead, wanted to isolate phages that infect Bacillus subtilis then soil would be a good source etc. However, in this lab, we have already isolated a set of bacteriophages for you.
Phages are too small (~ 200 nm) to be seen using light microscopy, but they can be detected if grown on a lawn of bacteria using an overlay plating technique. In this procedure, phages and their host cells are mixed in a small tube of soft agar and then poured (overlayed) on top of an agar base plate. Soft agar contains a lower concentration of agar and thus allows the phages to diffuse more freely. The plates are then incubated at the optimum growth temperature for the host bacteria.
During incubation, most of the bacteria multiply to produce a thick covering (a lawn). Some of the bacteria will become infected with a virulent phage. This phage will replicate to large numbers and ultimately kill, or lyse the host cell (lytic cycle). The phages released from the lysed cells will go on to infect neighboring cells, and so on. Cycles of infection and bacterial cell lysis continue until a clear area, called a plaque, is evident within the bacterial lawn. Once the bacteria stop growing due to crowding and lack of nutrients, the phages can no longer successfully infect the bacteria and the plaque will not increase in size.
The overlay plating technique is commonly used to determine the phage titer (the number of phage particles/mL). Theoretically, each isolated plaque originated from one phage. Therefore, the plaques can be counted to determine the number of phages in the original suspension (the titer). Each plaque can be designated as a plaque-forming unit or pfu. The titer is usually given in terms of pfus per mL. In this exercise, a phage suspension will be prepared from the previous experiment and its titer will be determined using the overlay plating technique.
1. In a test tube rack, 6 small plastic dilution tubes. Label the tubes #1-#6.
2. Using a P1000, transfer 0.99 mL of sterile broth into tube #1. Aliquot 0.45 mL
of sterile broth into each of tubes #2-#6.
3. Using a P20, add 10 μL of the provided phage suspension into tube #1. * You will want to draw out your dilution scheme in the space provided below.
4. Mix tube #1 well by tapping or vortexing.
5. Using a P200, remove 0.05 mL from tube #1 and put this aliquot into tube #2. Mix tube #2 well by tapping or vortexing, and continue the serial dilution out to tube #6, as illustrated below. Be sure to use a new pipet tip for each dilution.
6. Label 3 LB agar plates with your initials and section number. Number the plates #4, #5, and #6.
7. Collect a sterile tube containing 5 mL of soft agar from the water bath.
8. Add 100 μL of Escherichia coli culture to the soft agar. Then, add 0.1 mL from phage dilution #4 into the same soft agar tube. DO NOT PLACE THE TUBE BACK INTO THE WATER BATH.
9. Roll the tube between the palms of your hands quickly to mix and pour the entire contents onto the LB agar plate labeled #4.
10. Swirl the plate gently but quickly, ensuring complete coverage before the agar solidifies. This is the overlay technique.
11. Repeat this procedure for phage dilution #5 and #6.
12. After the agar has solidified (about 10 minutes), invert the plates and place them in the tray on the side bench to be incubated at 37°C.
Note the circular clearings in the lawn of bacterial growth. These are called plaques and similar to colonies are derived from a singlar bacteriophage. We can count these plaque forming units (pfus) to determine the original phage titer.
