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Bacteriophage Lab: Determining titer of the coliphage by plaque count. Bacteriophages, also called phages, are viruses that attack bacteria. Phages are highly host specific; meaning that any given phage will attack a particular species or group of species of bacteria. Pages are commonly named in reference to their host, thus a phage which attacks the bacterium Staphylococcus is called a staphylophage; one, which attacks Escherichia coli, is called a coliphage. The virus life cycle consists of an intracellular phase and an extracellular phase. In its extracellular phase, the phage exists as an infectious particle, or virion. A virion consists of one or more molecules of nucleic acid, either DNA or RNA, contained within a protein coat, or capsid. Viruses that attack animal cells may have a lipid and protein membrane surrounding the capsid. A virus, when introduced to the correct host bacterium, will adsorb to the host cell wall surface and inject its own genetic material into the host. This begins the intracellular phase of the virus life cycle. Depending on the virus involved, it will then enter either a lytic or lysogenic cycle. If it enters a lysogenic cycle, the virus nucleic acid will either become part of the bacterium’s genetic material or will form a circular ring of DNA (called a plasmid) within the bacterial cell’s cytoplasm. The viral nucleic acid will then replicate and divide each time the host cell does. This will continue until the phage virus is triggered to enter the lytic cycle. In the lytic cycle, the viral nucleic acid takes over the cell’s metabolism to replicate its own nucleic acid, to dictate the manufacture of viral coat protein, and to assemble these pieces together. Following this replication process, the host cell then ruptures (lyses) and releases the new virus particles to continue their life cycle. The T-series coliphages are frequently used to demonstrate bacterial lysis by a virus. These phages are double stranded DNA viruses enclosed in a "naked" envelope (lane lacking a membrane). They possess "binal" capsid symmetry (a polyhedral "head" and a hexagonal "tail"). T –series phages are cultured using a sensitive host strain of bacteria, Escherichia coli B, and grown at 37° C in sterile medium. The concentration of infectious viral particles per milliliter of growth medium is referred to as the viral titer. The purpose of this lab activity is to demonstrate a simple method for determining bacteriophage titer. This is accomplished by making a serial dilution of bacteriophage stock culture. The dilutions of bacteriophage are then mixed with a soft agar medium and this mixture is layered on top of an agar plate. The bacteria grow as tiny colonies within the soft agar layer. These colonies appear as a translucent lawn of growth covering the surface of the plate. The phages form clear patches in the bacterial lawn, called plaques. The plaques form because the phages have bust open the bacteria at that site on the plate. By counting the number of plaques, and multiplying by the serial dilution factor, one can determine the titer, which is the number of phage particles in the original phage culture. Materials Needed E. coli culture Dilution of coliphage culture 6 agar plates (tryptic soy agar) 6 tubes of soft agar (melted and kept at 48° C) 10 tubes of dilution broth (9 ml per tube) sterile transfer pipets Procedure: 1. Label the 10 tubes of the dilution broth tubes as follows: 10-1 to 10-9 , and label the 10th tube "control". These will be the tubes for your serial dilution of the phage culture. Label your agar plates as follows: 10-5, 10-6, 10-7, 10-8, 10-9, control. Also write your names or initials on the agar plates. 2. Transfer 1 ml of the phage culture to the tube labeled 10-1. Use the pipet to mix the dilution by drawing the liquid up and down several times (if the tubes have a screw cap, you can cap the tubes and gently shake them to mix). It is very important that the dilution be thoroughly mixed, but not foaming! 3. Use a new sterile pipet to transfer 1 ml from the 10-1 tube to the 10-2 tube. Again, thoroughly mix the dilution. Continue diluting the sample in this manner until you have transferred to the 10-5 tube. Make sure you use a new sterile pipet with each step, and that you mix each dilution very well. NOTE: The next steps must be done rather rapidly. One person should pipet the phage dilutions, while the other person should pipet the E. coli and then pour the soft agar onto the plate. It is extremely important that you use a new sterile pipet for each step. 4. Inoculate each tube of soft agar with three drops (about 0.1 mL) of the E. coli broth culture. Lift the caps from the tubes of soft agar just enough to accommodate the pipet, leaving the tubes in the water bath at 48oC.This temperature maintains soft agar in the molten state and does not harm phages or bacteria. 5. Continue the dilution series by pipetting 1 mL from the 10-5 tube to the 10-6 tube. Remove one of the tubes of soft agar from the 48° C water bath. With same pipet, remove 1.0 ml of phage dilution from the 10-5 tube and add this to the tube of soft agar. After you add the phage, cap tube and mix thoroughly, and without hesitation, pour the contents of the tube onto the surface of the agar plate labeled 10-5. Immediately after pouring the tube, swirl the plate to distribute the soft agar over the entire agar surface (to the edges of the plate). 6. Repeat step 5, with dilution tubes 10-6 and 10-7, another tube of soft agar, and agar base layer 10-6. Continue this procedure with tubes for 10-7, 10-8, and 10-9. 7. For the control plate, pipet 1.0 ml of plain dilution broth into a tube of soft agar (that already has E. coli), and then pour the contents of the tube onto the plate labeled "control". 8. After the soft agar layer hardens, place the plates in the 37° C incubator (inverted) overnight. The next lab, the plaques can be counted to determine the phage titer. Theoretically, because of the even distribution of the E. coli and the phage across the plate, a single phage will adsorb onto a singe bacterium as it reproduces. The phage particles that are later released when the bacterium lyses then infect surrounding bacteria and repeat this lytic cycle. Eventually, this produces a clear area, or plaque, in the layer of bacteria on the plate. By picking a plate that has between 30 and 300 plaques, an accurate statistical estimate of phage titer can be made. The control plate containing only E. coli and sterile dilution broth in the overly should not show any plaques. A possible error in the titer can occur when more than one phage adsorbs onto a single bacterium or when two phages adsorb onto bacteria that are very close together and result in one plaque when there should have been two. These errors are more likely when a given sample has a high concentration of phage particles. But too low a concentration will increase sample measurement variability. For this reason it is essential to pick a plate of 30 to 300 plaques. The control plate is for comparison and to check the original sterility of the plate and your technique. This plate should appear uniformly translucent. To do the calculation of number of phage particles per mL, apply the following formula: phage/mL = (number plaques/plate) x (1/mL plated) x dilution factor For example, if you got 347 plaques when you plated out 0.1 mL of a 106 diluted phage suspension, the titer is 3.47 x 109/mL.