Bacteriophage Titer Lab
Lab 5: Bacteriophage
5 February 2013
Introduction:
A bacteriophage is essentially a virus that specifically infects bacteria. A bacterial cell infection progresses in much the same way as a eukaryotic cell. A plaque forms when a bacterial cells growing on soft agar burst from the viral infection and appears like a hole in the agar. Each plaque is created by the progeny of an individual phage and can thus be counted to determine the number of phage particles in a sample. The purpose of this lab is to employ a plaque assay method to determine the number of infected phage particles in the given sample.
Method & Materials: * (6) BHI plates at 37˚C * (5) tubes of 9.0mL TSB broth * (6) tubes of soft agar in a 50˚C water bath * T4 phage sample tube * 24hr. broth culture of Escherichia coli B * Sterile pipettes and droppers
1) (Phage Serial-Dilution) a) Label broth tubes with dilution factors (1:10, 1:100, 1:1000, 1:10,000, 1:100,000). b) Pipet 1.mL of stock to the first dilution. Vortex. c) Pipet 1.mL of the 1:10 dilution to the 1:100. Vortex. d) Continue the 10X dilution through to the final concentration.
2) (Phage Plating) e) Label each of the BHI plates according to the various dilutions and one plate “control” which will not include a phage. f) Add 2 drops of the Escherichia coli B to each of the soft agar tubes. g) Transfer 1.0mL of the first dilution to a soft agar tube, vortex gently and pour the contents over the appropriate BHI plate. Swirl to ensure the soft medium completely covers the face of the plate. h) Repeat the process with 1.0mL transfers of each of the dilutions to a soft agar tube then to the appropriate plate. i) The control plate will not receive a 1.0mL broth transfer rather just the soft agar containing only bacteria is poured directly on the BHI plate labeled “control” j) Incubate the plates at 37˚C for 24 hours then
5 February 2013
Introduction:
A bacteriophage is essentially a virus that specifically infects bacteria. A bacterial cell infection progresses in much the same way as a eukaryotic cell. A plaque forms when a bacterial cells growing on soft agar burst from the viral infection and appears like a hole in the agar. Each plaque is created by the progeny of an individual phage and can thus be counted to determine the number of phage particles in a sample. The purpose of this lab is to employ a plaque assay method to determine the number of infected phage particles in the given sample.
Method & Materials: * (6) BHI plates at 37˚C * (5) tubes of 9.0mL TSB broth * (6) tubes of soft agar in a 50˚C water bath * T4 phage sample tube * 24hr. broth culture of Escherichia coli B * Sterile pipettes and droppers
1) (Phage Serial-Dilution) a) Label broth tubes with dilution factors (1:10, 1:100, 1:1000, 1:10,000, 1:100,000). b) Pipet 1.mL of stock to the first dilution. Vortex. c) Pipet 1.mL of the 1:10 dilution to the 1:100. Vortex. d) Continue the 10X dilution through to the final concentration.
2) (Phage Plating) e) Label each of the BHI plates according to the various dilutions and one plate “control” which will not include a phage. f) Add 2 drops of the Escherichia coli B to each of the soft agar tubes. g) Transfer 1.0mL of the first dilution to a soft agar tube, vortex gently and pour the contents over the appropriate BHI plate. Swirl to ensure the soft medium completely covers the face of the plate. h) Repeat the process with 1.0mL transfers of each of the dilutions to a soft agar tube then to the appropriate plate. i) The control plate will not receive a 1.0mL broth transfer rather just the soft agar containing only bacteria is poured directly on the BHI plate labeled “control” j) Incubate the plates at 37˚C for 24 hours then