Preparation of the Filamentous Bacteriophage Ff

1. Preliminary Preparations

  • 250 mL MS+ (bottle with screw cap)
  • 1 L MS+ media (Erlenmeyer flask with foil cap)
  • 250 mL MS+ top agar (bottle with screw cap)
  • (2) 1 L MS+ bottom agar (Erlenmeyer flask with foil cap, 40-60 petri dishes)
  • 50 mL 50% glucose (bottle with screw cap)
  • 50 mL 2M CaCl2 (bottle with screw cap)
  • 250 mL 10mM Tris, pH 7.8 ± 0.2 (bottle with screw cap)

2. Bacterial Host Cells - Escherichia coli (strain Hfr3300)

  • Using aseptic technique, streak a loopful of cells onto an agar plate as depicted in the figure. Incubate overnight (ON) at 37 °C. This should produce single colonies of bacteria. Fresh plates should be made weekly. Long term storage of bacterial cells is in solution state at -80 °C.



  • Using aseptic technique, inoculate 30 mL of MS+ liquid media with a single colony of bacteria and shake ON at 37 °C. This bacterial growth should be done in an Erlenmeyer flask with a foil cap and is called an overnight (ON). An ON of bacteria can be used as plating bacteria for titering phage or as the bacterial solution used to inoculate a large scale phage preparation. For the former purpose, a fresh ON should be made every week. For the latter, an ON should be made the night before it is to be used.

    3. Titering the Phage Solution (All steps should be done using aseptic technique.)

  • Label 16 MS+ plates as follows: plate control, cell control, dilution buffer control, top agar control, and 2 each (A and B) with 10-2, 10-4, 10-6, 10-8, 10-10, 10-12.

  • Add 2 mL of 10 mM Tris, pH 7.8 to each of 12 dilution tubes and align in a test tube rack in groups of 6.

  • Add 20 mL of phage solution to the first tube in each series, agitate and let stand for 30 min. Agitate. This is a 10-2 dilution.

  • Make five more 10-2 dilutions in succession. (Add 20 mL of the first solution (10-2 dilution) to the second tube and agitate (produces 10-4 dilution).) The last dilution is 10-12.

  • Add 0.1 mL of plating bacteria to each of 13 agar tubes (to be used for 12 phage-containing tubes and cell control tube).

  • Add 0.1 mL of one phage dilution to a bacteria-containing agar tube. One bacteria-containing agar tube will not have a phage dilution and this is the cell control. Add 0.1 mL of the dilution buffer to an empty agar tube. This is the dilution buffer control.

  • Add 2.5 mL of melted (but not hot) top agar to each of the cell- and/or phage-containing agar tubes and quickly pour the contents of each tube onto the appropriately labeled plate. Add 2.5 mL of melted top agar to the top agar control plate. Allow plates to stand for about 20 min., so that the top agar is completely cooled and hardened.

  • Incubate the plates, inverted, at 37 °C, ON.

  • Remove the plates from the incubator and count the number of plaques on each plate. Using the equation below, calculate the number of infectious phage (PFUs, plaque forming units) in the original phage solution.
    (concentration of phage solution)(0.02/2)n(0.1 mL) = # of plaques on plate from n dilution

    For example, if there were 30 plaques on the 10-8 plate, then

    (concentration of phage solution)(0.02/2)4(0.1 mL) = 30 PFUs (30 infectious phage).

    Solve for the concentration of phage.

    concentration of phage solution = 3.0 X 1010 PFU/mL

    4. Preparation of Stock Solution

  • Using aseptic technique, inoculate 100 mL of MS+ media with 1 mL of E. coli ON.

  • Pick one plaque of phage from a titered plate and blow it into the cell suspension. (It works well to use a Pasteur pipette.)

  • Incubate with shaking, ON at 37 °C.

  • Pellet the cells at 7K, 10 minutes at 4 °C using the JA-20 rotor and the high speed centrifuge.

  • Decant the phage-containing supernatant and make it 0.5 M NaCl, 2% PEG (polyethylene glycol). Stir for 3-5 hours at 4 °C.

  • Pellet the phage precipitate at 10 K for 30 minutes at 4 °C using the JA-20 rotor and the high speed centrifuge.

  • Resuspend the phage pellet in 10 mL of 10 mM Tris, pH7.8, ON at 4 °C.

  • Titer the phage solution.


    5. Large Scale Phage Preparation

    5a. Growth

  • Using aseptic technique, inoculate 1 L MS+ media with 1 X 109 cells (approximately 5 mL of an ON). 1 OD = 2 X 108 cells/mL (i.e., if Abs550 = 0.800, then you have 1.6 X 108 cells/mL)

  • Using aseptic technique, infect the bacteria with fd stock at an MOI (multiplicity of infection) of at least 10. (I prefer to use an MOI of at least 100 or even 1000.) Shake the preparation at 37 °C, ON (about 20 h).
    MOI = (volume of phage solution)(phage concentration)/ (volume of cell solution)(cell concentration)

    Also, MOI = # of phage / # of bacterial cells
    5b. Isolation

  • Spin the 1 L culture in the JA-20 High speed centrifuge at 7K, 4 °C for 10 minutes using either the JA-14 rotor with 6 bottles containing approx. 175 mL each or the rotor that accommodates 4 1-liter bottles with screw caps. For the latter rotor, each bottle must be full (with either phage culture or water). Decant the supernatant (phage solution) into a 1 L graduated cylinder (or other 1 L glass container). Add CLOROX (a splash) and water to each cell pellet and, when loosened, discard the disinfected debris solution.

  • Make the phage solution 0.5 M NaCl, 2 % PEG (polyethylene glycol) by adding the appropriate amounts of NaCl and PEG. Stir the phage/NaCl/PEG solution at 4 °C at least 3 h. It is best to stir this solution ON and it doesn't hurt to stir it over the weekend. This step precipitates the phage.

  • Spin the phage solution in the high speed centrifuge at 10K, 4 °C for 20 minutes using a rotor that accommodates 1 liter of solution. Discard the supernatant and resuspend each phage pellet (4 °C, ON) in about 2 mL of 10 mM Tris, pH 7.8.

    5c. Purification

  • Pool the solutions of resuspended phage. Rinse the centrifuge bottles with 2 mL (total) of 10 mM Tris, pH 7.8 and add this solution to the pooled phage solution.

  • Spin phage solution in the high speed centrifuge at 10 K, 4 °C for 10 minutes using the JA-20 rotor and the appropriate number of polypropylene tubes with screw caps. This step pellets out excess cell debris.

  • Decant the supernatant (phage-containing) into another tube. Separate the phage solution into the appropriate number of polycarbonate 70Ti centrifuge tubes with cap assemblies. Be sure the cap assembly has an O-ring. Spin the phage solution in the L8-80M ultracentrifuge at 60 K, 4 °C for 100 minutes using the 70Ti rotor. Discard the supernatant and resuspend the pellets in 10 mM Tris, pH 7.8 (ON, 4 °C).

  • Spin the resuspended phage solution in the ultracentrifuge at 7 K, 4 °C for 10 minutes using the 70Ti rotor. Transfer the supernatant to a clean centrifuge tube(s) and spin the phage solution in the ultracentrifuge at 60 K, 4 °C for 100 minutes using the 70Ti rotor. Discard the supernatant. If the phage pellet looks clean (minimal dirt in the center of the pellet), the purification is finished. However, two more cycles of resuspend-pellet should be done to remove all of the salt. If the phage pellet still looks dirty, two more cycles of resuspend-pellet should be done with the addition of the 7 K spin.


    6. Determining the Concentration of the Phage Solution

  • Take a UV absorbance reading at 270 nm. Determine the OD according to the following equation.
    OD = (Abs270)(dilution)(path length)

    For a 1:10 dilution (1 mL into 9 mL), the dilution value is 10.
  • Calculate the concentration using the following equation.
    phage concentration = OD/3.84

    7. Recipes

    MS+ media

    10 g tryptone
    1 g yeast extract
    8 g NaCl
    1 L H2O
    After autoclaving this, add 2 mL sterile 50% glucose and 2 mL sterile CaCl2.

    MS+ bottom agar

    Same as above, except add 13 g agar before autoclaving.    		 MS+ top agar

    Same as above, except add 8 g agar before autoclaving.


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