Escherichia coli bacteriophage Q-β

1. Follow general procedures given below for phage propagation.
2. Use Escherichia coli (ATCC 23631) as host.

GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE

To recover phage from freeze-dried or frozen vial:

1. Before opening the phage vial, prepare an actively growing culture of the recommended host strain. The host should be 16-18 hours old. 
2. Pick one colony from the isolation plate and homogenize in 5 mL of the appropriate broth. Incubate at 37°C while shaking (160-180 rpm) until the growth reaches OD₆₀₀ of 0.1 to 0.4. 
3. Add approximately 1.0 mL of the recommended broth to a freeze-dried bacteriophage vial or 0.5 mL to a liquid cryovial. Infect each 5 mL culture with 100 µL of the bacteriophage. Shake at 160-180 rpm in 37°C overnight. After 16 to 18 hours, centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate with a 0.2 µm or 0.45 µm PES sterile filter.  The filtrate can be stored at 4°C. 
4. Prior to performing a spot titer, warm one or two plates at 37°C. 
5. Melt the soft agar (0.5% agar added to the recommended medium) and maintain at 43°C to 45°C until ready to use. It is best to allow the melted agar to remain at this temperature for about an hour to ensure that it has cooled to 43°C to 45°C. Warmer temperatures may kill the host.
6. Add 50 to 100 µL of the host culture from step 1 to each 9 mL (approximately) of soft agar from step 5 above. Immediately overlay the surface of each plate with 2.5 mL. Allow the overlay to harden for 10 to 20 minutes.
7. The phage lysate can be serial diluted in a 96 well plate in quadruplicate (if desired). Aliquot 90 µL of broth medium into each well. Add 10 µL of phage filtrate from step 3 to each well and mix. Transfer 10 µL from each well of the first dilution to each well of the second dilution and mix. Continue to the desired number of dilutions.
8. Spot 2 µL of each dilution on the plate from step 6. Up to 8 dilutions can fit on a 90 mm petri dish. After overnight incubation, lysis should be visible.  At the higher dilutions, individual plaques should be countable. To calculate pfu/mL, use the following formula: pfu/mL = average plaque count / [(dilution factor) (2x10^-3 mL)]
9. Spotting the phage on plates makes visualizing the lysis easier. If phage is added directly to soft-agar before pouring plates, hazy or tiny plaques may be difficult to see. Resistant host bacteria may also mask plaque formation.
   
    

To propagate phage:

1.  Determine the total volume needed and place this amount of broth in a flask.  Add a small amount of overnight host culture to the flask and incubate at 37°C while shaking until the growth reaches OD₆₀₀ of 0.1 to 0.4.   
2. Infect with the calculated volume of phage lysate using the following formula. Volume of phage to add (ml) = (8x10⁸ x total culture volume in ml x OD₆₀₀ x MOI) / phage titer (PFU/ml). Shake at 160-180 rpm at 37°C overnight.
3. Centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate with a 0.2 µm or 0.45 µm PES sterile filter.  The filtrate can be stored at 4°C.
4. Lysates should remain viable under refrigeration for long periods. They may also be frozen with or without cryoprotectant.  If available, liquid nitrogen storage is the best method for long term storage. Most phage can also be freeze-dried. ATCC uses double strength skim milk mixed half and half with the filtrate.
   Note: To achieve the highest PFU and total volume, the broth method detailed above has demonstrated the best results if the process is followed exactly. However, if any of the equipment are not available or if the technique is not possible for other reasons, the Adam’s Overlay method described below will provide adequate results for smaller volumes at satisfactory titer count. (Adams agar-overlay method as described in M. H. Adams' Bacteriophages, Interscience Publishers, Inc., New York, 1959).
   
    

Resistant bacterial growth may occur in areas of complete lysis.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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