Escherichia coli bacteriophage P1

1. Follow general procedures given below for phage propagation.
2. Use Escherichia coli strain K-12 (ATCC 25404) as host.

1. Prepare an actively growing broth culture of the recommended host strain 18-24 hours before opening the phage specimen. The host should be in early log phase.
2. Melt several tubes of soft agar (0.5% agar) and maintain at 45-55°C until ready to use.
3. Prewarm plates of the recommended medium in an incubator for approximately 15-30 minutes prior to the plating of the bacteriophage.
4. Add 100 µL of the host culture to a pre-melted 0.5% agar tube and pipette gently to evenly mix, avoiding the creation of bubbles.

1. Overlay the agar plate surface with 2.5-3 mL of melted 0.5% agar containing the host. Allow overlay to harden. 

5. Perform the broth serial dilution in quadruplicate using a 96-well plate.

1. Dispense 90 μL of broth medium into each well.
2. Add 10 μL of phage lysate to each of the four 10^-1 wells. Mix thoroughly by pipetting up and down at least 15 times.
3. Transfer 10 μL from the 10^-1 wells to the 10^-2 wells and mix. Continue the serial dilution until at least 10^-8.
4. Using a multichannel pipette, spot 2 μL of each dilution on the agar overlay. Allow to dry before moving the plates to the appropriate incubator.

6. Incubate plates agar side down at the appropriate temperature. Incubation times will vary depending on the phage. Most only need 12-18 hours. Incubating for too long can make the plaques very difficult to read.  

To propagate phage:

1. Open the host organism according to the information on the product sheet.
2. Pick one colony from the isolation plate and homogenize in 5 mL of the appropriate broth.

1. Note: It may be necessary to inoculate a larger volume or more test tubes based on the volume needed for the next amplification. 

3. Incubate at the appropriate temperature in a shaking incubator until it reaches OD₆₀₀ of 0.1–0.3.
4. Thaw or rehydrate the bacteriophage vial. Use 0.5 mL of the appropriate broth to rehydrate freeze-dried material.

1. Infect each 5 mL culture with 100 μL of the bacteriophage and shake at the appropriate temperature overnight. Prepare a fresh subculture of the host.
2. Centrifuge phage culture at 4000 x g for 10 minutes.
3. Filter the lysate with a 0.2 μm PES sterile filter and store the filtrate at 4°C.
4. Perform a spot titer as per the instructions above.
5. 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. We use double-strength skim milk mixed half-and-half with the filtrate.

Resistant colonies may be seen in areas of clear lysis. 

In the event plaques aren't seen, 2M MgCl₂ and 2M CaCl₂ may be added to the broth used throughout the propagation procedure to help increase phage aggregation potential.

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

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