Pseudoalteromonas espejiana bacteriophage PM2

1. Follow general procedures given below for phage propagation.
2. Use Pseudoalteromonas espejiana BAL 31 (ATCC 27025) as host.

GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE

To recover phage from freeze-dried or thawed frozen vial:

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.NOTE: Broth propagation methods may also be employed with most phage.

Phage is sensitive to heat, so care must be taken when adding to melted agar overlay.  Plaque size is variable (from 1 to 3 mm) and hazy.

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

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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