Clostridium sporogenes bacteriophage 71

1. Follow general procedures given below for phage propagation.
2. Use Clostridium sporogenes strain 213 (ATCC 17886) 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 48 hours before opening the phage specimen. Ensure that growth of the host is robust in broth and on agar before starting any bacteriophage workup. 

1. ANAEROBIC CONDITIONS:

1. Anaerobic conditions for transfer may be obtained by the use of an anaerobic gas chamber.
2. Anaerobic conditions for incubation may be obtained by any of the following:

1. Loose screw caps on test tubes in an anaerobic chamber
2. Loose screw caps on test tubes in an activated anaerobic gas pack jar
3. Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.

3. Always use freshly prepared pre-reduced media or pre-reduced media that has been previously prepared but stored under anaerobic conditions.

2. Melt several tubes of #51 soft agar (0.5% agar) and maintain at 55-60°C until ready to use.
3. Prewarm reduced #260 agar plates in an anaerobic incubator for approximately 15-30 minutes prior to the plating of the bacteriophage.
4. Add 300-500 µL of the host culture to a pre-melted 0.5% soft agar tube and pipette gently to evenly mix, avoiding the creation of bubbles.

1. Overlay the agar plate surface with 3-4 mL of melted 0.5% soft 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 reduced #51 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. Incubate for approximately 23-26 hours. Incubating longer than 26 hours will cause the plates to dry and the plaques harder to visualize. 

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.

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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