Salmonella enterica subsp. enterica bacteriophage Chi

1. Follow general procedures given below for phage propagation.
2. Use Salmonella enterica subsp. enterica serovar Bispebjerg (ATCC 9842) as host. 

GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE

To recover phage from freeze-dried or frozen vial:

1. Prepare an actively growing broth culture of the recommended host strain before opening the phage specimen. The host should be 18-24 hours old. 
2. Pre-warm plates of the recommended medium in an incubator. Overlay the surface with 2.5 mL of the melted 0.5% soft agar (same medium) that contains one or two drops of the freshly grown host. The soft agar should be maintained at 43°C to 45°C  until ready to pour. It may be advisable to use a warm bath. Allow overlay to harden. 
3. The phage can be serially diluted by passing 0.25 mL of the phage into a tube containing 2.25 mL of the broth medium. Repeat for as many passages as desired. 
4. Spot 0.1 mL of each dilution on the surface of the prepared plates. Allow to dry for approximately 15-30 minutes. Three to four dilutions can be placed on each plate. After 24 hours incubation, lysis should be visible. At the higher dilutions, individual plaques should be countable. 
   NOTE: Spotting the phage on plates makes visualizing the lysis easier. If the 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. Phage may be propagated by preparing plates with soft agar/host overlay as above and covering the surface with approximately 0.5 mL of the concentrated phage. Or, alternately, you may add the phage directly to the melted agar/host before pouring over the plates. For large amounts, large-size T-flasks can be prepared with the recommended agar, and approximately 12.0 mL of melted soft agar/host poured over the surface.  Phage is then allowed to run over hardened surface. Phage may also be added directly to melted soft agar before pouring as described above.
2. After 24 hours incubation, or when lysis is observed, the soft agar is scrapped off the surface of the agar plates. Centrifuge at about 1000 rpm for 25 minutes to sediment the cellular debris and agar. Conserve the supernatant. 
3. This supernatant is passed through a .22  μm Millipore filter and the filtrate stored at 4-8°C. 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. We use double-strength skim milk mixed half- and -half with the filtrate.
   NOTE: Broth propagation methods may also be employed with most phage. Unless otherwise noted, ATCC uses the Adams agar-overlay methods as described in M.H. Adams' Bacteriophage (Interscience Publishers, Inc., New York, 1959) for routine phage.

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