Staphylococcus aureus subsp. aureus bacteriophage 6

1.   Follow instructions below for titrating or propagating phage.
2. The preferred method of propagation for this phage is either broth culture or soft-­agar overlay, using Staphylococcus aureus strain 6 (ATCC 27704) as propagation host.

GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE

To titrate phage:

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. Pre­warm plates of the recommended medium in an incubator. Overlay the surface with 2.5 mL of melted 0.5% agar (same medium) that contains 0.1mL of the freshly grown host. The soft agar should be maintained 43 to 45°C until ready to pour. It may be advisable to use a water bath. Allow overlay to harden. 
3. Add approximately 0.25 mL of the recommended broth to a freeze-­dried phage vial, 0.5 mL to a liquid cryovial.
4. The rehydrated 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. 
5. 0.1 mL of each dilution is spotted on the surface of the prepared plates. Allow to dry. Three to four dilutions can be placed on each plate. After 18-24 hours incubation, lysis should be visible. At the higher dilutions, individual plaques should be countable.

To propagate phage (agar-overlay method):

1. Phage may be propagated by preparing plates with the soft-agar/host overlay as above and covering the surface with approximately 0.5 mL of the concentrated phage.  Or, alternatively, you may add the phage directly to the melted agar/host before pouring over the plates.  For larger 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 18 to 24 hours incubation, the soft agar is scraped 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 (see below).  Unless otherwise noted, ATCC uses the Adams agar-overlay method as described in M. H. Adams' Bacteriophages (Interscience Publishers, Inc., New York, 1959) for routine phage production.

For broth propagation:

1. An overnight broth culture of the above host is aseptically transferred into a flask of the recommended broth so that a 1:100 dilution of the culture is achieved.
2. To this, add the rehydrated phage.  Incubate for 6 hours with shaking.
3. Harvest, centrifuge, and filter as described above.

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