Staphylococcus aureus subsp. aureus bacteriophage 88

1. Follow general procedures given below for phage propagation.
2. Use Staphylococcus aureus subsp. aureus strain PS 88 (ATCC^® 33742™) as host.

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 ~4 hours old.
2. Add approximately 1.0 mL of the recommended broth to a freeze-dried phage vial, 0.5 mL to a liquid cryovial.
3. Pre-warm plates of the recommended medium in an incubator.  Overlay the surface with 2.5 mL of melted 0.5% agar (same medium) which contains one or two drops of the ~4 hour host.  The soft agar should be maintained 43-45°C till ready to pour.  It may be advisable to use a water bath.  Allow overlay to harden.
4. The re-hydrated phage can be serially diluted by passing 0.5 mL of the phage into a tube containing 4.5 mL of the broth medium.  Repeat for as many passages as desired.
5. 100 µL 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 overnight incubation, lysis should be visible.  At the higher dilutions, individual plaques should be countable. 
6. Many strains may also be titrated without a soft-agar overlay.  Pipette approximately 1.0 mL of the host onto the surface of each plate. After tilting plate to ensure the entire surface is covered, the excess liquid is aspirated off.  After the surface dries, the various dilutions of the phage are dropped onto the surface as before. 
   NOTE:  Spotting the phage on plates makes visualizing the lysis easier.  If 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.

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 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.  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.

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.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.