Bacillus subtilis bacteriophage SP 01枯草芽孢杆菌噬菌体ATCC 27370-B1 BioVector NTCC质粒载体菌种细胞基因保藏中心

Bacillus subtilis bacteriophage SP 01枯草芽孢杆菌噬菌体ATCC 27370-B1

Bacteriophage known to infect Bacillus subtilis subsp. subtilis strain 168 M.
Product category
Viruses
Product type
Bacteriophage
Strain designation
SP 01
Type strain
No

Host
Bacillus subtilis subsp. subtilis 168 M (27370)
Medium
Medium 265: Enriched nutrient broth
Temperature
37°C
Atmosphere
Aerobic
Handling procedure
Follow general procedures given below for phage propagation.
Use Bacillus subtilis strain 168 M (27370™) as host.

GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE
To recover phage from freeze-dried or thawed frozen vial:
Prepare an actively growing broth culture of the recommended host strain before opening the phage specimen. The host should be 18-24 hours old.
Add approximately 1.0 mL of the recommended broth to a freeze-dried phage vial, 0.5 mL to a liquid cryovial.
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 drop of the 18-24 hour host. The soft agar should be maintained at 43°C to 45°C till ready to pour. It may be advisable to use a water bath. Allow overlay to harden.
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.
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.
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.

To propagate phage:
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. 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.
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.
This supernatant is passed through a .22 um Millipore filter and the filtrate may be stored at 4-8°C for a brief time. The phage should be frozen with or without cryoprotectant if kept for more than a few days. If available, liquid nitrogen storage is the best method for long term storage. Most phage can also be freeze-dried, using 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.
Handling notes
This phage will freeze-dry, but there is a sharp decrease in the titer. Therefore, we use frozen storage for best results.



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