Escherichia coli bacteriophage MS2 ATCC 15597-B1 BioVector NTCC质粒载体菌种细胞基因保藏中心

Escherichia coli bacteriophage MS2 ATCC 15597-B1 BioVector NTCC质粒载体菌种细胞基因保藏中心 Designation: MS2 Deposited Name: MS2 Medium ATCC® Medium 271: Escherichia medium Growth Conditions Temperature: 37°C Atmosphere: Aerobic Propagation Procedure 1. Follow general procedures given below for phage propagation. 2. Use Escherichia coli strain C3000 (ATCC® 15597™) as host. GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE To recover phage from freezedried or thawed frozen vial: 1. Prepare an actively growing broth culture of the recommended host strain from a frozen stock before opening the phage specimen. The host should be 6 hours old or at least 24 hours old if needed. Repeated passage of this host may result in hazy plaques. 2. Add approximately 1.0 mL of the recommended broth to a freezedried phage vial, 0.5 mL to a liquid cryovial. 3. Prewarm 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 6 hour host, or a 24 hour old host if needed. 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. 4. The rehydrated 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 softagar 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 softagar 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 the softagar/ 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, largesize Tflasks can be prepared with the recommended agar, and approximately 12.0 mL of melted softagar/ host poured over the surface. Phage is then allowed to run over hardened surface. Phage may also be added directly to melted softagar 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 um Millipore filter and the filtrate may be stored at 48° 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 freezedried, using doublestrength skim milk mixed halfandhalf with the filtrate. NOTE: Broth propagation methods may also be employed with most phage. Unless otherwise noted, ATCC® uses the Adams agaroverlay method as described in M. H. Adams' Bacteriophages (Interscience Publishers, Inc., New York, 1959) for routine phage production. [Supplier来源] http://www.biovector.net