Phage Propagation噬菌体繁殖【University of British Columbia】

1. Grow host bacteria overnight at 37C. in Proteose peptone No 2 broth (PP2).
   
2. Add 0.1 ml phage stock solution and 0.1 ml of overnight bacterial culture into 4 ml of prewarmed overlay agar (0.6% agar, 1% PP2). Agar is kept at 45-49C.
   
3. Swirl and pour overlay agar onto PP2 plates. Allow to set and incubate at 37C. overnight.
   
4. Scrape off soft overlay with sterile spreader and some sterile broth. Get most of the overlay and broth into 5 mls sterile PP2.
   
5. Centrifuge at maximum speed in a clinical centrifuge for
   15 minutes.
   
6. Save supernatant. Add 0.1 ml. CHCL₃ and label.
   
7. Do a serial dilution of this phage stock and do plate counts of 10-8, 10-10, 10-12 dilutions to obtain the titre. (eg. repeat step 2, incubate and count.)