Categorized: Microbiological Techniques
- Grow host bacteria overnight at 37C. in Proteose peptone No 2 broth (PP2).
- 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.
- Swirl and pour overlay agar onto PP2 plates. Allow to set and incubate at 37C. overnight.
- Scrape off soft overlay with sterile spreader and some sterile broth. Get most of the overlay and broth into 5 mls sterile PP2.
- Centrifuge at maximum speed in a clinical centrifuge for 15 minutes.
- Save supernatant. Add 0.1 ml. CHCL3 and label.
- 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.)