1. Inoculate a single colony of an appropriate E. coli strain into 5 ml of rich A, medium in a sterile polypropylene culture tube. Incubate the culture overnight with vigorous shaking at 30°C.
2. Transfer 0.1 ml of the fresh overnight bacterial culture (prepared in Step 1) to a sterile 17 x 100-mm polypropylene culture tube with a loose-fitting cap. Infect the culture with approx. 106 pfu of bacteriophage X in 50-100 pi of SM.
3. Incubate the infected culture for 20 minutes at 37°C to allow the bacteriophage particles to adsorb to the bacteria.
4. Add 4 ml of rich A, medium, prewarmed to 37°C, and incubate the culture with vigorous agitation until lysis occurs (usually 8-12 hours at 37°C).
5. After lysis has occurred, add 2 drops (approx. 100 pl) of chloroform and continue incubation for 15 minutes at 37°C.
6. Centrifuge the culture at 4000g (5800 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C.
7. Recover the supernatant, add 1 drop (approx. 50 pl) of chloroform, and store the virus stock.
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