| Methods: Recipes:
* Special conditions include 37°C incubation and nutrient agar, which selects for E. coli growth. E. coli is the specific host of the phage to be isolated, allowing for the phage's "enrichment." Part I: Enrichment (Amplification of Phage) 1. Measure out 45 mL raw sewage into graduated cylinder and decant into Erlenmeyer flask 2. Pipette 5 mL DSPB and 5 mL E. coli into the sewage flask 3. Incubate flask at 37° C for 24 hours. Part II: Filtration (Isolation of Phage) 1. Aliquot sewage and E. coli mixture into 8 centrifuge tubes, filling each up to within 1/2 inch from the top. 2. Centrifuge the tubes at 2500 rpm for 10 minutes. 3. Without disturbing the pellet, pipette the liquid from each tube into a clean new set of tubes. 4. Centrifuge the second set of tubes at 2500 rpm for another 10 minutes. 5. Pipette the top 2/3 of each tube into a new set of tubes and centrifuge at the same settings. 6. Pipette the liquid into a membrane filter assembly and run vacuum until all the liquid is pulled into the container. 7. Aseptically transfer the final filtrate from the filtration assembly to a flask. Part III: Seeding (Growth of E. coli and Phage) 1. Liquefy four tubes (labeled 1 - 4) of 5 mL soft nutrient agar and maintain a 50°C temperature. 2. Inoculate tubes with 0.3 mL of fresh E.coli culture. 3. In tube 1, inoculate 1 drop of phage filtrate. To tube 2, 3 drops of filtrate. To tube 3, 6 drops of filtrate. To tube 4 (control), do not add any filtrate. 4. Pour tubes quickly onto corresponding hard nutrient agar plates (labeled 1 - 4) before soft nutrient agar hardens. 5. Incubate plates at 37°C for 24 hours. (If possible, check plates at 6 hour intervals.) Part IV: Reproduction of Results 1. Observe the plates and record results. Choose a plate with the most distinct plaques. If the plates appear too lysed, it may be necessary to produce a dilution series in seeding. 2. Using a flamed and cooled needle, scrape off a single plaque from the plate and inoculate into a fresh culture of E. coli. (If multiple plaques are chosen, proceed with the following steps for each plaque in separate tubes, filters, and plates.) 3. Incubate at 37°C for 24 hours. 4. Repeat filtration procedure in Part II. 5. If necessary, create a dilution series of 10X with saline and the filtrate. (original concentration, 0.1, 0.01, 0.001, etc. for as many as necessary.) 6. Pipette 0.15 mL (about 3 drops) of each sample of filtrate into soft nutrient agar tubes (5 mL) with 0.3 mL of E.coli, and pour onto nutrient agar plates. 7. Incubate plates at 37°C for 24 hours, and check for formation of plaques. 8. If more than one type of plaque is still observed (identifiable by different size or shape), repeat Part IV, attempting to be very careful in removing a single plaque from a plate. A single type of virus is likely obtained when plaques look uniform. Methods based on Microbial Applications Manual. 1 |