ENVR 421 Laboratory #5: Detection of bacteriophages in environmental waters Introduction Some bacteriophages occur naturally in the human and animal intestinal tract and are excreted in feces. Thus, phages can be indicators of fecal contamination in the environment. A variation of the plaque assay can be used to detect these viruses in environmental waters. Purpose The purpose of this laboratory exercise is to detect coliphages (bacteriophages that infect E. coli) in water from Morgan Creek and effluent from the OWASA sewage treatment plant. Generally, numbers of phages in the environment are low. In order to detect them, instead of using the double agar layer (DAL) assay, we will use a variation called the single agar layer (SAL) assay. The SAL can accommodate larger sample volumes than the DAL. Principle The SAL works on the same principle as the DAL, but does not use top and bottom agar. Instead, equal volumes of agar and sample are mixed in a bottle with bacterial host. The single layer of agar is poured into a large plate, and incubated until plaques form. Materials needed: • large petri dishes • E. coli host • 90 mL dilution bottle • Empty sterile sample bottle • graduated cylinders • Morgan Creek water • OWASA effluent • 3 100 mL bottles of TSA • MgCl2 stockProtocol Preparation of the host culture for this lab will be done for you by the TA. You will have three samples: Undiluted OWASA effluent, a 1:10 dilution of OWASA effluent, and Morgan Creek water. 1. Mix the effluent sample by shaking well 2. Make one dilution of the effluent sample by adding 10 mL to 90 mL of PBS in a dilution bottle 3. Measure 100 mL of OWASA water into an empty sterile dilution bottle 4. Measure 100 mL of Morgan Creek water into an empty sterile dilution bottle 5. Mix the three bottles vigorously by shaking 6. Add 0.5 mL MgCl2 stock solution to each bottle 7. Add 10 mL of host bacteria to each bottle 8. Set out 5 large petri dishes on the bench 9. Remove one bottle of TSA from the waterbath 10. When the TSA reaches the right temperature, add the Morgan Creek sample to the bottle of TSA. 11. Swirl to mix 12. Immediately pipette 40 mL into each petri dish 13. Set plates aside to solidify 14. Repeat with diluted effluent 15. Repeat with undiluted effluent 16. Once plates solidify, invert and incubate at 37°C 16-24 hours. 17. Count plaques using a light