I am doing my master's degree on food microbiology. I am trying to isolate Listeria bacteriophages from cattle feces and waste water. Even though I am successful at isolation step, i can not move forward. I am applying double plaque assay. I see clear zones on the agar. I pick a plaque and suspend it in 100 µL PBS (phosphate buffered saline) in order to make dilutions. I prepare dilutions up to 10-6 again by using PBS. However, i don't get any results. No clear zones appear after I apply double plaque assay with the dilutions. In short, I lose my bacteriophages. How can I overcome this problem ? Could you please help me ?
Bacteriophages will be there in waste sewage waters. First you have to subsequently spot test on host. If they are lytic phages then go for calculating titer value. Increase titer value by picking clear plaques on Soft agar (Double agar overlay method)
Kindly check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434219/
