Obtaining pure phage stock
Transfer phages from the clearing zone on the plate to 1 mL phage buffer or sterile sea water in a sterile tube by scraping off the surface layer of the soft agar containing the phages using a sterile loop.
Allow the phages to diffuse into the medium overnight at 4°C.
Vortex the tube and centrifuge the sample (10,000 x g, 10 min) to remove bacteria and agar.
Transfer the supernatant to a new tube.
This sample will typically contain 106–108 phages mL–1.
To isolate single phages, dilutions of this concentrate should be done, followed by plaque assay, and subsequent isolation of phages from single plaques.
Usually, this procedure is repeated 3 times to dilute out any contaminant phage associated with the phage of interest and increase the probability that only one specific phage is present in the final phage stock.
In the end, the phage concentrate is 0.2 µm filtered and kept in the fridge.
