Isolation of temperate phages by plaque agar overlay
Soft agar overlay tubes are melted in boiling water and placed in the 47°C water bath.
One tube of soft agar is removed from the water bath.
1.0 mL host culture and either 1.0 or 0.1 mL viral concentrate is added.
The contents of the tube is mixed well by rolling back and forth between two hands, and the tube contents are immediately emptied onto an agar plate.
The top agar is gently spread over the agar surface by sliding the plate on the bench surface using a circular motion.
The top agar is allowed to harden by not disturbing the plates for 30 min.
The plates are incubated (top agar side down) overnight to 48 h.
Temperate phage plaques will appear as turbid or cloudy plaques, whereas purely lytic phage will appear as sharply defined, clear plaques.
