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 .
