Enumeration of bacteriophages by plaque assay requires the mixing of host-specific bacteria with a lytic bacteriophage of interest in a soft agar overlay (top agar) to prevent the spread of viral infection in the medium; the mixture is then spread on a solid bottom agar. An infection of a single lytic phage particle with a bacterium results in the lysis of the host bacterium and the release of new phage progeny. The new phage progeny released from each bacterium will infect/lyse neighboring bacteria to form a \"plaque,\" which is a clear visible area (that can be counted) with the naked eye. If phages are not present in the mixture, the host bacterium will form a \"lawn\" in the soft agar overlay and grow to a stationary phase.

A universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification. Here, simple optimizations that increase efficiency and throughput in agar overlay based isolation and cultivation of virus-host systems are presented. The agar overlay is streamlined to minimize steps and materials. Serial purification of viruses from viral colonies (plaques) is optimized to eliminate steps by combining purification by serial re-streaking with the optimized agar overlay approach. Finally, recommendations are made for efficient archival and storage of virus plaques. In sum, this work presents: •Tube-free Agar Overlays: rapid plaque assays with fewer steps and materials•Molten Streaking for Singles: rapid tube-free serial purification of viruses•Archiving Plaques: saving virus purification for later.