Assessing the Effects of Host Density on Bacteriophage Evolution and Virus Yield

Begin with bacterial cultures at a range of host densities.

Inoculate the cultures with the same number of bacteriophages and incubate.

The bacteriophage attaches to the bacteria and delivers its RNA genome.

The viral RNA directs genome replication and viral protein synthesis. New virions assemble, and the host cell lyses, releasing progeny.

Add chloroform to lyse remaining cells, releasing intracellular virions.

Centrifuge and collect the viral supernatant. Transfer an aliquot to fresh bacterial cultures to continue viral evolution over multiple transfers.

Incubate fresh bacterial cultures with the evolving and the original viral population, which serves as a reference, to produce progeny. Centrifuge and collect the extracellular virus. Perform plaque assays using bacteria to compare extracellular virus yields between the original and evolving viral populations.

At optimal host density, frequent virus–host interactions sustain virus yields, while very low density limits these interactions, leading to virus extinction.

At suboptimal densities, limited host availability selects for adaptive variants with enhanced entry, increasing extracellular viral yield.