Evaluating Double Agar Overlay Assay and Flow Cytometry as Methods for Characterizing Competition between T4 and T7 Bacteriophages in Escherichia coli C600

Bacteriophages are the most abundant viruses found on earth with population densities approximately ten fold of prokaryotes in the same environments. As a result, interspecies competition likely plays a large role in regulating phage populations in the environment. T4 and T7, two of the most well characterized bacteriophages, infect Escherichia coli. Co-infection experiments using E. coli can be used to study the interspecies competition between these phages. Previous work studying competition dynamics through respective phage population fluctuations reported contradictory data in T7 and T4 co-infection experiments. However, variables such as time of incubation of cells with phage and multiplicities of infection varied thereby confounding comparisons. We sought to establish a reliable experimental system to address these discrepancies in order to study phage population dynamics during coinfection of E. coli. Mono-infection and co-infection experiments using T4 and T7 phage were performed. Phage progeny were enumerated by observing phage plaques in double agar overlay assays. T4 and T7 plaque sizes were expected to be different thereby enabling enumeration of each phage. We observed variable plaque morphology within pure phage populations of T7 of mono-infected culture. These data suggest that double agar overlay may not be a reliable method to enumerate T7 in co-infection experiments. We therefore pursued a flow cytometry based enumeration approach as an alternative method to characterize phage competition. Our data suggest that flow cytometry can be used to measure changes in phage populations. Preliminary results of co-infection experiments using flow cytometry suggest that T4 phage outcompete T7 phage during co-infection of E. coli C600 at a multiplicity of infection of 0.25 for each virus.