Improving high-throughput techniques for bacteriophage discovery in multi-well plates

Bacteriophages (also called phages) are viruses of bacteria that have numerous applications in medicine, agriculture, ecology, and molecular biology. With the increasing interest in phages for their many uses, it is now especially important to make phage discovery more efficient and economical. Using the host Mycobacterium smegmatis mc²155, which is a model organism for phage discovery research and is closely related to important pathogens of humans and other animals, we investigated three procedures that are an integral part of phage discovery: enrichment of environmental samples, phage isolation and detection (which can also be used for host range determination), and phage purification. Enrichment in 6-well plates was successful with most environmental samples, and enrichment in 24- and 96-well plates was successful with some environmental samples, demonstrating that larger sample volumes are preferred when possible, but smaller sample volumes may be acceptable if the starting concentration of phages is sufficiently high. Measuring absorbance in multi-well plates was at least as sensitive as the traditional plaque assay for the detection of phages. We also demonstrated a technique for the purification of single phage types from mixed cultures in liquid medium. Multi-well techniques can be used as alternatives or complementary approaches to traditional methods of phage discovery and characterization depending on the needs of the researcher in terms of time, available resources, host species, phage-bacteria matches, and specific goals. In the future, these techniques could be applied to the discovery of phages of aquatic mycobacteria and other hosts for which few phages have currently been isolated.