Optimized protocol for live imaging of Bacillus subtilis phage infection at a single-cell resolution

Time-lapse microscopy of growing bacteria allows quantitative observation of cellular features and dynamic processes. Here, we present a protocol for live imaging of Bacillus subtilis phage infection at single-cell resolution. We describe steps for preparing agarose-based solid media for microscopy, staining phage particles with a DNA-binding dye, and making agarose pads for single-cell growth analysis. This protocol is optimized for studying growth and infection of the oxygen-dependent bacterium Bacillus subtilis using the lytic phage Nf.