Visualization and quantification of phage-induced bacterial lysis by phosphotungstic acid-based SEM imaging

Abstract Phage biocontrol, which employs bacteriophages for medical and industrial applications, has attracted increasing attention as an alternative to conventional antimicrobial strategies. However, rapid screening and quantitative evaluation of lytic phages remain major challenges for practical implementation. We propose a method based on phosphotungstic acid (PTA)-derived image contrast in scanning electron microscopy (SEM). This approach enables direct visualization of phage-induced bacterial lysis at the single-cell level and quantitative evaluation of cell damage associated with lysis using tabletop SEM. When Escherichia coli was mixed with T4 phage at a phage-to-bacterium ratio of 10:1 (MOI = 10), high-brightness cells indicative of cell damage became detectable within 30 min. Because PTA-stained cells exhibited increased brightness in images, the image-based damaged cell index was quantified as the proportion of high-brightness bacterial area within the total bacterial area, and used as an indicator of cell damage. Across MOIs ranging from 0.01 to 10, the index increased with increasing MOI, and the resulting growth inhibition was consistent with turbidity-based growth curves. Transmission electron microscopy confirmed intracellular PTA redistribution during lysis progression, along with progeny phage particles in pre-lysis cells. These results demonstrate the utility of this method for rapid detection of phage-induced lysis and evaluation of lytic activity.