Automated and spatially resolved quantification of phage-mediated lysis intensity in spot assays using a...

Macroscale evaluation of bacteriophage host range frequently relies on visual interpretation of spot inoculation assays, in which clearing is recorded using qualitative or ordinal categories including +++, ++, +, and -. These categories are rapid and practical but compress continuous image variation and can introduce observer-dependent disagreement, particularly for weak and intermediate reactions. This study presents an open-source computer-vision platform that applies a defined image-reading procedure to arrayed phage spot assays by converting local clearing into a deterministic numerical measurement. The analytical signal is calculated from the relative grayscale-intensity difference between a predefined central spot region and spatially matched peripheral bacterial-lawn regions, providing local background normalization instead of reliance on absolute image brightness. Spatial positions are defined from plate anchors and proportional grid interpolation, allowing the same analytical logic to be applied to images with different dimensions after appropriate plate-specific calibration. The platform provides a continuous normalized spot-clearing score and an optional ordinal representation for compatibility with conventional reporting. Validation comprised 84 predefined spot reactions independently scored by four observers and four repeated computational analyses of identical image input. Manual scoring showed the greatest dispersion for weak and intermediate clearing, whereas automated re-analysis showed high repeatability. Automated scores also showed strong monotonic concordance with manual consensus. The platform is intended as a quantitative image-analysis layer for completed spot assays. The score does not measure plaque diameter or plaque number and does not independently establish productive phage infection, for which efficiency of plating (EOP) or other biological assays remain necessary.