Microtiter Plate Liquid-Culture Host Range Assay for Phages

A subset of four bacterial strains (S. Anatum strain FC1033C3, S. Newport strain USDA2, S.Typhimurium strain USDA1, and S. Enteritidis strain SGSC 2475) and four phages (Sasha, Season12, Munch, and Sw2) were selected to develop the parameters for the microtiter plate liquid-culture host range assay. Different initial bacterial inoculum levels were tested in combination with phages at starting concentrations of 10⁶ to 10⁸ PFU/mL. The low inoculum condition (~10⁵ CFU/mL) was achieved by adjusting fresh overnight cultures OD_550nm ~0.5 and diluting 1000-fold in TSB. For the high inoculum condition, fresh overnight cultures were adjusted with TSB to OD_550nm ~0.1 to achieve a concentration of ~10⁸ CFU/mL. Phage lysates were titered and adjusted to concentrations of 10⁷, 10⁸, and 10⁹ PFU/mL with phage buffer. For each assay, 180 µL of adjusted bacterial inocula in TSB were mixed with 20 µL of phage in sterile, untreated Falcon (Corning) 96-well transparent plates to achieve final phage concentrations of 10⁶ PFU/mL, 10⁷ PFU/mL, and 10⁸ PFU/mL. The plates were incubated at 37 °C with double orbital shaking in a Tecan Spark 10 M plate reader (Tecan Group Ltd., Männedorf, Switzerland) and growth was monitored by measuring OD_550nm at 30-min intervals for 12 h, which results in 25 total time points including the initial (time 0) measurement. Growth curves were obtained by plotting OD after baseline adjustment against time. All assays were performed with three biological replicates.