4.2. Stock Preparation and Subculture of Bacteria

Four standard bacterial strains (P. aeruginosa ATCC 27853, E. coli ATCC 25922, S. aureus ATCC 29213, and E. faecalis ATCC 29212) of Clinical and Laboratory Standards Institute (CLSI) were purchased from MicroBioLogics, Inc. (Saint Cloud, MN, USA). We conducted all the experiments in accordance with the ethical standards from American Type Culture Collection and performed all the experiments in the biosafety cabinet. Bacterial stock solutions were made by mixing 50 mL of glycerol (Sigma-Aldrich, St. Louis, MO, USA) and 50 mL of deionized water. A total of 400 μL of 50% glycerol-deionized water mixture was mixed with 800 μL of bacterial cation adjusted Mueller–Hinton broth (CAMHB; BD Biosciences, San Jose, CA, USA) mixture. These stock solutions were placed in microcentrifuge tubes (SPL Life Sciences, Seoul, Korea) and cryopreserved at −70 °C in a deep freezer. For the IBAST, subcultures were performed before the experiment. Stock solutions from aliquots were inoculated with one loop on Luria–Bertani agar plates (KisanBio Co., Ltd., Seoul, Korea) and were incubated in a 37 °C incubator overnight. After 20 to 24 h of incubation, several colonies were formed and used to generate target concentration of bacteria.