Antibiotic Disk Diffusion (Kirby–Bauer): Standard cation-adjusted medium for disk diffusion of nonfastidious bacteria such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, ensuring consistent Ca2+/Mg2+ levels (40 mg/L CaCl2·2H2O, 20 mg/L MgCl2·6H2O).

Broth Microdilution MIC Testing: Prepared at 2X to mix 1:1 with inoculum for final 1X in MIC assays, with defined cations to yield reproducible aminoglycoside and tetracycline results.

Antibiotic Potency and QC Assays: Used with reference strains (e.g., E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853) to verify lot-to-lot antibiotic performance.

Checkerboard Synergy Testing: Employed in combinational MIC/FBIC studies for drug–drug interaction profiling against organisms like Enterococcus faecalis and Klebsiella pneumoniae.

Time–Kill Kinetic Studies: Supports bactericidal time–kill curves under controlled Ca2+/Mg2+ conditions for agents targeting P. aeruginosa and Acinetobacter baumannii.

Inoculum Preparation for Agar-Based AST: Used to grow and standardize bacterial suspensions prior to plating on Mueller Hinton agar or performing gradient diffusion (Etest) with E. coli and S. aureus.

General Cultivation of Nonfastidious Bacteria: Nutrient base (3.5% Casamino Acids, 0.6% Beef Extract, 0.3% Soluble Starch) for routine growth of Enterobacterales and other nonfastidious species.

Supplemented Testing of Fastidious Species: Base broth for AST with added supplements when required (e.g., for Streptococcus pneumoniae or Haemophilus influenzae), maintaining standardized cation concentrations.