Medium-Induced Turbidity of Zinc Sulfate Heptahydrate (ZnSO₄·7H₂O) in Cation-Adjusted Mueller–Hinton Broth: Implications for Broth Microdilution Susceptibility Testing

Introduction: In antimicrobial susceptibility testing, complete solubility of the tested compound is critical. This study investigated the impact of zinc sulfate heptahydrate (ZnSO₄·7H₂O) on visible turbidity in cation-adjusted Mueller-Hinton broth (CAMHB) during broth microdilution assays, with and without bacteria.
Methods: Solutions of ZnSO₄·7H₂O were prepared in sterile deionized water and mixed with CAMHB at varying concentrations (4 to 2048 µg/mL). Visually detectable turbidity was assessed immediately and after 24 hours at room temperature and 37°C. Broth microdilution was conducted as a proof-of-concept assessment using Staphylococcus aureus ATCC 29213 and five clinical isolates, with agar dilution used for methodological comparison.
Results: ZnSO₄·7H₂O remained completely clear in deionized water at any concentration. In contrast, visible turbidity appeared in CAMHB at concentrations around 1000–1024 µg/mL, interfering with MIC determination. Although agar dilution mitigated turbidity issues, the findings suggest that medium-associated physicochemical changes at high concentrations of ZnSO₄·7H₂O may potentially affect the freely dissolved or biologically available fraction.
Conclusion: In this proof-of-concept study, medium-induced turbidity of ZnSO₄·7H₂O was identified as an important methodological consideration when performing susceptibility testing at high nominal concentrations, as the nominal concentration of ZnSO₄·7H₂O may not accurately represent its biologically available concentration in culture media. Further physicochemical characterization is required before high-concentration MIC data for zinc sulfate are interpreted with confidence. The findings should be interpreted as evidence of a potential methodological limitation in ZnSO₄·7H₂O susceptibility testing that should be considered in zinc susceptibility testing.