Minimum inhibitory concentration assay by broth microdilution

The minimum inhibitory concentrations (MIC) of eyesalve, single ingredients, dropout batches and allicin were determined as described by Wiegand, Hilpert and Hancock (2008) in MHB and in SWF. Treatments, or water as a negative control, were serially diluted with media in Corning Costar TC-Treated 96-Well Plates, following the scheme in Table ​Table5.5. Bacterial isolates were streaked onto LB agar to obtain single colonies. After 18–24 h incubation at 37 °C, three to five morphologically similar colonies were transferred to phosphate-buffered saline (PBS) and diluted to 0.5 McFarland standard (OD₆₀₀ 0.08–0.1). This suspension was diluted 1 in 100 in media, resulting in 5 × 10⁵ CFU/ml. The bacterial suspension was then added to the treatment dilutions, resulting in another twofold dilution. The highest final concentration of eyesalve, single ingredient or dropout batch tested was 50%. Plates were incubated for 18 h at 37 °C. Results were visually checked for turbidity, and MIC values are the lowest concentration where growth was not visible.

Final volumes and calculated percentages of Bald’s eyesalve in wells of 96-well plates used for MIC.