Cellstar 96 well microtiter plates

Staphylococcus aureus USA 300 JE2, Staphylococcus aureus USA300 LAC, Escherichia coli K12 were provided by Dr. Bayles’ research lab and Pseudomonas aeruginosa PA01 was provided by Dr. Gus Wang’s lab (both at the Departament of Pathologhy and Microbiology of the University of Nebraska Medical Center – UNMC). The MICs of the PAs were studied using the broth microdilution method. Bacterial cultures were made by the direct colony suspension method to 1.5 × 10⁸ colony forming unit CFU/ml and dilute for 2 mL into 40 mL of Muller Hinton Broth (MHB) to a final concentration of ~10⁵ CFU/mL. A stock solution of each PAs was prepared in ultrapure water at 1 mg/ml concentration and pH was adjusted to 7. Then, serial dilutions were made in MHB, in Cellstar 96-well microtiter plates (Greiner, Bio-One). Each well was inoculated with 10 μL of bacterial cultures. The plates were incubated statically for 16–24 h at 37°C. The lowest concentration of PA that prevented bacterial growth was considered the MIC. The O.D value was set at 600 nm and was recorded with an AccuSkan, MultiSkan FC (Thermo Fisher Scientific). Vancomycin and Gentamicin were used as positive controls and media was used as negative control. The assay was performed in triplicate.

MICs for Cit 1.1 and the Cit 1.1 analogues were measured using the following bacterial strains: S. aureus JE2 and E. coli K12. The MICs was determined using the broth microdilution method as previously described [52 ]. A stock solution of each peptide was prepared in milliQ water and then serial 2-fold dilutions were made in Difco™ Muller Hinton Broth (MHB) (BD Diagnostics, Becton Drive, NJ) in Cellstar 96-well microtiter plates (Greiner Bio-One, Kremsmünster, Austria). Bacterial cultures were prepared using the direct colony suspension method to 1.5 × 10⁸ colony forming unit (CFU)/mL (0.5 McFarland) and diluted from 2 mL into 40 mL of MHB. Each well was inoculated with 10 μL of bacterial cultures. Plates were statically incubated at 37 °C for 24 h. MIC values correspond to the lowest AMP concentration that yielded no observable bacterial growth based on analysis with the unaided eye or a microplate reader. The optical density (O.D.) value at 600 nm was recorded using an AccuSkan, MultiSkan FC (Thermo Fisher, Waltham, MA). Vancomycin (Sigma, St. Louis, MO) and gentamicin (Alfa Aesar, Ward Hill, MA) were used as positive controls. Blank media was used as a negative control. All assays were performed in triplicates using three independent measurements. All assays were performed in triplicates using three independent measurements.