This assay was realized by the microtiter broth dilution method described by the Clinical and Laboratory Standards Institute [50 ] using P. aeruginosa ATCC 27,853 (Museum of Microbiological Cultures, State Research Institute of Highly Pure Biopreparations, St. Petersburg, Russia).
In a 96-well plate, 125 μL of Mueller-Hinton broth (HiMedia, Mumbai, India) were added. Stock solutions of NPs with antibiotics were prepared by diluting the sample in Mueller-Hinton broth to a maximum 2-fold required CT concentration. Serial dilutions of CT at concentrations from 64 to 0.25 μg/mL were then obtained on the plate. The OD of an overnight P. aeruginosa suspension in Mueller-Hinton broth was measured on a UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 540 nm, and the suspension was serially diluted 1:100 in Mueller-Hinton broth to give approximately 1 × 107 CFU/mL.
The inoculum was prepared by cultivation in a liquid medium (Mueller-Hinton broth) for 18 h by adding 125 μL of inoculum to the wells of the plate. The plate also contained controls: 100% growth (bacteria only), sterility control (Mueller-Hinton broth only), and blank NPs at equivalent polymer concentrations. The plate was incubated for 24 h at 37 °C, and then the OD was measured on an ELx808 ™ Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA) at 630 nm.
In a 96-well plate, 125 μL of Mueller-Hinton broth (HiMedia, Mumbai, India) were added. Stock solutions of NPs with antibiotics were prepared by diluting the sample in Mueller-Hinton broth to a maximum 2-fold required CT concentration. Serial dilutions of CT at concentrations from 64 to 0.25 μg/mL were then obtained on the plate. The OD of an overnight P. aeruginosa suspension in Mueller-Hinton broth was measured on a UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 540 nm, and the suspension was serially diluted 1:100 in Mueller-Hinton broth to give approximately 1 × 107 CFU/mL.
The inoculum was prepared by cultivation in a liquid medium (Mueller-Hinton broth) for 18 h by adding 125 μL of inoculum to the wells of the plate. The plate also contained controls: 100% growth (bacteria only), sterility control (Mueller-Hinton broth only), and blank NPs at equivalent polymer concentrations. The plate was incubated for 24 h at 37 °C, and then the OD was measured on an ELx808 ™ Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA) at 630 nm.