Raman alpha 300r

A Raman alpha 300R (WITec, GmbH, Remscheid, Germany) confocal Raman microscope was used for acquiring spectra of purified protein sample. The equipment consisted of an excitation laser of 532 nm, 50X objective (Zeiss, NA = 0.8), 300 mm spectrograph equipped with 1200 gr/mm grating, and Thermo cooled charge-coupled detector (CCD). Raman-scattered photons were collected through a 50 µm fiber, and the spectra were obtained over the fingerprint region (600–1800 cm⁻¹). The specimen was placed on a quartz window, and a total of 10 spectra were obtained at different locations. Each spectrum was acquired for 5 s and averaged over 10 accumulations. The highest peak normalization was performed using MATLAB (version R2021a). The mean spectrum was computed by averaging the intensities at the Y-axis and keeping the X-axis constant.

As mentioned, colistin-sensitive and resistant E. coli strains have different generation times. To avoid any spectral variation due to differences in the growth stage, both resistant and sensitive cells were collected at the logarithmic phase. This translated in to collection of colistin sensitive cells at 12 h and resistant strains at 12, 18, 24, 36 h depending upon the antibiotic concentration. Cell number was adjusted by saline dilution to match the optical density. The culture medium was centrifuged at 12,000 RPM for 10 min at 4 °C and the supernatant was discarded. The cells were washed three times with saline. The pellet was transferred to the CaF₂ window for acquiring spectra using Raman alpha 300R (WITec, GmbH) microscope, equipped with a laser of 532 nm wavelength, 100× objective (Zeiss, NA = 0.8), 300 mm spectrograph with 600 g/mm grating and charged-coupled detector (CCD). Spectra were acquired with an integration time of 5 s with 10 accumulations. Spectra were collected at different spots from each pellet and repeated thrice to ensure reproducibility.