Nutrient broth media

Antibacterial activity was tested against Gram-negative bacteria Escherichia coli (E.coli) (ATCC 25922). For these tests, glass slides were coated as mentioned in Section 2.4, after which the samples were prepared according to EUCAST protocols [60 (link)]. Briefly, the covered glass slides with the applied treatments were left under UV light for 10 min for decontamination and then placed in 12-well plates in Nutrient broth media (VWR Chemicals, VWR International GmbH, Wien, Austria). At a confluence of 0.5 McFarland turbidity, the bacterial strain was added to the wells (10 µL at 1 mL media) and left for 24 h at 35 °C to develop. After this interval, the liquid was placed in a 96-well plate, and the optical density was read at 600 nm using an EPOC BioTek spectrophotometer (BioTek Instruments, Winooski, VT, USA). The results were compared to the untreated control.

Ticks were washed with 70% ethanol and rinsed 3 times with phosphate-buffered saline (PBS) (PanReac, AppliChem). Tick's exoskeletons were removed using sterile forceps and blades, and the internal organs were cut into small pieces and then transferred into tubes. Each tick was homogenized individually with PBS using an electric homogenizer. Each homogenate was inoculated into 3 ml of nutrient broth media (VWR Chemicals, USA) in a 15 ml tube and incubated for 24 h at 37°C with shaking (250 rpm) (10 (link), 22 (link)). The growing cultures were plated into different media, including blood agar base (VWR Chemicals, USA) and MacConkey agar (OXOID, UK) to allow the growth of a large spectrum of bacteria (23 ). After 24 h of incubation at 37°C, different colonies (1–2 colonies from each plate) were selected based on morphology (color, structure, shape, size). The bacterial isolates were stored with glycerol at −80°C for further analysis.