2.8. Radial Diffusion Assay

A radial diffusion assay (RDA) was employed to assess the antimicrobial activity of the peptides using a previously described method [21]. Briefly, a 100 μL aliquot of an overnight culture of P. aeruginosa 27853 was diluted in 25 mL of Mueller Hinton Broth and incubated aerobically at 37 °C in a ZHWY-11C shaking incubator for three hours to obtain a mid-logarithmic culture. The culture was centrifuged at 2594× g for 10 min, washed twice with 5 mL of 10 mM sodium phosphate buffer and the optical density at a wavelength of 600 nm (OD600 nm) adjusted to 0.4–0.5 in sterile 10 mM sodium phosphate buffer. An aliquot of 100 μL of this bacterial suspension (equating to approximately 5 × 10⁶ bacterial cells) was added to 10 mL of molten base agarose (21 mg MHB powder (Biokar Diagnostics, Pantin, France), 1 g agarose (Merck, Gillingham, UK), 20 μL Tween 20 (Merck, Gillingham, UK), 100 mL sodium phosphate buffer (10 mM), inverted, poured onto a square Petri dish (Sarstedt, Nümbrecht, Germany) and allowed to solidify. Wells of diameter 2.3 mm were punched into the agarose using a suction pump. A two-fold serial dilution (concentrations indicated in figures) of peptide was added to the plate, with 3 μL of each test concentration added per well. The insect AMP, cecropin A (100 μg/mL) (Merck, Gillingham, UK), and vehicle controls, were included as positive and negative controls, respectively. The Petri dish was incubated upright at 37 °C for three hours to allow diffusion of test compounds into the agarose. Then, 10 mL of molten high nutrient overlay agarose (4.2 g MHB powder, 1 g agarose, 100 mL distilled water) was poured over the base agarose, allowed to solidify and incubated aerobically at 37 °C overnight. The following day, 5 mL of conditioning medium (10 mL acetic acid (Merck, Gillingham, UK), 2 mL DMSO, (Merck, Gillingham, UK), 88 mL distilled water) was added to the Petri dish for 10 min with gentle rotation to prevent any further bacterial growth. The conditioning medium was replaced with Coomassie brilliant blue stain (2 mg Coomassie Brilliant Blue R250 (Merck, Gillingham, UK), 27 mL methanol, (Merck, Gillingham, UK), 15 mL 37% formaldehyde (Merck, Gillingham, UK), 63 mL distilled water) and incubated overnight at room temperature with gentle rotation. Inhibition of bacterial growth was indicated by circular areas devoid of colour. Measurement of the diameter of these areas was performed using a x8 measuring eyepiece (Flubacher, Wingate, UK). The Petri dish was imaged using a Syngene G:box and GeneSnap software. Minimal inhibitory concentration (MIC) values were determined by linear regression of the size of inhibition zones versus the log concentration of the peptides.