Labsystems varioscan flash

To compare the bactericidal activities of CSA-13 (0.7 μg/ml) and MNP-CSA-13 (5 μg/ml) and MNPs themselves (4.3 μg/ml) against P. aeruginosa PAO14, killing assay/CFU counting method was performed [58 (link)]. The concentrations of tested agents were calculated to added equal amount of free CSA-13 and CSA-13 molecules attached to the MNP surface. Briefly, P. aeruginosa was grown to mid-log phase at 37°C, re-suspended in PBS, and brought to 10⁸ CFU/ml (with the assumption that an optical density at 600 nm of 0.35 corresponds to 10⁸ CFU/ml). They were then diluted in PBS containing different concentrations of CSA-13, MNPs or MNP-CSA-13. After 1 h of incubation at 37°C the plates were transferred to ice and suspensions were diluted 10- to 1000-fold in PBS. Then, 10 μl aliquots were spotted on cetrimide-containing agar plates for overnight culture at 37°C, then CFUs were determined. In other set of experiment changes of chemiluminescence intensity of P. aeruginosa Xen5 after treatment with COL – colistin, CSA-13, MNP and MNP-CSA-13 measurements were determined as an additional method to assess bacteria viability. Chemiluminescence was evaluated using Labsystems Varioscan Flash (Thermo Scientific).

To assess the biological activities of synthesized compounds, we have chosen to determine their antibacterial activity against Gram-negative, multidrug resistant (MDR) Pseudomonas aeruginosa Xen 5 strain. Briefly, a bacterial culture was grown to mid-log phase at 37 °C, re-suspended in LB, and brought to 10⁸ CFU/mL. Then 100 μL of bacteria suspensions were added to tested agents at a concentration range 1–100 μg/mL. Colistin was use as a control, since it represents “last treatment option” in some infections caused by MDR Pseudomonas aeruginosa. Upon bacteria addition the chemiluminescence intensity were registered using Labsystems Varioscan Flash (Thermo Scientific Waltham, MA, USA) over a period of 1h.