This work was performed with the help of Dr. Evyenia Shaili (Department of Chemistry, University of Warwick) with guidance from Mr. Daniel McFeely (School of Life Science, University of Warwick).
2.2.15.1 Cell Maintenance
The bacterial cell lines used in this work, Staphylococcus aureus (strain R34) and
Sciences, University of Warwick. Bacterial stocks were made by plating cells on Lysogeny Broth (LB) agar plates and allowing growth at 310 K overnight. Colonies were then collected and dissolved in glycerol (which had been previously autoclaved) to an optical density at 600 nm (OD600) of 0.01, then placed in Eppendorf tubes and stored at 193 K until required.
2.2.15.2
In VitroGrowth Inhibition Assays
Bacteria stocks for growth inhibition assays were made by dissolving concentrated stocks of OD600 = 0.01 by dilution with LB to OD600 = 0.002. Stock solutions of Ir complexes were prepared by dissolving compounds in dmso (5 mM) and then diluting to desired concentrations by addition of LB medium. The bacterial solutions
were then plated onto 96-well plates (100 μL), and then solutions of Ir complexes were added (100 μL) to give a final OD600 = 0.001. The 96-well plate was covered with a clear gas permeable seal and placed in an iEMs plate reader with the following settings; incubation temperature = 310 K, shaking for 10 s every 5 min at a speed of 360 RPM; measurement under stepping mode every 30 min; filter = 600 nm; time = 22 h. Data were processed using Ascent software version 2.6. The experiments were performed as duplicates of duplicates and were undertaken on separate days.
The minimal inhibitory concentration (MIC) is defined as the minimum concentration of compound that inhibits visible growth of a microorganism over a given incubation period (22 h). This is the value that is reported as a measure of a compound’s antibacterial activity.
The minimum bactericidal concentration (MBC) is defined as the minimum concentration of compound that will kill a particular bacterium. This was evaluated
by plating an LB agar plate with bacterial solutions (10 μL) which showed no growth
over the initial 22 h incubation period. The agar plate was then incubated at 310 K overnight. The MBC was determined as the concentration of iridium complex that prevented colony growth overnight.
2.2.15.3 Cell Membrane Viability Assay
The membrane integrity of S. aureus after exposure to complexes was performed using the BacLight™DEAD/ALIVE bacterial viability kit (Invitrogen) according to the supplier’s instructions.16The kit works on the premise that the dye SYTO 9 can penetrate through both intact and compromised cell membranes and fluoresce green when bound to nuclear DNA. The dye propidium iodide (PI) only enters membrane compromised cells and fluoresces red when bound to nuclear DNA.17 The ratio of green/red fluorescence indicates the degree of membrane-damage when compared to prepared standards.
Briefly, 96 well plates were plated with cells (90 μL) at OD600 = 0.310 dissolved
with 0.85% w/v NaCl solution, followed by addition of iridium complexes (10 μL)
to give final a concentration of compound at 4 × MIC value. The well plate was
incubated at 310 K for 25 min. SYTO 9 and PI (1:1 mix) were added (100 μL) and
incubated at ambient temperature in the dark for 15 min before taking fluorescence readings (530 and 630 nm) on a Variascan Flash Multimode microplate reader by excitation at 470 nm at 298 K. Confocal microscopy images were also taken using an IX71 Olympus microscope with a 100× objective by Mr Ian Hands-Portman (School of Life Sciences, University of Warwick).
2.3
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