Cells were then incubated in KRH ± 20 μM MnTMPyP (a cell-permeative superoxide dismutase mimetic [21]) for 30 min at 37 °C using a shaking plate incubator (Labnet International, Oakham, UK) set at 100 rpm.
The cells were washed twice with glucose-free KRH and incubated in this medium ± 20 μM MnTMPyP for 30 min at 37 °C using a shaking plate incubator (Labnet International) set at 100 rpm.
Time-lapse microscopy was carried out on the same microscope using a heated incubator, heated stage plate and CO2 controller (Pecon) to maintain the cells.
Enterococci plates were incubated at 41 ± 2°C using an egg incubator and generator where electricity was not available, and Escherichia coli plates were incubated at ambient temperatures (30 ± 2°C).
The plate was then placed in a 37°C/5% CO2 incubator for 2 h, after which the fluorescence intensity was read at 532-nm excitation and 595-nm emission wavelengths using a Tecan GENios plate reader.
As a viability control, the model was replicated in static conditions using a conventional 12 well plate, and placed in the same incubator at the same time.
Small cultures were performed in 24 to 6 well multiwell plates using a linear shaker (Incutec K15-500) placed in a CO2 incubator with humidified atmosphere at 150 rpm.
After an initial reading of OD600 using a Synergy HT spectrophotometer, plates were covered with a lid and placed in an incubator at 30 °C for 18 h.
