Infinite 200 spectrophotometer

To assess in vitro growth, overnight V. cholerae cultures were diluted to an OD₆₅₅ of 0.01 and distributed into microtiter wells in triplicate. The OD₆₅₅ was recorded over time using an Infinite 200 spectrophotometer (Tecan). To assess host bacterial burden, replicates of ten flies were washed three times in ethanol followed by three washes in phosphate buffered saline (PBS) and homogenized in 500 μl of LB broth at 48h post-infection. The resulting homogenates were diluted, plated on LB agar, and incubated at 37 °C for enumeration of V. cholerae or plated on de Man, Rogosa, and Sharp (MRS) agar and incubated at 30 °C for enumeration of gut commensals. Colony forming units/fly were assessed after 24 and 48 hours. In graphs of bacterial burden, each point represents one cohort of ten flies. Colonization transfer experiments, including both enumeration and visualization of bacterial burden, were performed as previously described ^{24 (link)}.

For long-term cell viability assays, sample aliquots were taken every 24 h up to a maximum of 5 days and plated on TSB. Following incubation at 37°C, bacterial colonies were counted and colony-forming units determined. To monitor growth, S. aureus cultures were resuspended to an OD₆₀₀ of 0.06 in TSB (35 mM glucose) and dispensed into 96-well microtiter plates. Bacterial cultures were grown for 24 h at 37°C in a Tecan infinite 200 spectrophotometer under maximum aeration. The optical density (OD₆₀₀) was recorded every 30 minutes for the entire period of growth. In these experiments bacteria were either challenged with 30mM pyruvic acid or the pH of the media adjusted to pH 4.5 with concentrated hydrochloric acid followed by supplementation of sodium pyruvate.