Gaspaks

To determine the effects of different oxygen conditions on M. ulcerans growth, M. ulcerans initially grown aerobically at 30°C was exposed to either microaerophilic or anaerobic conditions at 30°C for 24 h, by placing those cultures within anaerobic chambers with appropriate GasPaks (BD) and oxygen indicators (Fig. 6A). Optical density was measured initially and after 24 h. M. ulcerans cultures were then placed back under aerobic conditions at 30°C for an additional 24 h (48 h from initial time point). The optical density for each treatment and time point was measured. To study the effects of temperature, M. ulcerans in exponential growth aerobically at 30°C was exposed to 37°C for 24 h and then placed back in the 30°C incubator for an additional 24 h. The optical density was measured for each treatment and time point. At each time point and condition, 1 mL of sample was transferred for serial dilution and growth measurement, and 5.0 mL of sample was transferred for RNA isolation and RT-qPCR to measure mycolactone gene expression. Each experiment was conducted with triplicate replicates and at least three times.

For successful settle-down of probiotics in gastrointestinal tract resistance to bile salt is one of necessary requirements (Klaenhammer and Kullen 1999 (link)). S. boulardii is known to grow at 37 °C and has been used as a probiotic in humans. To compare the bile salt resistance and growth efficiency at 37 °C of SJP-SNU and S. boulardii, the strains were cultured on MacConkey agar plates containing bile salt at 37 °C, with colony formation being observed for 5 days. To assess the anaerobic growth of SJP-SNU, the strain was streaked onto YM agar plates and incubated at 25 or 37 °C in anaerobic jars with GasPaks for 48 h (BD).