Using Microsoft Excel’s formulaic protocol, the TAPC-based doubling time = 1/LINEST(LOG(TAPC1:TAPCn,2),t1:tn) where the values TAPC1 through TAPCn are log-linear with respect to associated growth times t1 to tn; n was typically

6-8 points. All TAPC studies were performed using highly diluted stationary phase cells (initial colony forming unit [CFU] concentration or CI ≥ 103 CFU mL-1) in either LB or MM. Steady State ARS-1620 clinical trial oxygen O2 levels ([O2], units of μM) were measured using a Clark-type oxygen electrode (Model 5300, Yellow Spring Instruments) connected to a Gilson water-jacketed chamber (1.42 mL; circulating water bath attached, 37°C) Selleckchem EX527 containing a magnetic stirring bar. Air-saturated 37°C water was used for calibration. To determine steady-state [O2] in shaking/bubbled cultures, samples were withdrawn

with a syringe from bacterial culture flasks at various time points during mid-to late-log phase growth, and the oxygen consumption (e.g., selleck inhibitor [O2] dropping with time) determined without vortexing. The time lapse between sample withdrawal and the first [O2] data point was recorded and used to back-calculate the [O2] at the time of sampling. These same samples were then vortexed ca. 15 sec and [O2] measured again as a function of time. The rate of O2 consumption was calculated from the slope of cell density-normalized [O2] (TAPC plating was performed simultaneously on LB) as a function of time (apparent Km ~ 15 ± 6 μM) [18]. 96-well Microplate Protocol In order to avoid

water condensation SPTLC1 which might interfere with absorbance readings, the interior surface of microplate covers were rinsed with a solution of 0.05% Triton X-100 in 20% ethanol [12] and dried in a microbiological hood under UV light. About 270 μL of each bacterial cell concentration was pipetted into every well. Each initial concentration (CI) is equal to C0 ΦI where C0 is the cell density from liquid culture (either log or stationary phase). When C0 ≤ 108 CFU mL-1, the cells were sampled from an early-to mid-log phase culture. When C0 ≥ 109 CFU mL-1, the cells were sampled from a stationary phase culture. Typically, each 96-well microplate contained 2 replicates each of the 8 least dilute samples (Φ = 3×10-3 to 5×10-6; 16 wells), 4 replicates of the next 4 highest dilutions (Φ = 2×10-6 to 5×10-7; 16 wells), 8 replicates each of the following 2 dilutions (Φ = 2×10-7 or 1×10-7; 16 wells), and, lastly, 24 replicates of the 2 most dilute samples (Φ = 6×10-8 or 3×10-8; 48 wells). The 96-well plate was then covered with the Triton-treated top, placed in a temperature-equilibrated Perkin-Elmer HTS 7000+ 96-well microplate reader, and monitored for optical density (OD) under the following conditions: λ = 590 nm; the time between points (Δt) = 10-25 min; total points = 50-110; temperature = 37°C; 5 sec of moderate shaking before each reading (see Results section).