Spectronic 200 spectrophotometer

TIGR4 was inoculated 1:1000 (v:v) in 1 L of pre-heated (37°C) TSB-GYP in a GLS80 1 liter bottle (Duran, USA). Temperature was maintained constant at 37°C, the pH was continuously measured with a probe (InPro3030, Mettler Toledo) and kept at 6.9 by peristaltic pump controlled addition of 3M NaOH. Agitation was set at 100 rpm. Growth was monitored by aseptically drawing aliquots and measuring their OD₅₉₀ in a Spectronic 200 spectrophotometer (ThermoFisher). At the peak OD₅₉₀ (about 2.5, corresponding to 10^9 CFU/ml) bacteria were harvested by centrifugation, resuspended in PBS/10% glycerol and frozen at -70°C. TIGR4 bacteria were then thawed and inactivated by treatment with 1.5% formalin for 2 hours on a roller mixer at room temperature, then washed twice and resuspended in water.

A.calcoaceticus ATCC 23055 and A. calcoaceticus Carolina Biological (CB1) strains were purchased for use in this study. Four clinical isolates of A. calcoaceticus (M31602, T82482, X75393, and M53152) were obtained [24 (link)]. For comparison, Acinetobacter baumannii ATCC 19606 and Acinetobacter spp. ATCC 27244 were used. All strains were cultured on Leeds Acinetobacter agar, a selective and differential medium for Acinetobacter spp. [25 (link)] Single colonies were used to inoculate liquid cultures of brain–heart infusion (BHI). All bacteria were grown at 37 °C aerobically in 10 mL conical tubes and shaken overnight at 150 rpm. The bacterial growth of liquid cultures was assessed by optical density (OD_600nm) on a Spectronic 200 spectrophotometer (ThermoFisher, Waltham MA, USA).

Growth of mono‐ and co‐cultures was followed in replicate Balch tubes with 10 ml of culture volume under anaerobic conditions. Culture density was measured by tracking changes in optical density with a Thermo Scientific Spectronic 200 spectrophotometer at 600 nm wavelength. Maximum growth rate was determined as previously described (Turkarslan et al, 2011). Monocultures of Dv wild‐type and regulatory mutants were cultivated in sulfate respiration growth medium until late stationary phase when sulfate was exhausted (lactate fermentation) for SR state transition experiments. This ensured depletion of electron acceptor but not carbon source due to medium formulation with limiting sulfate concentrations. At this stage, 0.5 ml of inoculum was transferred to 10 ml of fresh SR medium and growth was tracked. For state transition experiments with co‐cultures alternating between ST and SR conditions, co‐cultures were initially grown in ST growth medium until mid‐log phase (OD₆₀₀ ~0.15) and 0.5 ml of inoculum was transferred into 10 ml of fresh SR growth medium. Cells were grown to early log density (OD₆₀₀ ~0.2), and 0.5 ml was transferred back into fresh ST growth condition medium. Alternating shifts between ST and SR conditions were continued as long as growth was observed.

MIC values were determined according to the broth microdilution protocol outlined by CLSI guidelines for antimicrobial susceptibility testing (33 ). Pneumococci were grown on BAB plates overnight at 37°C with 5% CO₂. Cultures grown overnight in BHI broth from plate sweeps were incubated for 18 h. Antibiotic susceptibility against a 2-fold serial dilution of each antibiotic, from 1 μg/mL to 0.03125 μg/mL, was tested. Each well was inoculated with 50 μL of an overnight-grown culture adjusted with BHI broth to an OD₆₀₀ of 0.1 using a Spectronic 200 spectrophotometer (Thermo Fisher Scientific). Wells were inoculated within 30 min of standardization. Plates were incubated for 18 h, and the OD₆₀₀ was measured using a SPECTROstar Nano instrument (BMG Labtech). The percentage of growth compared to the no-antibiotic control was calculated.

Total phenolic content was measured with a colorimetric assay using Folin-Ciocalteu’s phenol reagent [22 ]. An aliquot (0.2 ml) of appropriately diluted PEEC or dieckol was mixed with 2.6 ml of deionized water. At 0 min, 0.2 ml of Folin-Ciocalteu’s phenol reagent was added to the mixture. At 6 min, 2 ml of 7% (w/v) Na₂CO₃ solution was added. At 90 min, absorbance was measured at 750 nm using a SPECTRONIC 200 spectrophotometer (Thermo Fisher Scientific Inc., USA). Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g.

Haloarcula hispanica cells in exponential growth phase in pre-culture were inoculated into 60-ml screw-capped tubes containing 20 ml of Medium 307. The cultures were incubated in the dark with shaking at 180 rpm at 25, 30, 35, 40, 45, and 50°C. The optical density at 660 nm (OD₆₆₀) of the culture was monitored using a Spectronic 200 spectrophotometer (Thermo Fisher Scientific) with sterilized medium as the negative control. When the cultures reached the early exponential growth phase (OD₆₆₀ = 0.25–0.50), the cultures were centrifuged at 6230 × g for 3 min. The pelleted cells were mixed with 100 μl RNAlater (Life Technologies) and stored at –85°C until RNA extraction.