Ultrospec 2100 pro spectrophotometer

Growth was monitored by measuring optical density at a wavelength of 620 nm (OD₆₂₀) with an Ultrospec 2100pro spectrophotometer (Amersham Biosciences, Sweden). Cell dry weight determination was performed as previously described, as was the determination by normal and reverse phase HPLC of (R)-1-phenylethylamine, (S)-1-phenylethylamine, (R)-1-phenylethanol, (S)-1-phenylethanol, acetophenone, glucose, glycerol, acetate, succinate, and ethanol [12 (link)]. Pyruvate was detected by the same HPLC method as previously described for glucose and its metabolites [12 (link)], except that a UV spectrophotometric detector (Shimadzu SPD-6A) at 214 nm was used instead.

For WITS experiments, 300 µl of tissue homogenate was inoculated into LB broth containing streptomycin (200 µg/ml) and kanamycin (40 µg/ml) as a recovery method to enrich for low abundance strains. An UltroSpec 2100pro spectrophotometer (Amersham Biosciences, Piscataway, NJ) was used to obtain optical density readings of the resulting bacterial cultures. Genomic DNA (gDNA) was extracted from 2×10⁹S. Typhimurium from each sample in duplicate using a DNeasy blood and tissue kit (Qiagen, 69506) as per the manufacturer's protocol for Gram-negative bacteria.
All qPCRs were performed on an Applied Biosystems 7300 real-time PCR system. A 25 µl reaction contained 12.5 µl of FastStart SYBR Green Master Mix with Rox (Roche, 04913914001), 8 µl DNase/RNase-free water, 0.75 µl of forward and reverse (10 µM) primers (Table S1), and 3 µl of gDNA (1–10 ng). Standard curves were generated using gDNA from each W1–W8 strain. Reaction conditions were 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; followed by a dissociation stage of 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s.

The effect of the artificial sweeteners on bacterial growth was measured in transparent 96-well flat-bottom microtiter plates (Corning) containing 80 μL of the bacterial culture (OD₆₀₀ ≈ 0.2), 10 μL of 3-oxo-C12-HSL (at a final concentration of 5 × 10^–10 M), and 10 μL of the different concentration of artificial sweeteners using an Ultrospec 2100 pro spectrophotometer (Amersham, Berks, UK). The positive control contained 10 μL 3-oxo-C12-HSL and 10 μL LB medium in addition to the bacterial culture, and the negative control contained 20 μL LB in addition to the bacterial culture. The OD₆₀₀ was measured with continuous shaking of the plates maintained at 26 °C for 16 h.

After arrival in the laboratory, the RNAlater solution on the fixed cells was replaced by PBS (Invitrogen, Darmstadt, Germany). The cells were scraped off using cell scrapers (Sarstedt, Nümbrecht, Germany), transferred to 50 ml tubes, and pelleted by centrifugation (2500 g for 10 min at 4°C). An RNeasy Mini Kit (Qiagen, Hilden, Germany) was used according to the manufacturer’s instructions to isolate total RNA. RNA concentrations and quality were determined spectrophotometrically at 260 nm using an Ultrospec 2100 pro Spectrophotometer (Amersham Biosciences, Freiburg, Germany). The isolated RNA had an A260/280 ratio of >1.7. cDNA designated for the quantitative real-time PCR was then obtained with the First-Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) using 1 μg of total RNA in a 20-μL reverse transcription reaction mixture.

Protein concentrations were measured using Quick Start Bradford dye (Bio-Rad) and an Ultrospec 2100 Pro spectrophotometer (Amersham). Labeling efficiency of TMR-tagged dynein was measured using a Nanodrop ND1000 Spectrophotometer. Protein concentration was measured with A₂₈₀ absorbance, and was used to calculate the dynein molarity using the extinction coefficient of 797340 M^-1 cm^-1, corresponding to a monomer of the full snap-tagged dynein complex. The ratio of TMR dye concentration (calculated directly by the spectrophotometer) to dynein protein concentration showed that the monomeric labeling efficiency was always at least 85%, with no difference observed between wtDyn and mtDyn. This corresponds to a labeling efficiency of at least 97.8% in the dimer.

Optical density was measured at 620 nm using an Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences Corp., USA). Cell dry weight was measured in triplicate by filtering a known volume of the culture through a pre-weighed nitrocellulose filter with 0.45 μm pore size. The filters were washed with three volumes of water, dried in a microwave oven and weighed after equilibrating to room temperature in a desiccator.
Concentrations of glucose, xylose, xylitol, glycerol, succinate, acetate and ethanol were analysed by HPLC (Waters, USA). Aminex HPX-87H ion exchange column (Bio-Rad, USA) was used at 45°C with a mobile phase of 5 mM H₂SO₄ at a flow rate of 0.6 mL/min. All compounds were detected with a RID-10A refractive index detector (Shimadzu, Japan).

The strain was plated on TSA containing different concentrations of nitrate (0, 0.1, 0.5, 1, 10, and 50 mM) to examine nitrate reduction ability. Nitrite concentrations were determined using a colorimetric assay [11 (link)]. The reagents comprised 1 part 0.4% sulfanilic acid plus 30% of acetic acid mixed with H₂O and 1 part 0.6% N,N-dimethyl-1-naphthylamine plus 30% of acetic acid mixed with H₂O. The bacterial colonies grown on the TSA were collected with a cotton swab after 3, 6, and 10 days of incubation and suspended in 1 mL of distilled water. The optical density of these suspensions was measured at 660 nm (OD₆₆₀) using an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Piscataway, NJ, USA) and adjusted to approximately 0.1. A total of 1 mL of cell suspension was mixed with 20 µL of each reagent and incubated for 1 h at room temperature. The absorbance was measured at 540 nm using a spectrophotometer, and distilled water with reagents was used as a blank. Nitrite concentrations were calculated from a calibration curve that was constructed using several known concentrations of the nitrite solution.