Tri carb 3110tr

Aliquots of water were fixed for bacterial cell counts, following Porter and Feig (1980 ). Staining was carried out with 4′, 6-diamidino-2-phenylindole (Sigma-Aldrich USA), and slides were counted under an epifluorescence microscope (Olympus U-RFL, Olympus USA) using MetaMorph Microscopy software (Molecular Devices USA). 10 fields of view were counted per slide, with duplicate slides made for each river station.
Bacterial production was measured using ³H-leucine incorporation (Kirchman, 2001 (link)). These measurements were only initiated in January 2011, so no data are available for samples collected during November/December 2010. Water from the upstream- and downstream-most stations (the same stations used to measure polysaccharide hydrolysis) in each river, plus autoclaved control water, was amended with ³H-leucine to a final concentration of 20 nM. Samples were incubated for 1–2 h; following this incubation period, reactions were terminated using 100% trichloroacetic acid (TCA). Samples were then concentrated and washed with 80% ethanol before drying over night. Samples were then amended with scintillation liquid and allowed to sit for a 2-day period before analysis in a scintillation counter (Perkin Elmer TriCarb 3110 TR).

Radioactivity was measured by a liquid scintillation counter (LSC, Tri‐Carb^® 3110TR, PerkinElmer, Inc.). The radioactivity in liquid samples (i.e., cell lysate, tissues and plasma extracts) was counted directly after mixing the samples with 10 ml Emulsifier Scintillator Plus™ (PerkinElmer, Inc.) in low potassium glass vials. Blood, plasma, and tissue samples were solubilized with 3 M potassium hydroxide solution (approximately 1 ml/vial) in a glass vial, then bleached with 30% hydrogen peroxide solution, and finally mixed with 10 ml of Hionic‐Fluor™ (PerkinElmer, Inc.) for LSC analysis.

Aliquot of the twice Cs-eliminated drain water was adjusted to approximately 5 ml of 1 M HNO₃ solution (DW-Tc). 0.5 ml of the TEVA resin (Eichrom Technology, LLC) swollen with ultrapure water was packed into a column and conditioned with 1 M HNO₃. The DW-Tc was loaded into the TEVA resin column. The bottle which had contained DW-Tc sample was rinsed with ultrapure water 3 times and all rinse solutions were loaded into the column. After that, 2.5 ml of ultrapure water was passed through the column 4 times. The extracted Tc was recovered by passing through 1.5 ml of 8 M HNO₃. The recovery solution was appropriately diluted to measure Re with ICP-MS. The remaining recovery solution was also appropriately diluted and mixed with Ultima Gold LLT to measure β-ray of ⁹⁹Tc with liquid scintillation counter (PerkinElmer Tri-Carb 3110 TR).

The bacterial uptake of tobramycin was measured by two independent methods. In the radioactivity assay, a stock solution of 15 μCi ³H-labeled tobramycin/ml was prepared in pure water and stored at –20°C. Referring to the method described in our earlier report for assaying ³H-labeled estrogen uptake in E. coli cells (79 (link)), exponential-phase E. coli cell cultures were concentrated 4-fold by centrifugation and resuspension, thoroughly mixed with ³H-labeled tobramycin at a final concentration of 0.015 μCi per 100 μl, and then subjected to the rapid freezing treatment. After thawing in an ice-water bath, the cells were centrifuged (16,000 rpm, 15 s), washed once with the PBS buffer, resuspended in a lysis buffer (0.2 M NaOH, 1% SDS), and incubated at 90°C for 30 min. The cell lysates were then cooled, centrifuged quickly (800 rpm, 10 s), and mixed with a 5-volume scintillation cocktail before subjected to radioactivity measurement on a PerkinElmer Tri-Carb3110TR liquid scintillation analyzer. The nonradioactivity assay was performed as recently described by us (41 (link)), such that tobramycin as taken up by E. coli cells was extracted by cell lysis and then dropped on LB agar dishes to inhibit bacterial cell growth.

The primer extension ability of TkoPolD was measured by counting incorporated radioactivity into DNA strands using dNTP containing [methyl-³H]-dTTP as substrates, and the activities were compared among the WT, ΔPIP, ΔKR, and ΔPIPΔKR in the presence and absence of PCNA. The reaction was performed in 25 μl containing 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.1% Triton X-100 and 0.1 mg/mL BSA, 10 nM template primer substrate (prepared by annealing M13mp18ssDNA and a deoxyoligonucleotide, M13-63; 5′-dTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCGT-3′), 0.2 mM dNTPs including 0.13 μM [methyl-³H]-dTTP (PerkinElmer, MA), 20 nM PCNA, and 5 nM PolD, at 72 °C for 1, 2, and 4 min. The reaction mixture was pre-incubated for 3 min, and PolD was added to initiate the reaction. After incubation, aliquots (8 μl) were fractionated and spotted onto DE81 filters (GE Healthcare). The filters were washed with 5% Na₂HPO₄ solution thrice and dried. Incorporated radioactivity was measured with a scintillation counter Tri-Carb 3110TR (PerkinElmer).