Sinorhizhobium meliloti wild type strain RM1021 and irr deletion mutant were routinely grown in LB medium supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LBMC) in the presence of 200 μg/ml Streptomycin at 30°C for 48 hours. When mentioned, S. meliloti were grown in minimal media (MM) with composition as described previously81 (link). For making iron-free MM, FeCl3 was omitted from the MM. Metal free water (VWR ARISTAR® ULTRA) was used to make MM. All flasks and tubes were washed with 6 M HCl and then with metal free water before autoclaving. Escherichia coli strains were routinely grown in LB medium at 37°C. When required 100 μg/ml of neomycin, 50 μg/ml of kanamycin, and 25 μg/ml of chloramphenicol were used.
Aristar ultra
Manufactured by Avantor
Sourced in United States
ARISTAR®ULTRA is a high-purity, low-metal analytical reagent designed for use in sensitive analytical techniques. It is produced under stringent quality control measures to ensure consistency and reliability in laboratory applications.
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5 protocols using aristar ultra
1
Sinorhizobium meliloti Growth Conditions
2
Trace Metal Analysis in Infant Blood
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Samples were shipped by a courier company that ensured the appropriate temperature (at least −20°C) of blood samples at all shipping process stages. All samples were delivered to the University of Kentucky and were consigned and stored in the Clinical Center for Translational Science (CCTS) Lab and kept at −80°C. Frozen samples of the infants' blood were transferred to the Environmental Chemistry and Toxicology Lab, University of Kentucky, to analyze Pb and As concentrations.
The samples were digested in ultrapure nitric acid (Aristar Ultra; VWR) and analyzed to determine Pb and As concentrations using inductively coupled plasma mass spectrometry (Agilent 7500cx; Santa Clarita, CA, USA) based on previously validated methods [23 (link)]. Quality control procedures included analysis of procedural blanks, initial calibration verification, and analysis of standard reference materials (NIST SRM 955c, Toxic Metals in Caprine Blood; National Institute of Standards and Technology, Gaithersburg, MD, USA).
The samples were digested in ultrapure nitric acid (Aristar Ultra; VWR) and analyzed to determine Pb and As concentrations using inductively coupled plasma mass spectrometry (Agilent 7500cx; Santa Clarita, CA, USA) based on previously validated methods [23 (link)]. Quality control procedures included analysis of procedural blanks, initial calibration verification, and analysis of standard reference materials (NIST SRM 955c, Toxic Metals in Caprine Blood; National Institute of Standards and Technology, Gaithersburg, MD, USA).
3
Serum Metal Quantification by ICP-MS
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Whole blood from indicated mice were obtained via submandibular vein puncture or from the orbital sinus. Whole blood was collected in untreated sterile 1.5 mL Eppendorf tubes and allowed to coagulate for 1–2 hrs at RT. Coagulated samples were spun at 13,000×g for 15 min and serum was collected. Collected serum was further clarified with a second spin at 13,000×g for 15min. 10 μL of clarified serum was treated with 2 mL/g total wet weight nitric acid (20 μL) (Trace metal grade; Fisher) for 24 hr, and then digested with 1 mL/g total wet weight hydrogen peroxide (10 μL) (Trace metal grade; Fisher) for 24 h at room temperature. The samples were preserved at 4 °C until quantification of metals. Ultrapure water (VWR Chemicals ARISTAR®ULTRA) was used for final sample dilution to 3 mL. Samples were then analyzed using inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer) utilizing Bismuth as an internal standard.
4
Quantification of Bacterial Iron Content
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A single colony of bacteria was inoculated overnight (16 hours) in 50 ml of LB medium. Equal OD of cells from OP50 and OP50PQE was centrifuged at 4400 rpm for 15 min. The harvested pellets were the washed once with will Milli-Q water and then three times with 1 mM EDTA, followed by one wash with Milli-Q water (62 (link)). Total cellular iron content was then measured as described previously (63 (link)). Briefly, bacterial pellets were treated with total wet weight nitric acid (2 ml/g; trace metal grade, Thermo Fisher Scientific) for 24 hours and then digested with total wet weight hydrogen peroxide (1 ml/g; trace metal grade, Thermo Fisher Scientific) for 24 hours at room temperature. The samples were preserved at 4°C until quantification of metals. Ultrapure water (VWR Chemicals, ARISTAR ULTRA) was used for final sample dilution. Samples were then analyzed using ICP-MS (PerkinElmer, Nexion 2000) using 50–part per billion Bismuth as internal standard.
5
Inductively Coupled Plasma Mass Spectrometry for Metal Analysis
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Tissue samples were analyzed for metals by inductively coupled plasma mass spectrometry (Perkin Elmer Nexion 2000). The internal standard was 50 ppb Bismuth. Briefly, the tissues were digested with 2 mL/g total wet weight nitric acid (Trace metal grade; Fisher) for 24 h, and then digested with 1 mL/g total wet weight hydrogen peroxide (Trace metal grade; Fisher) for 24 h at room temperature. The samples were preserved at 4 °C until quantification of metals. Ultrapure water (VWR Chemicals ARISTAR®ULTRA) was used for final sample dilution.
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