Mini plasma torch

Shimadzu ICP 9000 (with Mini plasma Torch in axial reading mode) was used to measure the zinc content. Standard solution for AAS (Sigma Aldrich) was used to generate the calibration curve between 10 to 500 µg/L in pure water with 1% of HNO3 (Fluka). Samples were routinely incubated in HNO3 10% ON at RT. Briefly, 500 µl of each cytosolic crude extract was incubated with 90 µl of HNO3 65% ON at RT. The sample was centrifuged at 13,000 rpm for 5 min. Before to be measured pure water was added to the supernatant extemporaneously to obtain a final volume of 7 ml. Ytterbium solution standard for AAS (Sigma Aldrich) was used as an internal standard to prevent calibration drift and fluidic perturbation. The result was express in µg/L [Zn] per mg of protein.

Pigments at just below the Lethal Dose 20 (LD 20) concentration for J774A1 macrophages, i.e. 2.5 µg/ml and 500 µg/ml, were mineralized in aqua regia (Mixture of nitric acid and hydrochloric acid, optimally in a molar ratio of 1:3). Samples were diluted in 10% nitric acid prior to analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES) on a Shimadzu ICP 9000 with Mini plasma Torch instrument used in axial reading mode. A standard range of cobalt, zinc and aluminum (from 3.9 µg/L to 10 mg/L) was prepared for quantification.

The biovar I strain of F. tularensis subsp. holarctica used in the virulence assay, referred as CHUGA-Ft6, was isolated at Verdun Hospital (France) from a blood sample collected during routine care of a patient with typhoidal tularaemia. Identification at the species and subspecies level was obtained by PCR amplification and sequencing of the intergenic region between the 16SrRNA and 23SrRNA encoding genes^{35 (link)}. Bacterial cultures were performed either on chocolate agar plates supplemented with PolyVitex (CPV, Biomérieux, Lyon, France) or in liquid brain heart infusion medium supplemented with 2% PolyVitex (BHI-2%PV). When necessary, kanamycin (10 µg mL⁻¹) or sucrose (5% (w/v)) was added. Cultures were incubated at 37 °C, in a 5% CO₂-enriched atmosphere. Intracellular iron concentration was measured on stationary phase bacteria grown over 15 h in modified Mueller–Hinton medium into a shaking incubator (200 rpm at 37 °C). Briefly, the cells have been washed several times with phosphate-buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA) 10 mM before hydrolysis with HNO₃ at 65% ON at 95 °C and measurements by ICP-AES (Shimadzu ICP 9000 instrument with Mini plasma Torch in axial reading mode^{20 (link)}).