The wild-type P. aeruginosa strain used in this study was PAO1, with the ΔznuA deletion mutant made using PAO1 according to Choi and Schweizer53 (link) using primers listed in Supplementary Table S2 online. P. aeruginosa was grown in a semi-synthetic cation-defined media (CDM) containing 8.45 mM Na2HPO4, 4.41 mM KH2PO4, 1.71 mM NaCl and 3.74 mM NH4Cl, supplemented with 0.5% yeast extract (Difco) and vitamins (0.2 μM biotin, 0.4 μM nicotinic acid, 0.24 μM pyridoxine-HCl, 0.15 μM thiamine-HCl, 66.4 μM riboflavin-HCl, and 0.63 μM calcium pathothenate) and Chelex-100 (Sigma-Aldrich) treated. CaCl2 and MgSO4 were subsequently added to 0.1 mM and 2 mM, respectively. Metal concentrations of the CDM were ascertained by inductively coupled plasma-mass spectroscopy (ICP-MS) with Zn2+ present at 800 nM. For routine bacterial growth, media was inoculated to OD600 of 0.05 using overnight culture. Cells were grown to an OD600 of 0.6 on an Innova 40R shaking incubator (Eppendorf) at 240 rpm, 37 °C. Whole cell metal accumulation was performed as previously described5 (link) and analysed by ICP-MS on an Agilent 7500cx ICP-MS (Adelaide Microscopy, University of Adelaide).
Innova 40r shaking incubator
Manufactured by Eppendorf
Sourced in Germany
The Innova 40R is a shaking incubator designed for laboratory use. It provides temperature-controlled incubation with orbital shaking capabilities. The unit features a standard 18 x 18-inch platform and an operating temperature range of 5°C above ambient to 80°C.
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3 protocols using innova 40r shaking incubator
1
Characterization of Pseudomonas aeruginosa Zinc Uptake
2
Acinetobacter baumannii Strain Cultivation Protocol
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The strains used in this study were purchased from the Manoil Lab (Seattle, WA, USA) Acinetobacter baumannii AB5075_UW mutant collection (Table S2 ). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. A. baumannii strains were routinely grown in Luria Bertani broth (LB), containing 1% tryptone (BD Bacto), 0.5% yeast extract (BD Bacto), and 1% sodium chloride. For routine overnight culturing of A. baumannii strains, a single colony from LB agar was used to inoculate 4 mL of LB medium. Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.01 in either 200 μL for growth assays or 20 mL for all other analyses. For growth assays, cultures in LB media were incubated at 37 °C with shaking in a FLUOStar Omega Spectrophotometer (BMG Labtech, Ortenberg, Germany), with the OD600 values presented. The 20 mL cultures used for all other analyses were incubated at 37 °C in an Innova 40R shaking incubator (Eppendorf, Hamburg, Germany) at 230 rpm until they reached mid log-phase (OD600 = 0.7). Results are the mean (± SEM) of at least three independent experiments.
3
Growth Assay of Acinetobacter baumannii
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Acinetobacter baumannii AB5075_UW mutant derivatives were purchased from the Manoil Laboratory (30) and T26 transposon insertions confirmed by PCR. The A. baumannii ATCC 17978 czcA and czcE mutants were generated in our laboratory for a previous study (22) (See Tables S1 andS2 for list of strains and oligonucleotides used in the study, respectively). All chemicals were purchased from Sigma Aldrich (Australia) unless otherwise indicated.
A. baumannii strains were routinely grown in Luria Bertani broth (LB), containing 1% tryptone (BD Bacto), 0.5% yeast extract (BD Bacto) and 0.5% sodium chloride. For overnight culturing, a single colony from LB agar (1.5%) was used to inoculate 4 mL of LB medium. Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.01 in 200 μL LB for growth assays or 20 mL LB for all other analyses. For growth assays, cultures in LB media were incubated at 37°C with shaking in a FLUOstar Omega Spectrophotometer (BMG Labtech), with the OD600 values presented. The 20 mL cultures used for all other analyses were incubated at 37°C in an Innova 40R shaking incubator (Eppendorf) at 230 rpm until they reached an OD600 of 0.7.
A. baumannii strains were routinely grown in Luria Bertani broth (LB), containing 1% tryptone (BD Bacto), 0.5% yeast extract (BD Bacto) and 0.5% sodium chloride. For overnight culturing, a single colony from LB agar (1.5%) was used to inoculate 4 mL of LB medium. Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.01 in 200 μL LB for growth assays or 20 mL LB for all other analyses. For growth assays, cultures in LB media were incubated at 37°C with shaking in a FLUOstar Omega Spectrophotometer (BMG Labtech), with the OD600 values presented. The 20 mL cultures used for all other analyses were incubated at 37°C in an Innova 40R shaking incubator (Eppendorf) at 230 rpm until they reached an OD600 of 0.7.
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