Mueller hinton 2 agar

P. mesoacidophila ATCC 31433 was purchased from LGC Standards and was grown at 28°C in PS broth (1% [wt/vol] glucose, 0.5% [wt/vol] tryptone, 0.5% [wt/vol] meat extract, 0.5% [wt/vol] NaCl [pH 7.0]) except where indicated. Escherichia coli JM109 was purchased from Promega and was grown at 37°C in LB medium except where indicated. Antibiotics and metal salts were obtained from Sigma-Aldrich (Poole, United Kingdom) or Melford (Ipswich, United Kingdom). All antibiotic susceptibility testing followed the methods described by the BSAC (47 (link)), except that Mueller-Hinton II agar (Sigma-Aldrich) was used for disc diffusion assays and PS broth was used for broth microdilution assays. Breakpoints were taken from BSAC methods for antimicrobial susceptibility testing version 14 (30 ). For disc diffusion assays, antibiotic discs (a maximum of 3 per plate) were prepared using 5-mm filter paper discs impregnated with 5 μl of antibiotic stock solution. Metal susceptibility testing was performed only using broth microdilution assays, with 10, 1, or 0.001 mg ml⁻¹ metal salt as the highest concentration on the plate. Antibiotic and metal susceptibility testing was performed at least in triplicate. Equivalent replicates for P. mesoacidophila and E. coli JM109 were performed on the same day using the same antibiotic stock solution.

All solvents were bought from Carlo Erba Reagents S.A. (Barcelona, Spain). Folin–Ciocalteu reagent, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were acquired from Sigma-Aldrich (Madrid, Spain). Gallic acid (GA), ascorbic acid (AA), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ferric chloride, 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), sodium acetate, dimethyl sulfoxide (DMSO), and lactic acid were from Sigma–Aldrich (Steinheim, Germany). The culture media Mueller–Hinton broth (MHB) and Mueller–Hinton II agar were acquired from Sigma-Aldrich (Madrid, Spain) and Liofilchem (Roseto degli Abruzzi, Italy), respectively.