Moxifloxacin HCl (potency: 90.9%) was obtained from Bayer HealthCare (Leverkusen, Germany); gentamicin sulfate (potency: 60.7%) and cation-adjusted Muller Hinton broth (CA-MHB), from Sigma-Aldrich (St. Louis, MO, United States); human serum, from Biowest SAS (Nuaillé, France); cell culture media, from Gibco/Life Technologies Corporation (Paisley, United Kingdom); Mueller Hinton broth (MHB) and tryptic soy agar (TSA), from VWR (Radnor, PA, United States); and primers, from Eurogentec (Liège, Belgium).
Mueller hinton broth (mhb)
Manufactured by Avantor
Sourced in Portugal
Mueller-Hinton broth is a nutrient-rich medium used for the cultivation and susceptibility testing of microorganisms. It provides the necessary growth factors and nutrients to support the growth of a wide range of bacteria and fungi.
Automatically generated - may contain errors
Lab products found in correlation
Peptone water, by Merck Group (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hyclone water, by GE Healthcare (2 mentions)
Gentamicin sulfate, by Merck Group (1 mentions)
Moxifloxacin hcl, by Bayer (1 mentions)
Tryptic soy agar, by Avantor (1 mentions)
Human serum, by Biowest (1 mentions)
Dimethylsulfoxid, by Merck Group (1 mentions)
Potato dextrose agar (pda), by Avantor (1 mentions)
Sevenmulti, by Mettler Toledo (1 mentions)
U 2001 spectrophotometer, by Hitachi (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Curcumin, by Merck Group (1 mentions)
Agar, by Avantor (1 mentions)
N hexane c6h14, by Merck Group (1 mentions)
Isopropanol c3h8o, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Reference antibacterial and antifungal compounds, by Merck Group (1 mentions)
Rpmi 1640, by Avantor (1 mentions)
10 protocols using mueller hinton broth (mhb)
1
Antibacterial Susceptibility Assay
2
Synthetic Wound Fluid Preparation
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Synthetic wound fluid (SWF) comprised equal volumes of foetal bovine serum (Gibco) and peptone water (Sigma-Aldrich), (23) . Mueller-Hinton Broth (MHB) was obtained from VWR.
HyClone water (GE Healthcare) was used throughout.
HyClone water (GE Healthcare) was used throughout.
3
Antimicrobial Peptide Potency Assessment
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The standard broth microdilution assay was utilized to assess the MIC of designed peptides according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [18 (link)]. Briefly, bacteria were grown in unbuffered Mueller–Hinton Broth (MHB, VWR International, Leuven, Belgium) as well as buffered (containing 50 mM NaHCO3) medium to the mid-logarithmic phase and were quantified by OD600. The bacterial suspension was diluted in the same medium to reach a final density of 1 × 106 colony-forming unit per milliliter (CFU/mL). A volume of 50 µL of the bacterial suspension was added to 96-well plates containing 50 µL of MHB in the absence (viability control) or the presence of the peptides at different concentrations (0.625–80 µM). MIC was defined as the lowest concentration of each peptide resulting in the complete inhibition of visible growth after 24 h of incubation at 37 °C. MIC values were derived from 3 sets of independent experiments with different bacterial cultures.
4
Antibiotic Susceptibility Testing Protocol
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Antibiotic stock solutions of 15 compounds, belonging to 9 classes acting on 4 key biosynthetic pathways (Table 1 ), were prepared at a concentration of 4096 µg/mL, adjusted for potency, and kept at −20 °C or 4 °C, per recommendation. For in vitro susceptibility testing, the CLSI guidelines were followed [56 ] as well as the EUCAST documentation [57 ]. In detail, 100 µL of antibiotic solution was serially diluted in flatbottom 96-well plates, to which 100 µL of fresh cation-adjusted Mueller-Hinton broth (MHB) (VWR, Portugal) was added, along with 5 µL of cell suspension to obtain a concentration of 5 × 105 Colony Forming Units per mL (CFU/mL). The bacteria were incubated at 37 °C for 24 h, after which growth inhibition was observed. MICs were determined as the lowest concentration at which no bacterial growth was observed for three independent cultures, and the inoculum size was confirmed by plating on cation-adjusted Mueller-Hinton Agar (MHA) and determining the CFU/mL.
5
Synthetic Wound Fluid for Research
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Synthetic wound fluid (SWF) comprised equal volumes of foetal bovine serum (Gibco) and peptone water (Sigma-Aldrich)24 (link). Mueller–Hinton Broth (MHB) was obtained from VWR. HyClone water (GE Healthcare) was used throughout.
6
Antimicrobial Potential of Natural Compounds
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Curcumin, dimethylsulfoxid (DMSO), lysogeny broth (LB) agar, phenalenone, and RPMI1640 medium were received from Merck KGaA (Darmstadt, Germany). Potato dextrose agar (PDA) and Mueller–Hinton broth (MHB) were purchased from VWR International (Vienna, Austria). Rose Bengal (RB) was received from TCI Europe (Zwijndrecht, Belgium). Hypericum perforatum extract (ethanol) was prepared from the pharmaceutical drug “Johanniskraut 600 mg forte” (Apomedica, Graz, Austria) by dissolving the filling of the film-coated tablet in ethanol after mechanical removal of the lactose. The 96-well plates (flat bottom) were bought from SARSTEDT (Nümbrecht, Germany).
The U-2001 spectrophotometer for adjusting the McFarland standard was from Hitachi (Chiyoda, Japan). For measurement of the 96-well plates, a Tecan Sunrise Remote Plate Reader (Tecan, Männedorf, Switzerland) was used. The adjustment of pH values was carried out with the pH-meter Mettler Toledo SevenMulti (Mettler-Toledo GmbH, Vienna, Austria).
The U-2001 spectrophotometer for adjusting the McFarland standard was from Hitachi (Chiyoda, Japan). For measurement of the 96-well plates, a Tecan Sunrise Remote Plate Reader (Tecan, Männedorf, Switzerland) was used. The adjustment of pH values was carried out with the pH-meter Mettler Toledo SevenMulti (Mettler-Toledo GmbH, Vienna, Austria).
7
Silica Xerogel Synthesis from Volcanic Tuff
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Volcanic tuff (SiO2 71.5%, Al2O3 14.0%, Na2O 4.8%, K2O 4.2%, Fe2O3 2.8%, CaO 1.3%, TiO2 0.5%, MgO 0.5%, P2O5 0.3% and MnO 0.1%) was obtained from Kompass (Kayseri, Turkey). For silica xerogel synthesis, sodium hydroxide (NaOH) and hydrochloric acid (HCl) were supplied from Sigma-Aldrich (St. Louis, MO, USA). Isopropanol (C3H8O) and n-hexane (C6H14) were purchased from Merck (Darmstadt, Germany).
For antibacterial activity experiments, linezolid (C16H20FN3O4, MedChemExpress) was used as antibacterial agent and ε-poly-l -lysine ((C6H12N2O)n) was supplied from Chengdu Jinkai Biology Engineering Co. Ltd. (Chengdu, China). Mueller-Hinton broth and agar were obtained from VWR International (Radnor, PA, USA). Phosphate buffer saline from GenoChem World (Valencia, Spain) with pH 7.4 was used. All chemicals were used without further purification.
For antibacterial activity experiments, linezolid (C16H20FN3O4, MedChemExpress) was used as antibacterial agent and ε-poly-
8
Antioxidant, Enzyme Inhibition, and Antimicrobial Assays
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The chemical compounds used for the antioxidant methods, the reagents for LOX and AChE inhibition assays and reference antibacterial and antifungal compounds were purchased from Sigma-Aldrich (Madrid, Spain). All compounds were of analytical grade (purity higher than 95%). All culture media were acquired from VWR Chemicals (Barcelona, Spain): Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), Roswell Park Memorial Institute medium (RPMI-1640), Sabouraud dextrose agar (SDA), tryptic soy broth (TSB) and yeast peptone dextrose (YPD). Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ·cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used in this work.
9
Antimicrobial Efficacy of Thymol-Hydrogen Peroxide
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Thymol (CAS: 89-83-8, ≥98.5 %), hydrogen peroxide solution (CAS: 7722-84-1, 30 wt./wt. % in H 2 O containing stabilizer) and methanol (CAS: 67-56-1, >99.9 %, for HPLC) were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS) (10X) was purchased from Fisher Scientific and diluted 10 times before use. Ethanol absolute anhydrous (64-17-5, RSE for electronic use) was purchased from Carlo Erba reagents. Tert-butyl alcohol (tBuOH) (≥99.0 %, CAS number: 75-65-0) was purchased from Fisher Scientific and used without any further purification. Liquid CO 2 (CAS: 124-38-9) was supplied by Air Liquide. Mueller-Hinton broth prepared from powder, Mueller-Hinton agar plates and pharmacopoeia diluent (NaCl peptone broth at pH 7) were purchased from VWR Chemicals. The cell proliferation reagent WST-1 for the spectrophotometric quantification of cell proliferation and viability was supplied by Roche.
A suspension of cellulose nanofibrils (Exilva P) at 2 wt. % solid content with a hemicellulose content of 2.8 %.was provided by Borregaard. The suspension was diluted to a 1% solid content with an Ultra Turrax IKA T25 stirrer at 8 000 rpm during 5 minutes.
The microorganism strains Staphylococcus epidermidis ATCC 14990, Escherichia coli ATCC 25922 and Candida albicans ATCC 14053 were supplied as KWIK-STIK lyophilized strains (Microbiologics).
A suspension of cellulose nanofibrils (Exilva P) at 2 wt. % solid content with a hemicellulose content of 2.8 %.was provided by Borregaard. The suspension was diluted to a 1% solid content with an Ultra Turrax IKA T25 stirrer at 8 000 rpm during 5 minutes.
The microorganism strains Staphylococcus epidermidis ATCC 14990, Escherichia coli ATCC 25922 and Candida albicans ATCC 14053 were supplied as KWIK-STIK lyophilized strains (Microbiologics).
10
Spectroscopic Analysis of Bacterial Growth
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Stock solutions of all compounds (Table S1) were prepared at 6 mM either in water or DMSO (6% v/v). Five independent cultures of E. coli (ATCC 33876) and S. aureus (ATCC 6538 P) were grown on 90 ml of cation-adjusted Mueller-Hinton broth (VWR, Portugal) at 37°C, 250 rpm until early-exponential phase (~3 h). Bacteria were centrifuged at 3000 RCF for 10 min and resuspended in 6 ml of NaCl 0.9% to obtain an ABS 600 of 0.75 or 1.5 for E. coli and S. aureus, respectively. Afterward, cells were dispensed on a 96-well microtiter flat-bottomed polystyrene plate (60 µl/well), previously prepared with 60 µl of 3× concentrated growth media and 60 µl of stock solution. After a 2 h incubation at 37°C, 30 µl were transferred to an infrared transparent ZnSe 96-well plate (Bruker) in triplicate, which corresponded to mechanical replicates. ZnSe plates were dehydrated for 1 h in a vacuum desiccator with abundant silica and inserted in an HTS-XT module coupled to a Vertex-70 spectrometer (Bruker Optics). Spectra were acquired in transmission mode and consisted of 40 coadded scans at a 4 cm -1 resolution. Raw spectra were exported from the OPUS software (Bruker) as data point table files into MA-TLAB (MathWorks) for subsequent analysis.
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