Antimicrobial susceptibility testing (AST) was performed at the National Reference Laboratory for Antimicrobial Resistance (NRL-AR), Department of General Diagnostics, IZSLT, through minimum inhibitory concentration (MIC) determination by broth microdilution using the EU consensus 96-well microtiter plates (Trek Diagnostic Systems, Westlake, OH, USA). The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST; http://www.eucast.org , accessed on 28 April 2022) epidemiological cut-offs and clinical breakpoints (when available). The following drugs were tested: ampicillin, cefotaxime, ceftazidime, meropenem, azithromycin chloramphenicol, nalidixic acid, ciprofloxacin, colistin, gentamicin, sulfa-methoxazole, tetracycline, tigecycline, and trimethoprim. E. coli ATCC 25922 was used as a quality control strain.
96 well microtiter plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States, Germany, Japan, United Kingdom, Spain, Australia, Poland
96-well microtiter plates are a type of laboratory equipment used for various applications in scientific research and analysis. These plates consist of a rectangular array of small wells, typically 96 in total, arranged in a 12x8 format. The plates are designed to hold small volumes of liquid samples, allowing for multiple experiments or assays to be conducted simultaneously in a compact and organized manner.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (5 mentions)
Infinite m200 pro, by Tecan (4 mentions)
Spectramax plus plate reader, by Molecular Devices (4 mentions)
Microplate reader, by Molecular Devices (3 mentions)
Non fat dry milk, by Merck Group (3 mentions)
Sigmafast o phenylenediamine dihydrochloride, by Merck Group (3 mentions)
Nonfat dry milk, by Americanbio (3 mentions)
Synergy ht, by Agilent Technologies (3 mentions)
Enspire multimode plate reader, by PerkinElmer (3 mentions)
Model 680, by Bio-Rad (3 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (2 mentions)
Xtt labeling mixture, by Roche (2 mentions)
Spectrafluor plus, by Tecan (2 mentions)
3h thymidine, by PerkinElmer (2 mentions)
5 tetramethylbenzidine liquid substrate, by Merck Group (2 mentions)
Mtt solution, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Restriction endonuclease, by New England Biolabs (2 mentions)
Tetramethylbenzidine, by Merck Group (2 mentions)
196 protocols using 96 well microtiter plate
1
Antimicrobial Susceptibility Testing Protocol
2
MIC Determination of Antimicrobial Resistance
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AST was performed as minimum inhibitory concentration (MIC) determination by broth microdilution, using the EU consensus 96-well microtiter plates (Trek Diagnostic Systems, Westlake, OH, United States). The results were interpreted according to epidemiological cutoffs (ECOFFs) included in the Annex A of the EU Decision 2013/652/EU, and for sulfamethoxazole the tentative ECOFF of >256 mg/L according to the EURL-AR protocol for antimicrobial susceptibility testing of Escherichia coli, Salmonella, and Campylobacter2 . E. coli ATCC 25922 was used as quality control strain.
3
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was performed as minimum inhibitory concentration (MIC) determination by broth microdilution, using the EU consensus 96-well microtiter plates (Trek Diagnostic Systems, Westlake, OH, United States). The following antimicrobials were tested, as reported in the EU Decision 2020/1729/EU: amikacin, ampicillin, azithromycin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, sulfamethoxazole, tetracycline, tigecycline, trimethoprim (first panel) and cefepime, cefotaxime, cefotaxime + clavulanic acid, cefoxitin, ceftazidime, ceftazidime + clavulanic acid, ertapenem, imipenem, meropenem, and temocillin (second panel). Dilution ranges and interpretation of MIC values using epidemiological cutoffs (ECOFFs) were performed according to the EU Decision 2020/1729/EU and to the EFSA manual published in 2021 [European Food Safety Authority (EFSA) et al., 2021 ]. For carbapenems and temocillin, results were also interpreted according to EUCAST5 clinical breakpoints (Table 1 ). Escherichia coli ATCC 25922 was used as quality control strain.
The results of AST were further compared with the AMR genotypes to determine if the phenotypic resistance patterns were confirmed by the presence of the corresponding AMR genes.
The results of AST were further compared with the AMR genotypes to determine if the phenotypic resistance patterns were confirmed by the presence of the corresponding AMR genes.
4
Biofilm Formation Screening in Dishwasher Isolates
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The seven selected isolates from each dishwasher (Table 2 ) were screened for biofilm formation as single species and in four-species combinations as described previously (Ren et al., 2015 (link); Røder et al., 2015 (link)) with few modifications. Serial 10-fold dilutions of bacterial cultures were performed from overnight grown cultures (in LB media) where 1 ml of the dilutions were inoculated with 29 ml fresh LB media, incubated overnight at 37°C and shaking at 200 rpm. Cell cultures in exponential phase (OD600 between 0.3 and 0.7) were then selected, centrifuged at 8000 rpm (10 min, 21°C), washed with 1x phosphate buffer saline (PBS) and re-suspended in 10% w/v LB media (reduced). The optical density OD600 of each bacterial culture was then adjusted to 0.15 in the reduced LB media. Biofilm cultivation assay was performed using 96-well microtiter plates (NUNC, Roskilde, Denmark) and peg lids (NUNC-TSP lid system, Roskilde, Denmark) placed on top of the plates, also referred to as the Calgary method (Ceri et al., 1999 (link)). A total of 150 μl as mono-species or four mixed species (37.5 μl of each species) cultures were added to each well. Each plate contained the representative mono-species cultures. 150 μl 10% LB served as blank. Plates were incubated at 25°C for 24 h.
5
Resveratrol and RNE Cytotoxicity Assay
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L929 cell line was seeded into 96-well microtiter plates (Nunc, Roskilde, Denmark) in DMEM (100 μL) supplemented with 10% FBS and 1% penicillin–streptomycin solution. Cells then were incubated at 37 °C in a humidity of 95% and 5% CO2 for 24 h. Resveratrol in concentrations of 100, 200, and 400 µg/mL, RNE in concentrations of 100, 200, and 400 µg/mL, PVC, and PBS as the positive and negative controls, respectively were added to each well in triplicate. After 24 h of incubation time, 10 μL of MTT solution was added to each well. After 3 h of incubation at 37 °C, the supernatant was removed and 100 μL of DMSO was added. Finally, the absorbance at 570 nm was measured by a microtiter plate reader (BioTek ELX800, Winooski, VT, USA) and the viability was calculated by the below formula [21 (link)]:
6
ELISA to Measure Antibody Levels
7
Cytotoxicity Evaluation of ARA Extract
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The cytotoxic effect of ARA extract was evaluated using a MTT-based colorimetric viability assay (Roche Diagnostics GmbH, Mannheim, Germany). C2C12 cells (5 × 104 cells/well) were plated in different concentrations of ARA extract in 96-well microtiter plates (Nunc, Roskilde, Denmark) and then cultured for 24 h at 37°C in a 5% CO2 incubator. At culture termination, MTT (0.5 mg/mL) solution was added to each well and cultured for 4 h at 37°C in a 5% CO2 incubator. Solubilizing solution 100 μL was added to each well, and the plate was allowed to stand overnight in the incubator. The optical densities (OD) of solubilized formazan crystals were measured at 570 nm using a microplate reader (UVM340, Asys Hitech GmbH, Austria).
8
Nanobody Modulation of Factor XI
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HK (0.5 μg/ml) was immobilized overnight at 4°C in coating buffer (50 mM NaHCO3, pH 9.6) in Nunc Maxisorp 96‐well microtiter plates. Wells were blocked with block buffer (1.5% BSA in PBS) for 1 h at RT. FXI (4 nM) was pre‐incubated with the nanobodies for 1 h at RT. Plates were washed with washing buffer (PBS, 0.1% Tween20) and the pre‐incubated samples were incubated 2 h at RT. The plates were washed with washing buffer and incubated 2 h at RT with polyclonal anti‐FXI antibody (2 μg/ml). After washing the plates with washing buffer, these were incubated with a secondary swine‐anti‐rabbit‐HRP antibody (0.5 μg/ml) and incubated 1 h at RT. After rinsing with wash buffer, wells were colored with 100 μl TMB coloring solution, which was stopped with 50 μl H2SO4 (1 M). Absorbance was measured at 450 nm on a SpectraMax (Molecular Devices).
9
Indirect ELISA for Parasite Antibody Titers
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To determine parasite-specific antibody titers in sera an indirect ELISA assay was performed. Briefly, 96-well microtiter plates (Nunc, Denmark) were coated with FhTE (2 μg/well) in 50 mM carbonate buffer (pH 9.6). After blocking with 1% gelatin in PBS, three washes with PBS containing 0.1% Tween-20 were performed. Serially diluted sera in PBS containing 0.1% Tween-20 and 0.5% gelatin were added to the wells for 1 h at 37°C. After three washes, wells were treated 1 h at 37°C using goat anti-mouse IgM, IgA, IgG IgG1, IgG2a, IgG2b, or IgG3 peroxidase-conjugates (Sigma-Aldrich, United States) followed by o-phenylenediamine (OPD) and H2O2 as substrates. Plates were read photometrically at 492 nm in an ELISA auto-reader (Thermo Fisher Scientific). Antibody titers were calculated to be the log10 highest dilution, which gave twice the absorbance of control (mock) mouse sera with the minor dilution. Titers are shown as the arithmetic mean ± SEM.
10
Biofilm Formation Assay Protocol
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Biofilm formation was performed in LB medium (Biocorp), Christensen broth (Cullen et al. 2015 (link)) and in Artificial Urine (Stankowska et al. 2012 (link)) at 37 °C in 96-well microtiter plates (Nunclon). Overnight culture was diluted (1:100) and transferred into wells in volume 200 μl and incubated overnight at 37 °C with a cover. The broth was removed, biofilm was triple-washed with 0.9 % NaCl solution (200 μl). The 200 μl of 0.01 % (w/v) crystal violet solution was added, incubated at room temperature (RT) for 15 min. and washed five times with 0.9 % NaCl solution. The washed wells were filled with 200 μl of 95 % ethanol at RT for 15 min. Crystal violet absorbance was measured at the λ = 595 nm with an Infinite M200PRO microplate reader (Tecan). Assay was performed in at least 3 independent repeats. An average value of the absorbance was presented on the graph with standard deviation (SD) as error bars.
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