The minimum concentration capable of inhibiting visible bacterial growth was determined for each antimicrobial agent. A specific weigh for each antimicrobial agent was dissolved in PBS (pH =7.3) to yield stock concentrations. The stock concentrations corresponded to the upper limits of MIC values as indicated by CLSI antimicrobial susceptibility testing guidelines (2016). Bacterial density was fixed to approximately 106 CFU/mL. Next, 50 μL of the bacteria were added to wells having 50 μL of each antimicrobial agent concentration (twofold serial dilution) in 96-well plates, and the plates were incubated aerobically overnight at 37°C. The MIC values were determined by measuring OD600 using an Epoch ELISA plate reader (BioTek).
Epoch elisa plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Epoch ELISA plate reader is a compact and versatile instrument designed for high-performance absorbance measurements. It features a wide wavelength range and precise optical system to support a variety of ELISA-based assays. The Epoch provides accurate and reproducible data to support research and diagnostic applications.
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Lab products found in correlation
4 protocols using epoch elisa plate reader
1
Antimicrobial Susceptibility Testing
2
Antimicrobial Activity Assay of WLBU2
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Briefly, 25 μL of the WLBU2 at its sub-MIC level for each respective isolate (ie, concentration=25% of MIC) was added to a 50 μL inoculum of 105 CFU/mL planktonic bacteria, in respective wells of a 96 well microtiter plates, in triplicates. Next, serial dilutions of the antimicrobial agents at 25 μL volumes were added to the wells, and the plates were incubated at 37°C for 24 hrs. Absorbance was measured as an indicator of growth inhibition (OD600) using an Epoch ELISA plate reader (BioTek).20 (link) The CLSI, 2016 concentration values for resistance, intermediate susceptibility, and susceptibility, respectively, in μg/mL were 128, 64, and 32 for amoxicillin-clavulanate, 16, 8, and 4 for ciprofloxacin, 64, 32, and 16 for tobramycin, and 32, 16, and 8 for imipenem.
3
Cell Cytotoxicity Evaluation Assays
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To access cytotoxicity of extracts two mechanism-independent assays were used. For this purpose, L929 cell lines were cultured for 24 h in 96-well plates. Then 20 µL of PBS-diluted extracts (2–100%) were added to the cells and incubated for 24 and 48 h. All absorbance measurements were made on a BioTek Epoch ELISA plate reader (BioTek Instruments, Winooski, VT, USA). The percentage of intact cells was calculated in relation to untreated cells.
4
MTT Assay for Evaluating Cell Viability
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The MTT assay was used to assess potential reduction in cell viability after treatment of eukaryotic cells with WLBU2 alone and in combination with antimicrobial agents. Briefly, Vero cells and human skin fibroblasts were seeded in a flat-bottomed 96-well plate at 5000 cells (in 100 μL) per well, and incubated at 37ºC for 24 hrs under 5% CO2. Next, medium was removed, and 100 μL of fresh media containing different concentrations of the WLBU2 alone or in combination with the antimicrobial agents at the indicated CLSI resistance cutoff values were adder per respective wells. The plates were incubated at 37ºC for 24 hrs, and medium was replaced with fresh medium containing 30 μL (2.5 mg/mL) MTT solution (Sigma-Aldrich, St. Louis, MO, USA). The plate was incubated for 4–6 hrs at 37°C, under 5% CO2. After medium/MTT was removed, 100 μL DMSO were added to dissolve the formazan crystals. Cell survival rates were calculated by measuring absorbance at 540 nm using an Epoch ELISA plate reader (BioTek). Medium without treatment was used for the positive control wells. All tests were run in triplicates.
The true relative value of viable cells was calculated using the equation: (Sample A570-Background A650)/(Control A570-Background A650)×100.
The true relative value of viable cells was calculated using the equation: (Sample A570-Background A650)/(Control A570-Background A650)×100.
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