The time-kill kinetics were determined for the peptides at concentrations of 8 and 16 µM for Esc(1-21) and at concentrations of 32 and 64 µM for Esc(1-18) (2 × and 4 × MIC, respectively). From a logarithmic growth phase, bacteria were added to modM9 at the final concentration of 1 × 106 CFU/mL in a total volume of 200 μL in the presence or absence of peptides. Samples were inoculated in triplicate in a 96-well polystyrene plate (Becton Dickinson) and incubated at 28 °C for 24 h. A 100 µL aliquot was removed from each well, serially diluted, and plated on LB plates at 0, 15, 30, 45, 60, 75, 90, 120, 150, and 180 min. All plates were incubated at 37 °C for 24 h before the enumeration of the colonies. An antimicrobial compound is considered bactericidal if it kills ≥99.9% (3-log reduction) of the initial inoculum, as described by the CLSI [52 ]. The time-kill curve for the reference control kanamycin was determined at 32 and 64 µM for 2 × and 4 × MIC, respectively.
96 well polystyrene plate
Manufactured by BD
Sourced in United States
The 96-well polystyrene plate is a laboratory equipment item used for various applications. It is a flat, rectangular plate made of polystyrene material, with 96 individual wells arranged in an 8x12 grid format. The plate provides a standardized platform for conducting experiments, assays, or sample analysis requiring multiple samples or replicates.
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4 protocols using 96 well polystyrene plate
1
Time-Kill Kinetics of Antimicrobial Peptides
2
Antimicrobial Peptide Minimum Inhibitory Concentrations
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The minimum inhibitory concentrations (MICs) were determined with the broth micro-dilution method [48 ]. The strains were grown in the modM9 with increasing concentrations of peptides. An overnight inoculum was added to the modM9 at the final concentration of 1 × 106 CFU/mL in a total volume of 200 μL and in the absence or presence of peptides. Samples were inoculated in triplicate in a 96-well polystyrene plate (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 28 °C with constant agitation for 24 h.
The MIC was determined as the lowest concentration of each peptide at which no visible growth was observed. Minimum bactericidal concentrations (MBCs) were obtained by streaking 100 µL from the clear wells of the microplate onto agar LB plates, which were incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration of peptides at which no live bacteria were detected. To determine both the MICs and MBCs, we used peptide concentrations ranging from 1 to 16 µM for Esc (1-21) and from 8 to 128 µM for Esc(1-18). Kanamycin was used as a reference control [23 (link)].
The MIC was determined as the lowest concentration of each peptide at which no visible growth was observed. Minimum bactericidal concentrations (MBCs) were obtained by streaking 100 µL from the clear wells of the microplate onto agar LB plates, which were incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration of peptides at which no live bacteria were detected. To determine both the MICs and MBCs, we used peptide concentrations ranging from 1 to 16 µM for Esc (1-21) and from 8 to 128 µM for Esc(1-18). Kanamycin was used as a reference control [23 (link)].
3
Quantifying Biofilm Density in E. faecalis
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Biofilm density was measured as previously described (34 (link)), with some modifications. In brief, E. faecalis strains from overnight cultures in TSBG broth were diluted in the same medium to an OD600 of 0.1 and grown statically for 3 h or 24 h at 37°C in 96-well polystyrene plates (BD, Franklin Lakes, NJ). The plates were gently washed with PBS, and then the cells were fixed with Bouin’s solution (Sigma-Aldrich Co., St. Louis, MO) for 30 min. After two washes with PBS, bacterial cells were stained with a 1% crystal violet solution (Sigma-Aldrich Co., St. Louis, MO) for 30 min. Excess crystal violet was removed by rinsing thoroughly with distilled water followed by the addition of ethanol-acetone (80:20) to solubilize the dye and dissolve the biofilms. The absorbance at 570 nm was measured with a microplate reader (Thermo Scientific, Waltham, MA). Two independent experiments were performed in duplicate (8 wells per strain each in duplicate).
4
Static Biofilm Formation Assay
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A static biofilm formation assay was performed in 96-well polystyrene plates (BD) as previously reported [17] (link) with some modifications. Briefly, cells were cultured overnight in TSB. They were centrifuged at 4,500 g and re-suspended in 10% TSB. The OD600 value was adjusted to 0.5 and then diluted 100 fold in 10% TSB. Aliquots of 200 µL of this inoculated culture, containing either a single strain or a mixture of R. insidiosa and L. monocytogenes, were transferred to individual wells for biofilm formation. The plates were incubated at 30 °C for 24 h without shaking. After removal of the liquid culture, attached biofilms were stained with crystal violet. Excessive dye was removed and rinsed three times with PBS. The retained dye was dissolved in 33% acetic acid, and absorbance was measured at 570 nm to quantify total biomass.
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