Mtb or Msm Lux was grown to log phase and diluted to an OD600 = 0.006 in each well of non-treated 96-well plates (Genesee Scientific) containing 100 μL of meropenem (Sigma Aldrich) and/or amoxicillin (Sigma Aldrich) diluted in 7H9 + OADC + 5 μg/mL clavulanate (Sigma Aldrich). Msm media contained ADC rather than OADC. Cells were incubated in drug at 37°C shaking for 7 days (Mtb) or 1 day (Msm), 0.002% resazurin (Sigma Aldrich) was added to each well, and the plates were incubated for 24 hr before MICs were determined. Pink wells signify metabolic activity and blue signify no metabolic activity. (Kieser et al., 2015a (link)) Checkerboard MIC plates and fractional inhibitory concentrations were calculated as described in (Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, 2016 ).
Non treated 96 well plates
Manufactured by Genesee Scientific
Non-treated 96-well plates are a standard laboratory equipment used for various biological and chemical assays. These plates have 96 individual wells, each with a flat bottom, allowing for the containment and processing of small volumes of samples or reagents. The plates are made of a durable, non-treated material, making them suitable for a wide range of applications requiring a neutral surface.
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Lab products found in correlation
4 protocols using non treated 96 well plates
1
Checkerboard Assay for Antimicrobial Synergy
2
Sacculi Hydrolysis Assay Protocol
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Sacculi was prepared from stationary-phase cells of S. aureus strain HG003 using a previously published protocol52 (link), with the following modifications. Volumes of solutions were scaled appropriately for the number of cells harvested. After 1 M HCl treatment, the pellets were washed with dH2O, flash-frozen, and lyophilized to yield purified sacculi.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
3
Antibiotic Susceptibility Testing of Mycobacteria
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Mab, Msm, and Mtb were grown to mid-log phase and diluted to an OD600=0.003 in each well of non-treated 96-well plates (Genesee Scientific) containing 100 μl of antibiotic serially diluted in 7H9+OADC + 5 μg/ml clavulanate (Sigma-Aldrich). For MICs on Mab knockdown cells, cultures were induced for knockdown 18 hr prior with 500 ng/μl ATc. Wells with knockdown bacteria also contained 500 ng/μl ATc. Msm media contained ADC rather than OADC. Cells were incubated with drug at 37°C with shaking for 1 day (Mab, Msm) or 7 days (Mtb). Afterward, 0.002% resazurin diluted in ddH2O (Sigma-Aldrich) was added to each well. Plates were then incubated for 24 hr. MICs were determined by the concentration of antibiotic that turned wells blue signifying no metabolic activity (Kieser et al., 2015b (link)).
4
Sacculi Hydrolysis Assay Protocol
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