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Protocol: Checkerboard Assay
Ryan David Ward edited this page Feb 13, 2024
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1 revision
- Add 200µL of DRUG A (100x) and 300µL of LB into the top-right well of a deep well plate, yielding a 40x concentration in a total volume of 500µL.
- Fill the rest of the wells in the top row with 250µL of LB.
- Perform a serial dilution across the top row by transferring 250µL sequentially from one well to the next.
- Discard 250µL from the penultimate well.
- Repeat steps 1-4 in the bottom-left well using DRUG B instead of DRUG A.
- Add 250µL of LB to each well in the top row and the first column. This sets the maximum concentration of each drug on the plate to 20x.
- Discard 275µL from each well in the top row and 175µL from each well in the first column -- or -- proceed beginning from Step 9 in a new deep well plate.
- Using a multichannel pipette, transfer 25µL from each well in the top row to each well in its respective column of the deep well plate.
- Repeat Step 8 for the first column, transferring to corresponding rows.
- Add 200µL of LB to each well of the deep well plate, resulting in a 2x dilution.
- Transfer 100µL from each well in the deep well plate to a corresponding well in a 96-well plate reader plate.
- Inoculate a single colony of Acinetobacter baumannii from a fresh agar plate into 5-10mL of LB and incubate overnight (~16 hours) at 37°C with shaking at approximately 200 rpm.
- Measure the overnight culture's optical density (OD) at 600nm using a spectrophotometer.
- Dilute the culture in fresh LB to an OD600 of 0.2, approximating 1x108 CFU/mL.
- Add 100µL of the bacterial suspension (OD600 = 0.2) to each well of the 96-well plate reader plate, yielding a final volume of 200µL per well (OD600 = 0.1). The most concentrated wells will now have a 1x concentration of each drug.