Protocol for Checkerboard Assay.md

Protocol for Antibiotic Checkerboard Assay

I. Preparation of Antibiotic Serial Dilutions

1. 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.
2. Fill the rest of the wells in the top row with 250µL of LB.
3. Perform a serial dilution across the top row by transferring 250µL sequentially from one well to the next.
4. Discard 250µL from the penultimate well.
5. Repeat steps 1-4 in the bottom-left well using DRUG B instead of DRUG A.

II. Adjustment for Maximum Plate Concentration

6. 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.

III. Distribution of Dilutions

8. 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.
9. 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.
10. Repeat Step 8 for the first column, transferring to corresponding rows.

IV. Final Dilutions and Transfer to Plate Reader Plate

10. Add 200µL of LB to each well of the deep well plate, resulting in a 2x dilution.
11. Transfer 100µL from each well in the deep well plate to a corresponding well in a 96-well plate reader plate.

V. Bacterial Inoculation

12. 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.
13. Measure the overnight culture's optical density (OD) at 600nm using a spectrophotometer.
14. Dilute the culture in fresh LB to an OD₆₀₀ of 0.2, approximating 1x10⁸ CFU/mL.
15. 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.