A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

One day prior to the MIC assay, prepare cultures of the strains to be tested in LB with and without 2-[2-nitro-4- trifluoromethyl)benzoyl]-1-3 cyclohexanedione or NTBC. This demonstration uses the representative level of 300 micromolars of NTBC. The titration method for determining the appropriate concentration of NTBC is described in the protocol text. Incubate the cultures overnight at 37 degrees Celsius with aeration. On the following day, make LB plus NTBC and LB plus DMSO master solutions for the MIC assay.

To test one antibiotic for one strain, add 600 micromolar of NTBC to 2 milliliters of LB, and mix to make the NTBC master solution. NTBC is added at a concentration of 600 micromolars because it will be diluted two-fold when the inoculum is added, yielding a final concentration of 300 micromolars. Add an equivalent volume of the vehicle DMSO to 2 milliliters of LB and mix to make the no-NTBC master solution. These two master solutions will be used for creating antibiotic stock solutions as well as for setting up the dilution series in 96-well plates.

Next, prepare the antibiotic solutions in the LB plus NTBC or LB plus DMSO master solutions. Prepare the gentamicin plus or minus NTBC stock solution at 64 micrograms per milliliter. Make the kanamycin plus or minus NTBC stock solution at 256 micrograms per milliliter. Prepare the tobramycin plus or minus NTBC stock solution at 8 micrograms per milliliter.

After the 2X antibiotic solutions have been made, add 100 microliters of each antibiotic solution to four wells in a 96-well plate in row A. For example, gentamicin should be placed in A1 through A4. Kanamycin should be placed in A5 through A8, and tobramycin should be placed in A9 through A12.

Add 50 microliters of the LB plus NTBC or LB plus DMSO master solution to rows B through H of the 96-well plate. Ensure that one plate is LB plus NTBC and one plate is LB plus DMSO. Using a micropipette header, perform two-fold serial dilutions of the antibiotics by transferring 50 microliters of the solution from row A to row B. Mix the solution, change the pipette tips, and transfer 50 microliters of the solution from row B to row C. Repeat for the remaining rows.

After diluting row G, remove 50 microliters of the solution from that row and discard. Use row H as a no-antibiotic control for bacterial growth. Retrieve the overnight cultures. After washing all cultures to eliminate biomelanin present in the media, measure the OD₆₀₀ of the cultures. Dilute the overnight cultures in LB.

Using a multichannel micropipette, add 50 microliters of the diluted bacterial culture to the appropriate wells. Add bacteria to three wells for each strain and antibiotic concentration. Add 50 microliters of LB to the fourth well to act as a control for bacterial contamination. Cover the 96-well plates with parafilm and incubate for approximately 24 hours at 37 degrees Celsius.

On the following day, examine the plates visually for bacterial growth in the wells. The MIC is the lowest concentration of antibiotic in which no bacterial growth is seen for all three replicates of each strain.