100
- Plate format 8 × 12 (rows A–H, and columns 1–12). The for-
matting of strains versus antibiotic concentrations can be
planned according to the requirements of the particular proj-
ect. We typically set up the assay so that on one microtiter plate
we test eight strains against 11 concentrations of drug (includ-
ing zero), and 1 medium sterility control. - A stock solution of the antibiotic compound should be made
as recommended by the manufacturer and diluted in MHII
medium to twice the highest required final concentration. In
the example given here, a ciprofloxacin stock solution (1 mg/
mL in 0.1 N HCl) is diluted to 64 μg/mL in MHII medium. - Drug assay concentrations: Drug concentrations are usually
varied in twofold dilution steps. The drug concentration range
should be set to match the properties of the particular experi-
mental expectations for the bacteria-drug combination being
studied. For example, in the fluoroquinolone resistance evolu-
tion experiment a typical range might be as follows, in μg/mL:
32: 16: 8: 4: 2: 1: 0.5: 0.25: 0.12: 0.06: 0.00: and 0.00
(medium only sterility control). - Transfer 5–10 mL sterile MHII medium into a sterile 50 mL
reagent reservoir (Corning Incorporated, cat no. 4870). - Medium inoculation into microtiter plate: Using a multichan-
nel pipette (eight channels) inoculate 50 μL MHII medium
from the reagent reservoir into the wells of columns 2–11. - Inoculate 100 μL MHII medium into the wells of column 12
(this will be the medium sterility control). - Drug inoculation into microtiter plate: Inoculate 100 μL of
the 64 μg/mL ciprofloxacin solution into each of the column
1 wells. - Using a multichannel pipette (eight channels) transfer 50 μL
of the ciprofloxacin solution from column 1 into the column 2
wells. - Repeat step 9, successively transferring 50 μL from column 2
to column 3, and so on until column 10. - Using a multichannel pipette (eight channels) remove 50 μL of
the ciprofloxacin solution from column 10 and discard it as
waste. - At this stage columns 1–10 have 50 μL of MHII with drug,
column 11 has 50 μL MHII without drug, and column 12 has
100 μL MHII without drug. - Bacterial inoculum preparation: Suspend fresh colonies of each
strain of interest, grown on nonselective medium (incubation
18–24 h at 35 °C ± 2 °C) in saline (0.9% NaCl) to 0.5
McFarland (≅1.5 × 108 CFU/mL) (see Note 9).
Douglas L. Huseby and Diarmaid Hughes