101- Transfer 50 μL of each bacteria suspension to 10 mL of MHII
broth to make a set of final bacterial suspensions: ≅ 5 × 105
CFU/mL (acceptable range 3–7 × 105 CFU/mL). - Using a 12 channel multichannel pipette (using only 11 of the
12 channels) transfer 50 μL of a bacterial strain to column A
(wells 1–11). Well 12 is a medium control. The assay volume
in each well in 100 μL. - Repeat step 14 for each of the seven remaining bacterial
strains. If more strains are to be tested, apply the same proce-
dure to additional microtiter plates as appropriate. - Cover the plates with a sterile polyester adhesive film for micro-
plates (VWR, cat. no. 60941-064), and a microwell lid for
96-well plates (VWR, cat. no. 734-2185). - Incubate without shaking for 16–20 h at 35 °C ± 2 °C. Do not
stack the plates more than four high (to maintain the same
incubation temperature for all plates). - Read MIC visually. MIC is defined as complete inhibition of
growth as detected by unaided eye, using medium only as the
control. - Each strain or antibiotic should be assayed in duplicate (inde-
pendent plates) to test the robustness of the data.
4 Notes
- BBL Mueller-Hinton II Agar (BD) plates are prepared accord-
ing to manufacturer instructions. - BBL Mueller-Hinton II Broth (BD) is prepared according to
manufacturer instructions. Once the media has cooled
following autoclaving, antibiotics can be added. Bottles with
media supplemented with antibiotics may be stored at 4 °C
during the course of selection until required. It is helpful to
preprepare many small bottles containing 100–200 mL of
media supplemented with the various concentrations of antibi-
otic you intend to use during the whole course of the serial
passage experiment. - Ten parallel cultures are routinely used as a standard scale of
experiment. Using smaller numbers of replicates increases the
possibility that the full spectrum of mutational paths toward
resistance will not be observed, while increased numbers of
replicates increases the complexity and expense of the experi-
ment, particularly if multiple selection regimes are investigated
simultaneously. - For some antibiotics and concentrations it is possible that 24 h
of growth will be insufficient to achieve full density cultures
Determining Trajectories of Bacterial Resistance Evolution