Fig. 1.
Broth microdilution method – preparation of a concentration pattern.
A – add Mueller-Hinton broth.
B – prepare a series of microdilutions of the first antibiotic.
C – prepare a series of microdilutions of the second antibiotic.
D – add antibiotics together.
E – re-add of Mueller-Hinton broth.
Fig. 2.
The “time-kill” method – a series of dilutions.
A – prepare six 96-well plates (t0, t1, t2, t4, t6, t24) with 0.9% NaCl
B – add the bacterial suspension to test tubes 1st–4th.
C – transfer the suspension portion to the appropriate plate at predetermined times.
D – obtain a dilution of 1:10.
E – transfer a portion of the suspension to the plates using the “drop plate” method.
Fig. 3.
CombiANT method – the insert and experimental protocol.
A – the insert design: antibiotic reservoirs (a-c), interaction imaging area (d).
B – the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020)
Fig. 4.
Assessment of the synergistic effect of antibiotics by the method of constant coefficients.
Fig. 5.
Assessment of the synergistic effect of antibiotics by cross-method.
Fig. 6.
Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.