JNAR Protocol

Minimum Inhibitory Concentration (MIC) by Broth Microdilution

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 27, 2026· 1 contributions

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JNAR-ID: JNAR-75274822

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PublishedLast updated Jul 27, 2026

Objective

To determine the minimum inhibitory concentration (MIC) of an antibiotic against bacterial strains using a broth microdilution method in a 96-well plate format, allowing for serial two-fold dilutions and multiple bacterial inoculum concentrations to assess dose-response and growth inhibition.

Classification

Experimental contextIn vitro

Biological modelBacteria

OtherAntibiotics

Reproducibility status

Reproducible

No reproducibility blockers or risks reported. The protocol runs end-to-end.

11 healthy

⏱ Total —5 sections11 steps

Prepare the following materials: Sterile 96-well microplate, Single-channel pipettes, Multichannel pipette, Sterile pipette tips, Sterile Milli-Q water, Sterile reagent reservoir, Tube rack, Plate sealing film, Biological waste container.

Preparation of Antibiotic Working Solution

Calculate the volume of antibiotic stock solution required to prepare 1 mL of working solution at a final concentration of the desired starting concentration

Plate Preparation

Fill a sterile reagent reservoir with sterile Milli-Q water (or the appropriate diluent).

Add 50 µL of sterile diluent to every well in columns 2–12.

Leave column 1 empty, as it will receive the starting antibiotic solution.

Preparation of Two-Fold Serial Antibiotic Dilutions

For each row (A–H): Add 100 µL of the antibiotic working solution to well 1.

Critical stepAccurate starting volume is critical for correct dilution series.

6.1

Transfer 50 µL from well 1 to well 2 and mix thoroughly.

6.2

Transfer 50 µL from well 2 to well 3 and mix thoroughly.

6.3

Continue the serial two-fold dilution across the row until well 11.

6.4

After mixing well 11, discard 50 µL from this well to ensure equal final volumes across the plate.

6.5

Do not add antibiotic to column 12, which serves as the antibiotic-free growth control.

Repeat this procedure independently for each row (A–H).

Critical stepEach row must be independent to avoid cross-contamination and ensure valid comparisons.

Addition of the Bacterial Inoculum

Prepare a fresh bacterial inocula before beginning the assay.

Critical stepInoculum concentration directly affects MIC determination and must be accurate.

For this protocol, each row receives a different bacterial dilution.

9.1

Add 50 µL of the corresponding bacterial inoculum to every well of each row.

Bacterial Inoculum per Row

Row	Inoculum (CFU/mL)
A	10^10
B	10^9
C	10^8
D	10^7
E	10^6
F	10^5
G	10^4
H	10^3

9.2

Mix gently without creating bubbles.

Plate Sealing

Seal the microplate carefully using sterile adhesive sealing film.

Critical stepPoor sealing can cause evaporation or oxygen variation, affecting bacterial growth.

The plate is now ready for incubation under the appropriate conditions for the bacterial species being tested