What is a two-fold dilution series?

Each step halves the drug concentration of the one before it. Starting at 64 µg/mL gives 32, 16, 8, 4, 2, 1, 0.5 and so on. This geometric spacing brackets the MIC efficiently across a wide range with few wells.

Why does the final concentration halve again after inoculation?

In broth micro-dilution you add an equal volume of bacterial suspension to each well. Adding the same volume of inoculum dilutes the drug by a further factor of two, so the final tested concentration is half the prepared concentration.

The minimum inhibitory concentration is the lowest drug concentration that visibly prevents bacterial growth after incubation. You read it as the first clear well in the series, reported at the post-inoculation final concentration.

How many dilutions should I run?

Enough to bracket the expected MIC with breakpoints above and below. A typical CLSI panel runs 8 to 11 two-fold steps, for example 0.06 to 64 µg/mL. Include a growth control well with no drug and a sterility well with no inoculum.

Does the planner enforce CLSI breakpoints?

No. It only builds the dilution arithmetic and volumes. Interpreting a result as susceptible, intermediate, or resistant requires the organism- and drug-specific breakpoints from CLSI or EUCAST tables.

MIC Dilution Series Planner

Broth micro-dilution is the reference method for measuring a drug’s minimum inhibitory concentration, and it lives or dies on getting the serial dilution arithmetic right. This planner builds the full two-fold series, the volume to pipette at each step, and the final tested concentration after inoculation.

How it works

A two-fold series halves the concentration at every step:

C(n) = C(start) / 2^n        for n = 0, 1, 2, ...

Between wells you transfer a fixed volume into an equal resident broth volume, which is what produces the two-fold drop. After the series is laid out, an equal volume of bacterial inoculum is added to every test well, so the concentration the organism actually sees is halved one more time:

final = prepared / 2

That post-inoculation value is the concentration you report the MIC at.

Plate layout and tips

A standard panel runs across one row of a 96-well plate: 8 to 11 wells of decreasing drug, plus a growth control (broth and inoculum, no drug) and a sterility control (broth only, no inoculum). Always read the MIC as the first well showing no visible turbidity. Keep the inoculum near 5 × 10⁵ CFU/mL — too heavy an inoculum inflates the MIC. Interpretation against susceptible / intermediate / resistant breakpoints comes from CLSI or EUCAST, not from the arithmetic here.