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 / 2That post-inoculation value is the concentration you report the MIC at.
Standard 96-well plate layout
A typical CLSI broth micro-dilution panel occupies one row of an 8×12 plate:
| Well | Role | Contents |
|---|---|---|
| A1 | Highest concentration | Drug + broth (prepared) |
| A2–A10 | Decreasing concentrations | Two-fold dilution series |
| A11 | Growth control | Broth + inoculum, no drug |
| A12 | Sterility control | Broth only, no inoculum |
The growth control confirms the organism grew in the absence of drug (validates the assay). The sterility control confirms no contamination was introduced during plate preparation.
Inoculum preparation
Getting the inoculum concentration right is as important as the dilution arithmetic. The standard target is 5 × 10⁵ CFU/mL in the final well volume:
- Prepare a suspension at approximately 1 × 10⁶ CFU/mL (typically by adjusting a colony suspension to a 0.5 McFarland standard ≈ 1–2 × 10⁸ CFU/mL, then making a 1:100 or 1:200 dilution in broth).
- Adding an equal volume of this to each well produces the final 5 × 10⁵ CFU/mL at the same time it delivers the two-fold concentration drop.
A too-heavy inoculum (above 5 × 10⁵ CFU/mL) inflates the MIC, a well-documented source of false “resistance” results. A too-light inoculum risks under-detecting the endpoint.
Reading and reporting the MIC
The MIC is the lowest concentration that gives no visible turbidity (growth) after incubation (typically 16–20 hours at 35–37°C for most clinically relevant bacteria). Report the MIC at the final post-inoculation concentration, not the prepared concentration.
Interpretation against “susceptible,” “intermediate,” or “resistant” categories requires the organism- and drug-specific breakpoints from CLSI (M100 tables) or EUCAST (available at eucast.org). These breakpoints are not embedded in this tool — they are species- and drug-pair specific and are updated annually.
Common errors and how to avoid them
- Evaporation during incubation concentrates wells at the plate edges; seal plates with adhesive film or incubate in a humidified chamber.
- Carry-over between wells during pipetting transfers drug into the next well; use fresh tips at each transfer step.
- Reading turbidity in colored media can obscure endpoints; use a plate reader or hold the plate over a mirror for better contrast.
- Not vortexing or sonicating sparingly-soluble drugs leads to inaccurate concentrations in the first wells.