Antimicrobial susceptibility (MIC)
The reference method: a two-fold dilution series of the drug in cation-adjusted Mueller–Hinton broth in a 96-well plate, inoculated to about 5 × 10⁵ CFU/mL and read after 16–20 h as the first well without visible growth.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Broth microdilution
The reference method: a two-fold dilution series of the drug in cation-adjusted Mueller–Hinton broth in a 96-well plate, inoculated to about 5 × 10⁵ CFU/mL and read after 16–20 h as the first well without visible growth.
How it differs from the standard method
- Adjust the suspension to 0.5 McFarland from several fresh colonies and dilute it so the wells hold about 5 × 10⁵ CFU/mL; use it within 15 min.
- Include a growth well, a sterility well and a QC strain row on every plate.
- Choose a range that covers the breakpoints and the QC strain's range.
- Read buttons or turbidity against a dark background; skipped wells or trailing growth mean the test should be repeated or read by the method's specific rules.
1The lowest concentration that stops growth
The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that prevents visible growth of a standard inoculum of an organism under standard conditions. It is a number with a method attached: the medium, the number of cells, the temperature and the reading time are all part of its definition, and an MIC measured any other way cannot be compared with the breakpoints that turn it into a clinical category.
Broth microdilution
Ten or so two-fold dilutions of the drug in broth in a 96-well plate, each inoculated with about 5 × 10⁵ CFU/mL. The MIC is the first clear well.
Disk diffusion
A paper disk of drug on a lawn of bacteria. The drug diffuses outwards; the diameter of the zone without growth is read against zone breakpoints.
Gradient strip
A strip carrying a continuous concentration gradient. The elliptical zone meets the strip's scale at the MIC, read on agar without a dilution series.
2Two-fold steps, one dilution of noise
Dilution MICs are read on a two-fold scale (…0.125, 0.25, 0.5, 1, 2…), so a result already carries a factor of two of resolution, and repeat tests of one strain commonly differ by one dilution. A reading only means something when the controls do: a growth well without drug must grow, a sterility well must stay clear, and a quality-control strain of known MIC, tested the same day, must fall inside its published range.
3The inoculum effect
For many drug–organism pairs the MIC barely depends on the number of cells. For some it does strongly: β-lactam antibiotics against bacteria that make a β-lactamase are the classic case, because more cells make more enzyme and destroy more drug.
4Why disk zones move
From a disk, drug diffuses into the agar and its concentration falls with distance, while the lawn grows. Growth is stopped only where the drug exceeds the MIC before the lawn has become too dense to inhibit, at a critical time a few hours after incubation starts.
- Breakpoint
- The MIC or zone diameter that separates categories; set by EUCAST or CLSI from pharmacology, clinical outcome and the distribution of wild-type MICs, and revised over time.
- S, I, R
- Susceptible; susceptible, increased exposure (EUCAST's I, susceptible at higher or more frequent dosing); resistant.
- ECOFF
- Epidemiological cut-off: the upper end of the wild-type MIC distribution, separating organisms with and without acquired resistance mechanisms.
Key terms
Practise these as flashcards →Minimum inhibitory concentration: the lowest drug concentration that prevents visible growth of a standard inoculum under standard conditions.
A turbidity reference for bacterial suspensions; 0.5 McFarland is about 1–2 × 10⁸ CFU/mL for E. coli.
The standard medium for susceptibility testing of non-fastidious bacteria, with defined calcium and magnesium, which affect aminoglycoside and tetracycline activity among others.
A rise in MIC with the number of cells tested, strong for some β-lactams against β-lactamase producers.
A reference strain (such as E. coli ATCC 25922) tested in every run; its MIC or zone must fall in a published range for the run to be valid.
A clear well between wells that grew at lower and higher concentrations; a sign of a pipetting error or contamination.
Two MIC methods agree essentially when their results are within one two-fold dilution of each other.
When to use it
- Guiding treatment of a clinically significant isolate, where the result is categorised with current breakpoints from EUCAST or CLSI.
- Measuring the potency of a new compound or the effect of a resistance gene in the laboratory, where an exact MIC matters more than a category: broth microdilution.
- Routine testing of many drugs against one isolate cheaply: disk diffusion; an MIC for one or two drugs on agar without setting up dilutions: gradient strips.
Compared with alternatives
Antibiotic selection plates use one fixed dose to keep only resistant cells; an MIC measures how much drug a particular strain tolerates.
Comes first: breakpoints and expected resistances depend on the species, so the isolate has to be identified before its MIC can be interpreted.
The same dose–response idea for mammalian cells: a fitted IC50 from a continuous readout instead of a visible growth cut-off on a two-fold scale.
Finds resistance genes and mutations directly; useful for surveillance and outbreaks, but a gene is not always expressed and an MIC is still the functional test.
Follows growth over time rather than at one reading, which shows slowed growth and regrowth at sub-inhibitory concentrations that an end-point MIC hides.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The reference method: a two-fold dilution series of the drug in cation-adjusted Mueller–Hinton broth in a 96-well plate, inoculated to about 5 × 10⁵ CFU/mL and read after 16–20 h as the first well without visible growth.
- Adjust the suspension to 0.5 McFarland from several fresh colonies and dilute it so the wells hold about 5 × 10⁵ CFU/mL; use it within 15 min.
- Include a growth well, a sterility well and a QC strain row on every plate.
- Choose a range that covers the breakpoints and the QC strain's range.
- Read buttons or turbidity against a dark background; skipped wells or trailing growth mean the test should be repeated or read by the method's specific rules.