2.5.3. Minimum inhibitory concentration
(MIC) determination
The MICs of coffee samples were determined by a broth dilution method in microtiter plates (CLSI, 2006). Twofold serial
dilutions of the products under study were carried out using
Mueller–Hinton broth (200 L/well after addition of the inoculum). The tested concentration ranges (mg lyophilized/mL) for
each coffee sample were: Arabica and Robusta spent coffee
(0.08–160), Arabica coffee brew (0.06–122) and Robusta coffee
brew (0.12–260). Following incubation of microtiter plates at
37 °C ± 1 °C for 24 h (bacterial strains) or 30 °C ± 1 °C for 72 h
(fungal strains), turbidity was measured in a spectrophotometer reader at 595 nm (SPECTRA MR Dynex Technology, Chantilly,
VA, USA). Positive (Mueller–Hinton broth) and negative wells
(Mueller–Hinton+ ampicillin 0.2 g/mL or amphotericin-B 0.2 g/
mL) were used as controls. Each experimental condition
was tested in triplicate. MIC was considered as the lowest
concentration of coffee sample that inhibited the growth of
microorganism.