4.6.3. Determination of the Minimum Inhibitory Concentration (MIC)

A. alternata: The 96-well microplate dilution method reported by Hassan and Cutter [51] was used for (+)-α-pinene, (−)-β-pinene, verbenone, rosmarinic acid and copper sulphate, while for borneol, and the two camphor enantiomers that were not soluble in the liquid growth medium, the protocol described above (Section 4.6.1) was used to determine the MIC. For the microdilution method, the fungus was grown on PDA plates for one week, then conidia were collected by scraping the agar surface and suspended in potato dextrose broth (PDB). Conidia were enumerated by a hemocytometer and concentration was adjusted to 1 × 10⁶ conidia/mL. The antimicrobial compounds were dissolved in methanol to obtain a 10% solution. This solution was serially diluted 1:1 in PDB until the concentration of 0.039% [51]. One hundred µL of each dilution were placed in the microplate wells, then 100 µL of the conidial suspension was added to each well. Each dilution was tested in triplicate. The positive control was represented by 100 µL of the conidial suspension plus 100 µL of PDB, while the negative control consisted of 200 µL of sterile PDB. A control to test methanol inhibition activity was also carried out. Plates were incubated at 25 °C for 48 h and fungal growth was determined both by visual reading and by a spectrophotometer at 450 nm. The MIC value for the two camphor enantiomers and borneol was considered as the highest dilution of each compound that determines a significant reduction of the growth of A. alternaria colony with respect to the control (SDW).

P. viridiflava: The 96-well microplate dilution method was used for all compounds. The bacterium was grown for 48 h in plates containing NGA medium, then the bacterial suspension was prepared in nutrient broth amended with 0.25% glucose (NGB) following the procedure described in Section 4.6.2. On the basis of the results obtained from the test of in vitro antimicrobial activity, only the compounds that were active against P. viridiflava (i.e., (+)-α-pinene, verbenone and rosmarinic acid) were assayed for the MIC determination. Only the enantiomer (+)-α-pinene was tested, since inhibition activity had resulted not different for the two forms of this compound. The dilutions of the different compounds and next steps of the procedure were the same of that previously reported for A. alternata, using NGB as a growth medium. The positive control was represented by 100 µL of the bacterial suspension plus 100 µL of NGB, while the negative control consisted of 200 µL of sterile NGB. A control to test methanol inhibition activity was also carried out. Plates were incubated at 27 °C for 24 h, then the bacterial growth was first recorded by a visual reading of the plates. Then, 30 µL of a resazurin solution (0.015%) was added to each well and plates were incubated for 3 h at 27 °C. The color of the suspensions contained in each well was recorded.