2.7. Antimicrobial Activity

The assay of antibacterial and antifungal activities of the extracts from the yellow, orange, red and purple flowers obtained from P. grandiflora was performed using the broth microdilution method according to EUCAST (the European Committee on Antimicrobial Susceptibility Testing) recommendations and the modified method by Malm and Grzegorczyk [34,35]. The following reference strains from ATCC (American Type Culture Collection) were used in the study: Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC BAA-1707, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876 (representative of Gram-positive bacteria), Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27852 (representative of Gram-negative bacteria), Candida albicans ATCC 10231, Candida glabrata ATCC 90030 and Candida krusei ATCC 14243 (representatives of yeast fungi). All the used microbial strains were first subcultured in Mueller-Hinton Agar (MHA for bacteria) or Mueller-Hinton Agar with 2% glucose (MHA + 2% glucose for fungi) and incubated at 35 ± 1 °C for 18 ± 2 h. Microbial colonies were collected and suspended in sterile physiological saline to obtain an inoculum of 0.5 McFarland standard, corresponding to 1.5 × 10⁸ CFU (colony forming units)/mL for bacteria and 5 × 10⁶ CFU/mL for fungi. The extracts were dissolved in sterile distilled water to obtain the final concentration 100 mg/mL. The 2-fold dilutions of extracts in Mueller-Hinton Broth (MHB for bacteria) or in Mueller-Hinton Broth with 2% glucose (MHB + 2% glucose for fungi) were prepared in 96-well polystyrene plates to obtain final concentrations ranging from 32 to 0.125 mg/mL. Next, 2 µL of a particular bacterial or fungal inoculum was added to each well containing 200 µL of the serial dilution of extracts in the appropriate culture medium. After incubation at 35 ± 1 °C for 18 ± 2 h, the MIC (minimum inhibitory concentration) was assessed spectrophotometrically with the lowest concentration of extracts showing complete bacterial or fungal growth inhibition. The MBCs (minimum bactericidal concentrations) or MFCs (minimum fungicidal concentrations) were determined by removing 5 μL of the bacterial or fungal culture used for the MIC determinations from each well and spotting this onto an appropriate agar medium. The plates were incubated at 35 ± 1 °C for 18 ± 2 h. The lowest extract concentrations with no visible bacterial or fungal growth were assessed as MBC or MFC, respectively. Vancomycin (range of 0.06–16 μg/mL), ciprofloxacin (range of 0.015–16 μg/mL) and fluconazole (range of 0.06–16 μg/mL) were included as the reference antimicrobial substances active against Gram-positive bacteria, Gram-negative bacteria and yeasts. The experiments were performed in triplicate. Of the three MIC, MBC and MFC values, the most common representative value was presented.