*3.3. Microorganisms*

Semen samples were obtained from 27 males following 2 days of sexual abstinence. The specimens were taken by masturbation into a sterile wide mouth container. The samples were liquefied at 37 ◦C for 30 min. All experiments were performed within 1 h after sampling. Only ejaculates showing normal semen parameters (concentration > 20 × 10<sup>6</sup>/mL, motility > 40%, viability > 40%, and morphology > 4%) and free from leukocytes were used. The experiments were approved by the Ethic Committee at the Specialized Hospital Sv. Svodar Zobor, protocol no. 030809/2015. Tryptone Soya agar (TSA, Merck, Darmstadt, Germany) and Blood agar (BA, Merck, Darmstadt, Germany) were inoculated with the semen samples, and after incubation (24 h, 37 ◦C), individual colonies were selected for further confirmation with MALDI-TOF MS Biotyper (Brucker Daltonics, Bremen, Germany) [78]. The isolates were maintained in Mueller Hinton Agar (MHA, Merck, Darmstadt, Germany) and cultured 24 h before the experiment to reach a concentration of 10<sup>5</sup> cfu/mL.

#### *3.4. Antimicrobial Susceptibility Testing*

The antimicrobial susceptibility test was performed with the disc diffusion method against (10 mcg) chloramphenicol, tetracycline, tigecycline, and tobramycin. The discs were obtained from Oxoid (Basingstoke, UK). The results were interpreted according to EUCAST [36].

#### *3.5. Disc Di*ff*usion Method*

A suspension of the tested culture (0.1 mL of 10<sup>5</sup> cells/mL) was spread onto Mueller Hinton Agar (MHA, Oxoid, Basingstoke, UK). Filter paper discs (6 mm) were impregnated with 15 μL of the EO and placed on the inoculated plates. The agars were incubated at 4 ◦C for 2 h and subsequently placed into an incubator at 37 ◦C for 24 h. The diameters of the inhibition zones were measured in mm. All the tests were performed in triplicate [79]. The results were evaluated as follows (disk diameter included): ≥15 mm was strongly inhibitory, <15–10 mm was moderately/mildly inhibitory, and <10 mm was not inhibitory [78–82].

#### *3.6. Determination of Minimum Inhibitory Concentration*

The broth microdilution assay was used for determination of the minimal inhibition concentration (MIC) according to the Clinical and Laboratory Standards Institute [83]. All tests were performed in Mueller Hinton Broth (MHB, Oxoid, Basingstoke, UK). The bacterial strains were cultured overnight at 37 ◦C in MHA. The tested strains were suspended in MHB to give a final density of 10<sup>6</sup> cfu/mL confirmed by viable counts. The EO solution was prepared in dimethyl sulphoxide (DMSO, Penta, Prague, Czech Republic). An amount of 50 μL of MHB was added to each 96-well micro-titer plate, and 100 μL of MHB was added to the 10th well for sterility control. For the growth control, MHB with 5% DMSO was added to the 9th well. Fifty microliters of EOs initially dissolved in 5% DMSO

were added into the first well. A serial 2-fold dilution was performed by transferring 50 μL of the suspension to the subsequent wells up to the 8th well; bacterial inoculum of 0.5 McFarland was diluted in the ratio of 1:100 and added into the 1st–8th wells in order to acheive the final concentration of 5×10<sup>5</sup> cfu/mL. Bacterial cell viability and MIC values were determined by observing the turbidity. The lowest concentrations of the EOs with clear suspension were considered as the MIC values. The test was performed in triplicate alongside cefoxitin (30 mcg), used as a positive control.

## *3.7. Statistical Analysis*

The basic variation (disc diffusion method) in statistical values from obtained data were calculated with Statgraphic, Tukey HSD test. Mean, standard deviation, minimum, maximum, coefficient of variation, and frequency of size of inhibition zones were calculated for the antimicrobial activity of essential oils.
