*4.8. Evaluation of Antibacterial Activity*

The antibacterial activity of the essential oil from chirimoya leaves was assessed against Gram-negative and Gram-positive bacteria (Table 4) by the microdilution broth method according to the procedure described by Valarezo et al. [40]. The bacterial strains were incubated in Müeller-Hinton (MH) broth. Tetracycline was used as a positive control and DMSO was used as a negative control. Results are reported as minimum inhibitory concentration (MIC).

For *Campylobacter jejuni* (ATCC 33560) the broth microdilution method was carried out according to Valarezo et al. [40] with some specific requirements as described briefly. Fluid thioglycollate medium was used for reactivation of the strain, supplemented with 5% of Horse serum. An aliquot of a cryogenic reserve was resuspended in thioglycollate and incubated for 48 h at 37 ◦C in a microaerophilic atmosphere provided by an Oxoid CampyGen (2.5 L sachet). Sample solutions were made by dissolving 80 mg of EO in 1 mL of DMSO. Two-fold serial dilutions were employed to obtain decreasing concentrations of EO from 4000 to 31.25 μg/mL and cation-adjusted Muller Hinton II broth (pH 7.3) with 5% lyssed horse blood [41] as media for the antibacterial assay. The inoculum was prepared from thioglycollate culture and adjusted to 0.5 McFarland. Final concentration of bacteria was 5 × <sup>10</sup><sup>5</sup> CFU/mL. The microplate was incubated for 48 h at 37 ◦C in microaerophilic atmosphere (5% CO2, Oxoid CampyGen). Erythromycin was used as positive control with a MIC value of 15.65 μg/mL and DMSO as negative control. MIC was determined by visual examination of growth and through addition of a 1% solution of TTZ as bacterial viability indicator after incubation time to confirm the visual results. A blank with the same range of concentrations of EO was prepared simultaneously and measurements at 405 nm were made to discard reduction of TTZ by contamination. Optical density (OD) for TTZ reduction was measured in a microplate reader (EPOCH 2, BioTek, Winooski, VT, USA).
