Disk Diffusion Assay for Clostridium perfringens

Disk diffusion assay was performed according to the Clinical and Laboratory Standards Institute (CLSI) [37 ] with slight modification as previously described by Gharaibeh et al. [35 (link)]. Briefly, sterile filter paper discs (Whatman disc, 6 mm diameter) (Oxoid, Basingstoke, UK) were impregnated with 15 µL of the EOs and kept at room temperature for 1 h. Freshly prepared Mueller Hinton broth culture of a C. perfringens NetB-positive toxin isolate (used for challenge) was adjusted to 0.5 McFarland turbidity, which is equivalent to 1 × 10⁸ CFU/mL, by using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 600 wavelength with 0.132 reading. Then the cultured broth was streaked evenly into a Mueller Hinton agar medium (Oxoid, UK) using a cotton swab in three directions. The EO discs and the antibiotic discs (Table 1) were applied on the Mueller Hinton agar surface and incubated at 37 °C for 24 h under anaerobic conditions.