Sterile filter paper discs

The swab paper disc method was modified to assess each LAB's antimicrobial activity against E. coli TISTR 073, S. aureus TISTR 029, and Ent. aerogenes TISTR 1540 according to the method of Phupaboon et al. [17] (link) and Alsaraf et al. [27] (link).
About 0.2 mL of the cultured-pathogenic strain (10⁵ CFU/mL) which obtained after incubation in nutrient broth (HiMedia, Maharashtra, India) for 18 h was spread with a sterile swab on nutrient agar, and then sterile filter paper discs (6 mm, Whatman, Maidstone, UK) in diameter were placed on the agar. Each of sterile bioactive protein hydrolysate in the supernatant (40 µL) was spotted on the paper disc. Streptomycin disc (15 µg) was used as a positive control. After incubation, the diameter of the inhibition zone around the disc was measured by calipers and expressed as millimeters (mm) obtained from triplicates.

The antimicrobial activity of the CEOs was done by the disc diffusion method as previously described by Puškárová et al. [44 (link)]. The bacterial inoculum was prepared in a Mueller Hinton broth by adding a single colony of bacteria from an overnight culture plate. Then it was incubated at 37 °C until the turbidity matched with 0.5 McFarland. The lawn culture was done in a pre-warmed sterile Mueller Hinton agar plate placed at the same temperature using sterile cotton swabs. The sterile filter paper discs (6 mm) prepared from Whatman No. 1 were pressed gently onto the surface of the agar plates and essential oil at 25% strength (diluted in DMSO) was pipetted onto the discs. The plates were then incubated at 37 °C for 24 h and the ZoI along with disc was measured in mm. Neomycin was used as positive control and DMSO was used as a negative control. The volume that comprised 10 mg pure essential oil was used to test antibacterial activity as each essential oil varied in density. The antibacterial activity of the CEOs is displayed in Table 5.