Blank disc

The antibacterial assay was performed using disc diffusion (Kirby-Bauer) method as described by Chew et al. [15 (link)]. This is a qualitative assay which is commonly performed to evaluate the antimicrobial activity of phytochemicals or extracts. Briefly, the density of bacteria was standardized to 1 × 10⁸ coliform units (cfu)/mL using Miles and Misra technique [18 (link)] and was swabbed onto Mueller Hinton Agar (Oxoid) surface. 1 mg of crude extract or 0.5 mg of fractions were dissolved initially in 100 μL methanol and loaded onto sterile blank disc (6 mm diameter; Oxoid). The discs were then impregnated onto inoculated agar. 30 μg vancomycin (Oxoid) and blank disc loaded with 100 μL methanol without extract or fraction were served as positive and negative controls, respectively. The plates were left at 4 °C for an hour to allow the diffusion of extracts before they were incubated for 16–20 h at 37 °C. Antibacterial activity was indicated when clear inhibition zones observed around the discs. The diameter of the inhibition zones was measured (Fig. 3) and the results were expressed as mean of three independent experiments. The test was repeated three times.Fig. 3

Inhibition zone of fractions exhibit antibacterial activity in disc diffusion assay

Disc diffusion method for antimicrobial susceptibility testing was carried out according to the standard method by Bauer et al. (1966) [12 (link)] to assess the presence of antibacterial activities of the plant extracts. A bacteria culture was inoculated to the entire surface of Mueller-Hinton agar plate using sterile swab. The plates were dried for 15 minutes and used for the sensitivity assay. The discs were infused with 10 μL (100 μg) plant extract/disc and placed on the Mueller-Hinton agar surface. Each test set comprises ten implanted extract discs, antibiotic controls, chettaphanin I, and negative controls. The standard antibiotic discs were Amoxicillin/clavulanic acid 30 μg, Doxycycline 30 μg, and Sulfa-trimethoprim 25 μg for S. intermedius. Amoxicillin/clavulanic acid 30 μg, Doxycycline 30 μg, and Fosfomycin 50 μg discs were for S. suis. The negative controls, ethanol and water, were saturated onto the blank disc (Oxoid, UK) and applied along the test. Each test plate had four treated discs placed in equidistance to each other. The plates were incubated at 37°C for 24 hours and examined for zone of inhibition (ZOI). Three replicates were carried out for each bacteria isolate. Data were expressed as mean ± standard deviation from three isolates of S. suis and S. intermedius.