Paper disc diffusion method

The antifungal activity of the EOs of C. lansium seeds was first investigated using the paper disc diffusion method³³. All strains were streaked onto the SDA plates. A single colony was streaked again to ensure the viability and purity of the strains. Then about 5 single colonies were picked up into 5 mL saline solution. Microbial suspensions of 0.5 McFarland standard were prepared equivalent to approximately 10⁶ CFU·mL⁻¹ yeast and evenly dispensed onto the MHA plates to form the microbe lawn. Pieces of papers were placed on the medium with sterile tweezers and keep the space between the paper more than 24 mm. The diameters of MHA plates and the paper were 120 mm and 6 mm, respectively. The antifungal drug FLZ (2.5 mg·mL⁻¹) was used as a positive control. Ten microlitres of EOs of C. lansium seeds, the constituent chemicals (sabinene, β-phellandrene and 4-terpineol) and positive control were added onto the paper. The plates were incubated at 30 °C for 24 to 48 h. The antifungal activity of the samples was evaluated by measuring zones of inhibition of microbial growth surrounding the paper discs. The zones of inhibition corresponding to diameters were measured with an antibiotic zone scale (Cecon-Brasil) in mm. All the experiments were carried out in triplicate.