2.5.2 Vapour diffusion method
Antibacterial activity of EO in vapour phase was evaluated by disc volatilization method at three different volumes (10, 20 and 30 μl per disc). In brief, solidified medium was inoculated over the surface of MHA/SDA with 0.1 ml of microorganism suspension. A paper disc (diameter 9 mm, Sigma, Steinheim, Germany) was laid on the inside surface of the upper lid and 10 μl of EO was placed on each disc.
The plate inoculated was immediately inverted on top of the lid and sealed with Para film to prevent leakage of the EO vapour. After the incubation period, the effectiveness of the EO was calculated by measuring the diameter of the inhibition zone. All tests were carried out in triplicate.