Antimicrobial Activities of Essential Oils of Nepal

Journal of Essential Oil Research: JEOR, Jan/Feb 2005 by Yonzon, Minoba, Lee, Dong Jin, Yokochi, Toshihiro, Kawano, Yasuhiro, Nakahara, Toro

Cultures of test microbes: Strains of bacterium, yeast and fungus were selected on the basis of common human and plant pathogens, which were obtained from Institute for Fermentation Osaka, Japan (IFO). The selected bacterial strains were Gram-positive bacteria: Staphylococus aureus (IFO14462) and Corynebacteriumamycolatum (IFO15207), a Gram-negative bacterium: Escherichia coli (IFO15034). Both Staphylococus aureus and Escherichia coli were clinical isolates and international reference standard strains for disk-susceptibility of many antibiotics. Corynebacterium amycolatum were isolated from human skin and does not contain mycolic acid. These bacterial strains were maintained on Nutrient Agar slant (DIFCO Laboratories, Detroit, MI). A loopful amount of bacterial colonies from agar slants were inoculated into the flasks with 120 mL PY medium (10 g polypeptone, 2 g yeast extract, 1 g MgSO^sub 4^7H^sub 2^O in one liter water, pH 7). The flasks inoculated were incubated for 20-24 h at 37°C for the former two strains and 30°C for the last strain.

The selectedyeast strain was Candida albicans (IFO 1594), which causes bronchomycosis. The strain was maintained on GPY agar slant (10 g glucose, 5 g polypeptone, 3 g yeast extract, 3 g malt extract, 15 g agar in one liter water, pH 5.6). Among fungi, Aspergillus ochraceus (IFO 31221), isolated from cucumber roots and ochratoxigenic, was selected. The strain was maintained on Potato Dextrose Agar Medium (DIFCO Laboratories, Detroit, MI). Loopful amounts of yeast and fungal cells from agar slants were inoculated into the flasks with 120 mL of GPY liquid medium and the flasks were incubated at 25°C for 40-48 h. After incubation the culture broths were diluted to give an OD650 of 0.2 using 1% Tween 80 aqueous solutions for agar seeding of antimicrobial activity test.

Testing method of antimicrobial activities: The Petri plate-paper disk method was performed for the determination of antimicrobial activity of the oils. Agar media specified previously were used for the growth according to the species of microbes. A base layer of 10 mL molted agar medium was initially poured into the Petri plates (9 cm diameter). The diluted culture broths were transferred into the molten agar medium (approximately 1:10, vol/vol) at 40°-45°C and mixed well. Approximately 4 mL of the seed agar medium suspendedwith test microbes was then poured over the base layer. In some tests, larger Petri plates (15 cm diameter) were used. Paper disks at 6 mm diameter were soaked with 15 µL of test oils (designated as neat in the Table) or 15 µL of the diluted ones with ethanol at 1:1(vol/vol) ratio and 1:2 (vol/vol) ratio and placed on the seeded agar medium. Ethanol was used as the negative control (15 and 30 µL), while novobiocin (1.0 % w/v in ethanol) was used as the positive control for bacterium. All the procedures were performed under sterile conditions. Plates were incubated in an upright position at different temperatures as mentioned above. Diameters of inhibition were measured after 24 h for bacterium and 48 h for yeast/fungus. All the tests were run in duplicate and the average value was adopted.