Nutrient broth no 2

The strains used for mutagenicity experiments were histidine-dependent (His-) mutation tester strains TA98 and TA100, and their genetic properties were tested regularly; uvrB mutation test, rfa (Δ) mutation test, histidine requirement test, R-factor identification test, and natural regression. For a 2.5% Nutrient broth solution as the culture medium of the strain, 3 g nutrient broth no.2 (Sigma-Aldrich Co., St. Louis, MO, USA) was added to 120 mL distilled water and autoclaved. Then, 30 µL and 20 µL of TA98 and TA100 strains are inoculated into 20 mL medium, respectively. The flask inoculated with the strain was operated in a rotator at 80 rpm, 37 °C, for 11 h. Cultured strains are refrigerated until used for experiment.

The two essential oils used in this study, nutmeg and fir needle essential oil, were obtained from Roth (Roth, Germany). In order to produce the desired materials, emulsions of these oils had to be obtained using Tween 80, produced by Roth (Germany), as well as ethanol and glycerin, produced by Sigma-Aldrich (Darmstadt, Germany). Deionized water was used during the assays. All these reagents were of reactive grade and were used without additional purification. The antimicrobial assessments were performed utilizing Nutrient Broth No 2 and Agar (microbiological grade) Sigma-Aldrich (Germany).

Laboratory reagents including ethanol, water, Folin-Ciocalteu’s phenol reagent. gallic acid, quercetin, phosphate-buffered saline (PBS), neutral red, dimethyl sulfoxide (DMSO), benzo[a]pyrene (BaP), 4-nitroquinoline n-oxide (4nqo), sodium azide (SA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS), agar, glucose, histidine/biotin, nutrient broth no. 2, NaCl, Lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The co-factors for the NADH regenerating system and the S9 fraction of rat liver was purchased from Wako (Osaka, Japan) and Mol-Tox (Annapolis, MD, USA). EZ-CYTOX kit for cytotoxicity test is available from DoGenBio Co. (Seoul, Korea), and the ELISA kit for inflammation test was purchased from Abcam (Cambridge, UK). All other chemicals and reagents used were of analytical grade.