) to secure sufficient cell density. The growth of contaminating microorganisms was inhibited by supplementing the growth medium with cycloheximide 100 mg, bacitracin 25000 units, polymyxin B sulphate 5000 units, vancomycin 20 mg, nalidixic acid 5 mg, and nystatin 100000 units (Oxoid, UK).28 The solid selective media were then prepared by melting the basal medium, cooling to 56°C in a water bath, adding appropriate amounts of stock solutions of the antibiotics and 5% horse serum (PAN-Biotech, Gmbh, Germany). The biotyping of the bacteria was done by CO2 requirement, H2S production, urease and oxidase positivity, growth in the presence of dyes (thionine
Inhibitors,research,lifescience,medical and basic fuchsine), and reaction with monospecific anti-A and anti-M sera (Arcomex, Jordan).29 Strains identified as B. melitensis or B. abortus were
stored in 2YT medium at -20°C. Only 16 isolates of B. melitensis resistant to tetracycline by susceptibility test were used in the present study. Plant samples Collection The arial parts of Inhibitors,research,lifescience,medical plant samples including the leaves and buds of Rosmarinus officinalis L., Origanum syriacum, Thymus syriacus, Salvia palaestina Benth, Mentha piperia and Lavandula stoechas L. (Labiatae), Inhibitors,research,lifescience,medical were collected during the flowering season from their natural habitat in Syria (table 1). The samples were cleaned from impurities, such as contaminating plants, dust, and other pollutants. The collected plants were air dried and were cut to pieces. Inhibitors,research,lifescience,medical Table 1 Plants and their families, collection sites, and parts used Essential Oil Extraction Extraction of essential oils was carried out using water steam distillation device (Clevenger-type
apparatus) according to the European Pharmacopoeia method.30 The device was attached to condenser and cold water recycler (Hydrodistillation technique). Distilled water was added (1:10 v/v) to each Inhibitors,research,lifescience,medical sample, and distilled for 2 h. The supernatant contained essential oil which was dehydrated by chemical structure filtering through anhydrous Na2SO4. The essential oil thus prepared was collected in airtight vials and stored in refrigerator. Antibacterial Susceptibility Assay The test isolates was grown in Muller-Hinton Broth (MHB, Merck) medium at 37°C for 22 h. The bacterial number in the final inoculum was adjusted to 106 CFU/ml. A bacterial lawn was prepared by pouring 0.1 ml of of bacterial suspension onto each plate of Muller-Hinton Agar medium (MHA, Merck), spread by a sterile cotton swab, and allowed to remain in contact for 1 min. 5% concentration of each essential oil were prepared in order to impregnate the paper discs. The sterile filter paper discs containing tested essential oils (6-mm diameter) were then placed on the bacterial lawn. The Petri dishes were subsequently incubated at 37°C for 24 h and the inhibition zone around each disc was measured in mm.