MATERIALS AND METHODS Bacterial strains and culture conditions Two S. aureus strains were used in the present study; S. aureus 8325-4 (SigB-) and SH1000 representing a SigB+.strain. Overnight cultures were grown in Luria Broth (LB) at 37°Cwith shaking at 150 rpm. Exposure to antibiotics was carried out as detailed below. Antibiotics Ciprofloxacin were purchased from Sigma-Aldrich CO. 10 mg/ml stock solution of antibiotic were prepared freshly with 0.1N HCl and stored at -20°C. During the experiment we diluted with sterile water 1:10 and 1:100 depending on the different drug concentration.
Then, the flask is put on shaker table and mixed at 150 rounds per minute before allowing them to settle for 10 minutes. After settling, the water sample is poured from side spout which is connected to the bottom of the flask. Some researchers reported better reproducibility with modified flask where stopcock is installed at the bottom of the flask, instead of side spout to pour the water sample (Blondina, et al., 1997) (Sorial, et al., 2004a) (Sorial, et al., 2004b). The dispersed oil in the removed water sample is extracted into methylene chloride for further analysis. Then, the oil concentration is evaluated using 340, 370 and 400 nm light absorbance (Environmental Protection Agency,
Prepare 0.6% noble agar by adding 0.6 g of noble agar to 100 ml of de-ionized water. Both agar solutions can be made in 100 ml glass bottles with closable lids for long-term storage. 8. Autoclave the noble agar mixtures to sterilize. These mixtures can be made in advance and stored at 4 °C but should be heated again at the time of the experiment until agar has completely dissolved.
When a carboxylic acid and alcohol was being mixed an ester was formed. In this first reaction (part A) the acid used was salicyclic acid while the alcohol that was used was methyl alcohol. A small amount of concentrated sulphuric acid about 3-4 drops was added and used as a catylst. Sulphuric acid helps to remove the water from the products formed. The mixture was placed in a water bath (90ºC) for 10 minutes to allow the reaction to be completed.
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
Extraction of Crude extract Propolis samples were refrigerated about -20c°. after refrigeration the propolis samples were grinded and macerated (30g of propolis, making up the volume to 100 mL with 70% ethanol) and was filtered. The filtrate was evaporated to dryness at 80°C under reduced pressure. 3. Characterization of Crude Extract 3.1 Physical Test 3.1.1 Organoleptic Test The color, odor and physical state of extract will be determined.
One millilitreer of the lichen extract (1 mg/mL) in a volumetric flask was diluted with distilled water (46 mL), and the content was mixed in a volumetric flask after adding. oOne millilitreer of Folin-Ciocalteu reagent was added and the content of the flask was mixed. thoroughly . After 3 min, 3 mL of 2% sodium carbonate (2 %) was added and then was allowed to standleft for 2 h with intermittent shaking. The reaction absorbance of prepared mixture absorbance was measured at 760 nm in a spectrophotometer (Jenway UK).
From dilutions 10-4 to 10-6 , 0.1 ml of each sample was spread onto nutrient agar plates containing 0.0016% (w/v) sodium azide and were kept for incubation at 37ºC for 24-48 hrs. The colonies showing the morphological characters like small, pin point, circular, translucent, convex and moist were selected. Each of selected colonies was cultured on to blood agar plates and pike streptococcal agar plates. The purity of the selected isolates was assured and the strains showing hemolysis on blood agar plates and gram positive cocci in chains were selected for
2.3. Preparation procedure for DS loaded PC-SA combined beads The ionotropic gelation method was used for the preparation of DS loaded PC-SA bead. The gum (PC) was dissolved in distilled water and initially boiled for 10 minutes, then cool and keep stirring for 24 hours at 400 RPM. Then filtration
Extraction of gum from Basil seeds Mucilage was extracted using distilled water. 100 gms of Basil seeds were added to a specific proportion of distilled water at a desired temperature for 2 hours. The soaked seeds were blended for entire extraction period. Slurry was maintained at a constant temperature and continuously stirred using a magnetic stirrer. Later, mucilage was separated from seeds using a rubber spatula on a mesh screen.