Then 1 ml from these bacterial solutions were added to 1 ml of MHII containing different concentration of ciprofloxacin 0.5x, 1x, 5x, 10x, 20x, 30x, 50x,75x and 100x MIC to each well in 12-well plate. Colony counts (CFU/ml) were determined at different time points (T0, T3, T24) by using appropriate dilutions of each culture. Using spot-plating method (32) 10µl was spotted on LB agar plates. However after 24 hour exposure the bacterial cell from dilutions of ≤ 1x102 were washed twice with sterile PBS prior to plating in triplicate. These were then incubated overnight at 37°C.
Twenty tablets were weighed accurately and powdered. An amount of the powder equivalent to 5 mg of amoxicillin trihydrate (content of one tablet) was dissolved in 60 ml of diluent. The solution was stirred for 10 min using a magnetic stirrer and filtered into a 100 ml volumetric flask through 0.45µ nylon membrane filter. The residue was washed 3 times with 10 ml of diluent and then the volume was completed to 100 ml with the same solvent. This solution was diluted with diluents to gae a concentration of 0.1 mg/ml solution each of Amoxicillin trihydrate.
After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes. These were centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was read at 540 nm at room temperature against appropriate blank. Blank consist of 1 ml distilled water, 0.5 ml of 30% TCA, and 0.5 ml of 0.8% TBA. TBARS values were expressed as n moles malonaldehyde (MDA)/mg protein.
The final volume was recorded. A pH probe connected through Microlab was calibrated using buffer solutions of pH 4.00, 7.00, and 10.00. The calibrated pH probe was used in order to measure the pH of the titrated solution of the unknown weak acid. These same steps were repeated except 2 mL of the strong base were titrated into the weak acid solution instead of 4 mL. This process was repeated 10 times.
Approximately 2 gm, nearest to 0.1 mg, oven dried cornhusk fibres, were weighed out accurately in weighing bottle and transferred to a 100 ml beaker. 40 ml of cold (10-15˚C) 72% sulphuric acid was added gradually to the fibres in small increments while stirring the mixture and macerating the fibres with a small glass rod. The beaker was kept in a bath at 2 ± 1˚C for dispersion of material. After the specimen was dispersed, beaker was covered with a watch glass and kept in a bath at 20 ± 1˚C for 2 hours. Mixture was stirred frequently to ensure complete
H and S were diluted 20x and P1 and P2 were diluted 2x to make up a total volume of 1ml each. 50ul of each diluted sample was pipetted into 8 wells of the microplate and 50ul of each protein standard was pipetted into 2 wells. 50ul were incubated with 50ul of alkaline copper reagent. 50ul of alkaline copper reagent was added to every well containing water, buffer, sample or standard and was incubated for 30 minutes. 100ul of Folin reagent was added to the wells and incubated for another 20 minutes.
Filtered the solution through 0.45 µm nylon filter Buffer having pH 3.70 used as Mobile phase A and mixture of methanol, acetonitrile and tetrahydrofuran (50: 50: 2 v/v/v) were used as Mobile phase B. Diluent: Prepared a mixture of buffer and methanol (80: 20 v/v). Placebo preparation (Placebo I): Weighed accurately and transferred 255 mg of placebo to a 100 mL volumetric flask. Add 40 mL of methanol and sonicated for 10 mins and add about 30 mL of diluent and again sonicate for 15 mins. Allow to equilibrate at room temperature (RT) and dilute to volume with diluent. Filter the solution through 0.45 µm nylon filter (25 mm) by discarding first few mL of the filtrate.
Then five millilitres of sample “A” was placed in the test tube labeled “A”. This was then repeated with the next three samples. 20 drops of Biuret reagent were then added to each test tube. The colour of each test tube was recorded and if proteins were present that was recorded for each test tube. Finally, the pH was recorded for each sample using pH
Hepcidin solution (20 μL) was added to the plasma before extraction with the beads, whilst in parallel another sample of plasma underwent extraction and then the same amount of hepcidin was added. The percentage of recovery was calculated as (AreaPRE / AreaPOST) x 100, where AreaPRE represents the samples with hepcidin added before bead treatment and AreaPOST the samples with hepcidin added after bead treatment. The values obtained were above 90 % in all cases. 4.4. Dietary intervention The functional juice aronia-citrus juice (ACJ) included in this study was a mixture of citrus juice (95%) and 5% aronia extract (Aronia melanocarpa), based on a drink model developed before and reported by Gonzalez-Molina et al .
H2SO4 • Catalyst: potassium sulphate (K2SO4), copper (II) sulphate pentahydrate (CuSO4.5H2O) • NaOH (40%) • 0.1 N HCl solution • Boric acid (4%) • Indicator: methyl red (200 mg make up to 100 ml with %95 ethanol ) 1 gram Feta Cheese was used as a sample in this experiment. First of all, the experiment was started with the digestion phase which was performed by mixing 10g potassium sulphate and 0.5 g CuSO4 solution that acts as catalyst, and added 20ml conc. H2SO4. The digestion unit temperature was set to 420±10°C and preheated for 15 minutes. Then, it was heated continuously at least 2 hours till a clear colorless solution was obtained.
Tn 4351 was originally isolated from bacteroides fragilis  . The transposon was successfully introduced into Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadiansis, Flexibacter strain SFI and Sporocytophaga myxococcoides by conjugation . Tn 4351carries two antibiotic resistance gene. One of the codes for resistance to erythromycin and clindamycin which is expressed in bactroides but not in E.Coli. The other gene codes for resistance in tetracycline and is expressed in aerobically grpwn E. coli, but not in anaerobically grpwn E. coli or in bacteroides.
32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate. 50 μL of these dilution solutions were separated on the TLC plate coated with SNISG. The plate was developed with petroleum ether: ethyl acetate (4:1) and the movement of solvent was usually controlled at 1 cm from the upper edge. After completion, the plate was dried until no solvent smell remained. It was sprayed with an ethanol solution containing 10% sulfuric acid, and heated at an infra-red drier until obvious color came up, as shown in Fig.2 (B.ab).
15. Add another 25cm3 of Methanol and Ethyl acetate to the solutions. Stir gently for 20 minutes using a stirring rod this is to allow more of the active ingredients to mix with the solution. 16. Take two funnels and place one in two separate clean measuring beakers making sure the bottoms of the funnels don’t touch the bottom of the measuring beakers.
15 mL of Solution A and B were mixed together to form solution F. Eight cuvettes were labeled distinctly as 1a, 2a, 3a, 4a, 1b, 2b, 3b, 4b, where “a” cuvettes were used for the concentration experiment and “b” cuvettes were used for the temperature experiment. Cuvette 1, the blank tube was prepared and the spectrophotometer was set to 405 nm. The enzyme was added, upon being ready to start the experiment, to tube 1 which then became tube “1a.” 3 mL of solution F was added to each cuvette, both “a” and “b.” The “b” cuvettes were then placed in their specific temperatures, 1b in the fridge, 2b in room temperature, 3b in a 32 degrees Celsius water bath and 4b in a 60 degree water bath. The temperature was recorded using a thermometer that was placed in the surroundings of the tube. The cuvettes were retrieved from their respected conditions.