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.
After that, cells were incubated at 37oC and CO2 with 5% during 24 hours. At the end of 24 hours, the plasmid with Pin1 gene was transfected to HEK-293 by the help of PEI. Then, cells were harvested after 24 hour and 48 hour incubations. After harvesting, medium was discarded and washed with 200 μl 1X PBS buffer at the side of the well. Then, the PBS was poured slowly.
Top agar was dispensed into 50ml test tubes and then autoclaved for 15 minutes at 121℃ as well. Stored the top agar at -5℃ refrigerator when it cooled down. Preheated top agar for 20 minutes to liquefy before use. The optimal dispense temperature for top agar was 45℃. 3.3.4 Preparation of CaCl2 solution The molecular weight of CaCl2 was 111 g/mole.
MATERIALS AND METHODS Preparation of human erythrocytes The 10 ml blood samples were withdrawn from three healthy volunteers with informed consent in heparin test tubes. After centrifuging at 1250 g for 10 minute, the plasma portion and buffy coat were removed and the remaining erythrocytes were washed 3 times using phosphate-buffered saline (PBS). The washed packed RBCs had a hematocrit of about 80%. RBCs loading with FVIII Stepwise hypotonic dialysis was used to load factor FVIII (Lyophilized powder from human plasma produced by Biotest, IBRF) in RBCs. Two ml of washed pack cells and 250 unit of FVIII were put into a dialysis bag (Sigma).
Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
Serial dilutions were made by sequentially adding 1 ml from 10-1 till 10-7. The serially diluted suspensions (10-1 to10-7) were spread plated on nutrient agar and incubated at 37 ºC for 24 h. The isolated colonies with distinct colony morphology were selected, further streaked to get pure cultures and stored in glycerol at -20oC. All the discrete isolated strains were tested for their plant
The minimum inhibitory concentration of abietic acid was studied by broth micro dilution method using 96-well microtitre plates.9 Test compound was dissolved in DMSO (1%) with the addition of Tween-80(0.5%) and diluted in Muller Hinton Broth to get a concentration range of 100-1000μg/mL. The solution was then two fold diluted in Muller Hinton Broth (100 μL), inoculated with bacterial strains and then incubated at 37°C for 24 h. The bacterial growth was measured as turbidity with a Cyberlab microplate reader at 405 nm. The minimum inhibitory concentration was defined as the lowest concentration of test compound that inhibits the growth of the test bacteria. DMSO assayed as the negative control at a concentration of 1% did not inhibit any of the strains tested. All tests were assayed in triplicate in three independent experiments and median values were used for MICs calculation.
1. Label each well of a tissue culture treated 6-well plate appropriately for each cell line or condition being investigated. 2. Prepare 2x cell culture medium by dissolving 1 g of powder medium and 0.2 g of sodium bicarbonate in de-ionized water to a final volume of 50 ml. 3.
Step-III: Synthesis of Cr(II) Complexes: The Schiff's base complexes were synthesized by mixing the Schiff's base (1.5 g) in ethanolic solution of Chromium chloride [CrCl2]. This reaction is refluxed in a waterbath for two hours and their volume were reduced to 70% of it’s original volume and residue was obtained. The coloured product obtained was filtered under suction, washed with ethanol. The product were recrystallized from ethanol. Their yields ranges from 50-55%, the product obtained were light green colour and melting point was 2100C.
The test tubes with and without enzyme (control) were incubated at 370C for 1 hour. After incubation, the enzyme was denatured by heating the tubes at 1000C for 5 minutes. Both the test treated and untreated samples (control) were assayed for antibacterial activity by agar well diffusion method15. 7. Antibiotic Sensitivity