One that fluoresced green under UV light, and one that did not fluoresce green under UV light. The third colony was taken from the LB/kanamycin plate, its ability to fluoresce is unknown. For each culture 1 mL of cell suspension was taken and added to a separate micro centrifuge tube. The cell suspensions were then centrifuged at 10,000 RCF for one minute to pellet the bacterial cells. The supernatant was then removed without disturbing the pellet.
On the second day of incubation, the plate was removed from the incubator and placed over a hot plate heating Iodine solids. The smoke of the Iodine stained the plate to display the presence or absence of a halo around the bacteria 2.12 Lipid Hydrolysis This test was done by making a single line streak inoculation on a tributyrin agar plate and allowing incubation. After the incubation period, the plate was observed for the presence or absence of a halo around the bacteria. 2.13 Gelatin Liquefaction A gelatin deep was deep stabbed and incubated. After incubation the tubes were placed in 4ºC for 30 minutes.
2.1. Isolation of fibrinolytic enzyme from Paenibacillus sp. IND8 The optimized conditions of fibrinolytic enzyme production by Paenibacillus sp. IND8 were described previously . This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate.
It was equilibrated with tissue for 10 minutes by flushing into the organ bath. After that, the steps above were repeated to test tissue response using 5ml of 1 x 10-5M and 1 x 10-4M of mepyramine. The experiment was repeated by replacing mepyramine with SIPBSDrug A as the antagonist. Lastly, concentration-response curve with Hill-Langmuir equation and Schild Plot were plotted using Bio-Graph. KB and pA2 values for mepyramine and SIPBSDrug A were calculated based on Schild plots and Gaddum
The solution was stirred at room temperature for 8h. The solvent was blown out with nitrogen. The residue was added to 1 ml of water containing 0.1% TFA and purified on RP-HPLC. Massspec of the final product clearly indicates presence of RB modified on PEI by series of peaks matching different polymer compositions (see Fig. 6).
Then, the test media is then incubated at 37 ° C, for 18-24 hours. Rinsing reusable instruments The samples were rinsed with 40 ml of pyrogen-free water using a glass beaker that is free from pyrogens. Endotoxin testing using STV A total of 0,2mL from the water obtained from the rinsing was placed in the STV containing LAL reagent and was shaken for 20 to 30 seconds. Then STV was placed in an incubator at 37 ° C for 60 ± 2 minutes. STV was then observed by reversing the reaction tube in one smooth motion.
Then plates will be allowed for incubation at 37°C and by using hemocytometer, after staining with 0.4% Trypan Blue every 24 h, number of cultured cells in the different wells will be counted to calculate the doubling time. d. Cell viability in presence of caspase-3 inhibitor The viability of MCF-7 cells treated with caspase-3 inhibitor and evaluation of drug will be done by using MTT assay. After incubation with caspase-3 inhibitor for 1 h, cells will be treated with different concentrations of drug(s) for upto 48 h after which viability will be
The solution was cooled to a lower temperature and mixed with 2µl of EtBr. This solution was poured into a trough which contained a comb, for polymerization. Then this stacked gel was placed in the tank which contained TBE buffer and then the comb was removed carefully for the formation
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.
2hrs. If more than 2 hours, it will lead to the star activity. Enzyme should be the last component which added to reaction before incubate it under 37°C. This is because the activity of the enzymes can be maintained at optimal temperature, 37°C. The mix components through pipetting the reaction mixture should be invert up and down slowly so that the mixture components are equalised.