Methodology Chapter (2) Dissertation Example | Topics and Well Written Essays - 1500 words

Methodology Chapter (2) - talk ExampleFigure 2.1 In vitro do of sildenafil on murine embryo culture 2.1.2 In vivo effects of sildenafil on murine embryo development This part of the experimentation was not conducted ascribable to requirement for changes in the license. However, the in vivo effects on the development of oocyte would have been examined by injecting sildenafil on womanish mice for a limit of four days with the following three doses 0.5 mg/kg, 1.5mg/kg and 2.5mg/kg. ulteriorwards, the female mice would be mated naturally with males then one-day old zygotes would be harvested from sacrificed females. The number of oocytes and corpora lutea recorded would be use to determine the invest of ovulation. Thereafter, the embryos would be grown to blastocysts again in a media not supplemented with sildenafil. The quality of development of the embryos would then be determined by examining their development rate, and assessing the numbers of the blastocyst cells (figure 2 .2). Figure 2.2 In Vivo effects of sildenafil on murine embryo development 2.1.3 Expression of PDE5a in the mouse pre-implantation embryo The experiment was conducted by examining the presence of PDE5 mRNA in murine embryos at the successive stages of embryo development. The embryos used for use up were obtained from mice that had been mated naturally. On the other hand, the blastocysts had been cultured in standard conditions. 2.2 Sildenafil Citrate purification from commercially available sildenafil tablets In both the in vitro and in vivo experiments, sildenafil had been cleansed from commercially available Viagra tablets by leaving 20g sephadex G-25 overnight to swell in 100ml of distilled water. A editorial of 80ml was then applied with the sephadex gelatine and equilibrated in 100ml of distilled water at room temperature. A Viagra tablet of 38mg was then placed in 91.2ml of distilled water and slowly mixed with a magnetic stirrer at room temperature for a period of twenty m inutes. It was then filtered for 20 minutes under(a) a temperature of 4 degrees Celsius. The liquid in which sildenafil has been dissolved was then applied on the pillar. The column was rinsed with the Viagra solution just before it was rose-cheeked with 400ml of water to wash away any possible small molecules. This way, only sildenafil was left precipitated to resin. The column was then applied with 1% Formic acid to rinse the sildenafil off. The absorbance of the rinsed solution was then discovered and according to Francis et al. (2003), the rinsed sildenafil had a sharp peak absorbance at 40ml (Figure 3.1). Figure 3.1 Elution of pure sildenafil from sephadex column After sildenafil was rinsed, the column was washed again with 160ml or distilled water to eliminate the formic acid. After the column was free of formic acid, it was then washed with 320ml of 0.2% sodium azide in order to preserve it for later use. The rinsed sildenafil was then frozen at temperatures of -80 degrees Celsius. It was then dried by freezing it in a high vacuum. This was done by first sublimating the contents for a period of 8 hours at 0.37 mbar and under a temperature of -53 degrees Celsius, then desorbing it at 0.001 mbar for 3 hours. The weight of the crystallized contents was determined by examining the rate of absorbance of sildenafil over the whole sildenafil that had been eluted, assuming that there had been a recovery rate of 60%. The eluted sildenafil was then dissolved in 0.1% formic acid in order to obtain the convention concentration of refined