The Fungal Species
The fungal species about 100 stains which were previously isolated at the laboratory of biology department king khalid university will be used in this study. Fungi will be cultured on potato dextrose agar pda medium for 7 days at 27 °c. Identification of these species will be done basically on their microscopical and cultural characteristics. The identity will be confirmed by amplification of its gene using universal primers.
The fungal genomic dna extraction will be carried out using the qiagen dneasy plant/fungi mini kit protocol according to the nstructions. The its region of fungal dna will be amplified using the fungal specific-primer set: Its1-f cttggt cat tta gag gaa gta a and its4 r tcc tccgct tat tga tat gc as described by white et al.
1990 pcr reaction will be performed in a final volume of 50 ?L containing 10 mm tris-hcl 50mm kcl 1.5 m m mgcl2 each dntps at a concentration of 0.2 mm and 1.25 iu of taq polymerase. The amplification will be carried out by pcr. The initial denaturation temperature is 95 °c for 5 min followed by 40 cycles at 94 °c for 1 min 55 °c for 1 min 72 °c for 1 min; final extension at 72 °c for 10 min and holding at 4 °c.
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The amplified products will be examined by electrophoresis in 1.5% agarose gels in tae buffer. Then the pcr product will be purified and will be sent for sequencing at macrogen company korea. The its sequence of fungus isolate will be used for blast search in the embl genbank database.
The sequence of the isolate will be further aligned and compared to publish its region sequences searched with the taxonomy browser of the national center for biotechnology information ncbi http://www.ncbi.nlm.nih.gov and retrieved from genbank. Screening for mycogenic biosynthesis of ag-nps all the identified fungal species will be screened for the biogenic synthesis of ag-nps. For the biosynthesis of silver nanoparticles the biomass of each isolated fungal species will be grown aerobically in cazpeks broth medium the inoculated flasks will be incubated on orbital shaker at 27 1 °c and with agitation at 150 rpm for 5 days.
The fungal biomass then will be harvested after incubation by filtering using filter paper whatman no. 1 followed by three times of washing with distilled water to eradicate the residues of the medium from the biomass. Ten g fresh weight of mycelia will be added to 100 ml of sterilized double distilled water for 48 h at 27 1 °c in a 250 ml erlenmeyer flask with shaking again at 150 rpm. After the incubation the cell filtrate will be obtained by filtration through filter paper whatman no. 1.
The filtrates will be inoculated with 1 mm silver nitrate agno3 solution and incubated at room temperature in dark abdel-hafez et al. 2016 the production of the nanoparticle will be checked visual by the changing the color into brown color. Cell-free filtrate without addition of silver nitrate will be severed as control. Purification of silver nanoparticles after formation the silver nanoparticles the agnps solution will be centrifuged at 10.000-14.000 rpm for 15-20 min.
The supernatant will be excluded and the pellets will be dispersed with distilled water. This dispersion will be again centrifuged. The procedure will be repeated 3 times to clean agnps the free entities and unbound biological molecules. The purified formed pellets will be dried at 50-60 °c and stored in a brown-glass container for further characterization. Characterization of the biosynthesized silver nanoparticles the obtained silver nanoparticles will be characterized using different advanced tools including uv -visible spectroscopy at absorption curve range between 410-480 nm.
Determining of size and shape of agnps by electron microscopy sem will be carried out. Particle sizing experiments will be carried out by means of laser diffract meter using zeta sizer nano-series nano zs the crystallinity of agnps will be confirmed by their xrd pattern. Ft-ir spectra will be recorded in the range 4000–500 cm?1. Uv-visible spectrometry measurement: Biotransformation of metal ions will be affirmed by uv–visible spectroscopy measurement.
Labomed uv–vis double beam within the wave length ranged from 200 to 600 nm will be used mourato et al. 2011 x-ray diffraction xrd measurement: Xrd technique will be used for examination of quality of the prepared nanoparticles. Xrd pattern of the obtained nanoparticles on glass material will be estimated in wide selection of bragg angles 2? At a scanning rate of 20 min-1. Fourier transform infrared ft-ir spectrometry analysis: Sample containing nanoparticles will be scanned by ft-ir spectrometry using a spectrophotometer. Ft-ir spectra will be scanned in rang 4000–400 cm–1 in ftir spectrometry at a resolution of one cm-1.
Transmission electron microscopy tem the morphology and size of produced nanoparticles will be determined using tem. Antimicrobial activity of the characterized nanoparticles antimicrobial activities of ag-nps will be performed by the agar well diffusion method in muller hinton agar plates selvamohan et al. 2012 human pathogenic bacterial species such as escherichia coli pseudomonas sp. Proteus mirabilis klebsiella pneumoniae and staphylococcus aureus will be used for the assay.
The bacterial species will be grown in muller hinton broth at 37 oc for 24 h. The bacterial growth will be prepared on agar medium and wells will be cut using sterile cork borer. In to the wells agnp will be applied at different concentration and incubated at 37 oc. The plates will be examined for appearance of inhibition zone and then their diameter will be measured and will be compared with standard antibiotic such ciprofloxacin.
Optimization of silver nanoparticles for large scale production and stable mycofabrication of agnps using fungi it is necessary to investigate the ideal physical and chemical parameters required for the production of effective and small sized agnps mishra et al. 2014 different parameters such hydrogen-ion-concentration ph temperature t °c concentration of silver nitrate agno3 and time t of reaction will be studied.
The absorbance of the resulting solution after color change will be measured using uv–vis spectrophotometer. For each condition respective controls will be maintained.the length of the text: 6244 (no spaces: 5243)get new reportthe uniqueness of the text: 65.1 %we strongly recommend not to use this text for academic purposes
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