SCREENING METHODS: Scanning electron microscopy(2)

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Once the sample is vapor fixed, the planchette will be plunged into liquid nitrogen -210 °C and then transferred on to the “peltier-cooled” stage of the freeze dryer, and freeze drying of the specimen will be kept for 10 h. Finally, the freeze dried specimen will be sputter coated with 5-10 nm gold before viewing in the SEM. Conventional SEM microscopy are frequently selected to visualize the ultrastructural damage on both cell wall and cytoplamatic membrane of entire microbes when fixed material can be used. The SEM is advantageous over several other microscopy methods as it is three-dimensional and almost the whole of the specimen is sharply focused.

Furthermore, besides having a combination of higher magnification, larger depth of focus and greater resolution, the preparation of samples is also relatively easier, compared to the TEM method (Sasidharan et al., 2010).

After SEM finding further confirmation can be obtained from TEM study. To study the antifungal activity through TEM method the hyphal specimens (1×3 mm, with approximately 1 mm thickness of underlying agar blocks) of test fungal strains will be excised from the margin of actively growing PDA culture treated with plant extract using a sterilized razor blade.

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