SCREENING METHODS: Scanning electron microscopy(1)

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electron microscopy(1)

After treatment with plant extract, SEM observation will be carried out on fungal pathogens. The method involves the preparation of a Petridish containing 15-25 ml potato dextrose agar, seeded with 1 ml of the fungal conidial spore suspension at a known concentration (105 spores per ml). 1 ml of the extract at the concentration of IC50 (obtained from the hyphal growth inhibition test), was dropped onto the inoculated agar and will be further incubated for another 4-7 days at 28 °C. A vehicle-treated culture can be used as a control. Using cork borer (6-10 mm) segments will be cut from cultures growing on potato dextrose plates at different time intervals from intial day to end of experiment for SEM examination (Sasidharan et al., 2008) (plate-7).

The specimen then placed on double-stick adhesive tabs on a planchette and the planchette placed in a petri plate. In a fume hood, a vial cap containing 2% osmium tetroxide in water will be placed in an unoccupied quadrant of the plate. After being covered, the plate will be sealed with parafilm, and vapor fixation of the sample proceeded for 1 h.
plate7Screening Methods in the Study-10
Plate 7 : Scanning electron microscope images of P. aeruginosa cells after treatment with methanol extract (suspended with 5ml distilled water) of A. dracunculus. (a) The region of inhibition zone (arrows) and shrinking and degradation of the cells. (b) The damaged cells of P. aeruginosa (arrows) in inhibition zone (Source-Benli et al., 2007)

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