SCREENING METHODS: Confocal laser scanning microscopy(part 2)

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microscopy(part 2)

To study the antifungal activity through CLSM method the plant extract with MIC concentration will be prepared. Two days old fungal culture will be developed by culturing the fungal strains on PDA with the treatment of plant extract either by disc diffusion or agar cup method. Controls without the plant extract are also cultured. The 48 h fungal culture will be gently transferred into a 12-well microtiter plate and rinsed with PBS for 15 s, incubated at 28oC for 24 h.

The viability of the fungal cells will be assessed by Molecular Probes LIVE/DEAD BacLight Bacterial viability kit which comprises SYTO-9 and propidium iodide (PI). After incubation with the dyes, the polymethylmethacrylate discs with biofilms will be placed on glass slides and live/dead ratio of cells will be quantified using the CSLM system (Thein et al., 2007). The advantages of confocal microscopy with SEM and TEM, is that it can be performed directly in solution, thus the sample is not subject to artefacts i.e. sample fixation or drying.

Herbal medicines make an enormous contribution to primary health care and have shown great potential in modern phytomedicine against numerous ailments and the complex diseases and ailments of the modern world.

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