**4.1 Scanning electron microscopy**

After treatment with plant extract, scanning electron microscopy SEM observation will be carried out on fungal strains. First of all, the plate containing 25 mL PDA medium will be seeded with 1 mL of the fungal conidial spore suspension containing 105 spores per mL from a 120-h-old culture. The extract 1mL, at the concentration of IC50 (obtained from the hyphal growth inhibition test), is then dropped onto the inoculated agar and will be further incubated for another 7 days at 28 C. A vehicle-treated culture can be used as a control. Five to ten mm segments will be cut from cultures growing on potato dextrose plates at various time intervals 1, 2, 3, 4, 5, 6, and 7 days for SEM examination (Sasidharan et al., 2008b). 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. Once the sample is vapor fixed, the planchette will be plunged into slushy nitrogen -210 C and then transferred on to the "peltier-cooled" stage of the freeze dryer, and freeze drying of the specimen will be proceeded for 10 h. Finally, the freeze dried specimen will be sputter coated with 5–10 nm gold before viewing in the SEM. 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). From the SEM micrograph (Fig. 4) we can observe the changes caused by the plant extract on fungal surface.

#### **4.2 Transmission electron microscopy (TEM)**

Further confirmation of SEM finding can be obtained from TEM study. To study the antifungal activity through TEM method the hyphal specimens (1×3 mm2, with approximately 1 mm thickness of underlying agar blocks) of test fungal strains will be excised from the margin of actively growing SDA culture treated with plant extract using a sterilized razor blade. The specimens are then fixed with modified Karnovsky's fixative (Karnivsky, 1965) consisting of 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.05 M sodium cacodylate buffer solution (pH 7.2) at 4°C overnight. Subsequently, the fixed specimens are washed with the solution three times for 10 min each. The specimens were then will be post-fixed in the solution with 1% (w/v) osmium tetroxide at 4°C for 2 h and then will be washed briefly with

Screening Methods in the Study of Fungicidal Property of Medicinal Plants 115

Further verification of SEM and TEM finding can be obtained from CLSM study. To study the antifungal activity through CLSM method the plant extract with MIC concentration will be prepared. 48 h fungal culture will be developed by culturing the fungal strains on SDA agar for 48 h. Controls without the plant extract or antimicrobials also will be included as control groups. The 48 h fungal culture will be gently transferred into a 12-well microtitre plate and rinsed with PBS for 15 s. The discs will be then immersed in 1 ml of the plant extract or antimicrobial agents and incubated at 37oC in an aerobic incubator for 24 h. Subsequently, the extract or antimicrobial will be removed and the viability of the fungal cells will be assessed by Molecular Probes LIVE/DEAD BacLight Bacterial viability kit which comprise SYTO-9 and propidium iodide (PI) (Molecular Probes, Eugene, OR). 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). CLSM has become a precious tool for a wide range of studies in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in

The above mentions methods demonstrated the great potential in the development of antifungal testing to study the fungicidal properties of medicinal plants to develop fungicide. The main advantages of the presented methods are the following: easy; rapid; cheap and accurate. Our discussion demonstrates that the use electron microscopy is vital to reveal the cell injury caused by plants extract on fungal strains. The cell changes occurring in surface and cytoplasm of fungal cells following exposure to the plant extract

This project was partly supported by USM Short Term Grants (304/CIPPM/639040) from Universiti Sains Malaysia. Kwan Yuet Ping was supported by MyPhD fellowship from

Bauer, R.W.; Kirby, M.D.K.; Sherris, J.C. & Turck, M. (1966). Antibiotic susceptibility testing

Camel, V. (2000). Microwave-assisted solvent extraction of environmental samples. *Trends in* 

Choi, M.P.K.; Chan, K.K.C.; Leung, H.W. & Huie, C.W. (2003). Pressurized liquid extraction

surfactant solutions. *Journal of Chromatography A,* 983, 153-162.

by standard single disc diffusion method. *American Journal of Clinical Pathology,* 45,

of active ingredients (ginsenosides) from medicinal plants using non-ionic

could be visible using a combination of SEM and TEM studies.

Ministry of Higher Education, Government of Malaysia, Malaysia.

*Analytical Chemistry,* 19, 229-248.

**4.3 Confocal laser scanning microscopy (CLSM)** 

thickness up to 100 micrometers.

**5. Conclusion** 

**6. Acknowledgment** 

493–496.

**7. References** 

distilled water twice each. The postfixed specimens will be *en bloc* stained with 0.5% (w/v) uranyl acetate at 4°C overnight and then will be dehydrated once in a graded ethanol series of 30, 50, 70, 80, and 95% and three times in 100% ethanol for 10 min each. The specimens will be further treated with propylene oxide twice for 30 min each as a transitional fluid and then will be embedded in Spurr's resin. Ultra-thin sections (approximately 50 nm in thickness) will be cut with a diamond/ glass knife using an ultra-microtome. The sections will be mounted on copper grids and will be stained with 2% uranyl acetate and Reynolds' lead citrate (Reynolds, 1963) for 7 min each. Finally the sections will be observed with a transmission electron microscope. From the TEM micrograph we can observe the changes caused by the plant extract on fungal cytoplasm (Fig. 5).

Fig. 4. SEM micrographs of *Aspergillus niger*

Fig. 5. TEM micrographs of *Candida albicans*

#### **4.3 Confocal laser scanning microscopy (CLSM)**

Further verification of SEM and TEM finding can be obtained from CLSM study. To study the antifungal activity through CLSM method the plant extract with MIC concentration will be prepared. 48 h fungal culture will be developed by culturing the fungal strains on SDA agar for 48 h. Controls without the plant extract or antimicrobials also will be included as control groups. The 48 h fungal culture will be gently transferred into a 12-well microtitre plate and rinsed with PBS for 15 s. The discs will be then immersed in 1 ml of the plant extract or antimicrobial agents and incubated at 37oC in an aerobic incubator for 24 h. Subsequently, the extract or antimicrobial will be removed and the viability of the fungal cells will be assessed by Molecular Probes LIVE/DEAD BacLight Bacterial viability kit which comprise SYTO-9 and propidium iodide (PI) (Molecular Probes, Eugene, OR). 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). CLSM has become a precious tool for a wide range of studies in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers.
