Hi Dear Grace Gallano Madanlo,

Refer to your query, please find my published work in the below link;

Article Selection of green carrier solvent for the effective creatio...

Article Development of nanoemulsion formulation of mustard oil, its ...

Please let me know if you need any assistance.

Best Regards,

Dr. Aloke

Thank you po, Dr. Aloke Purkait. Your answer is a great help, however, our study will use disc diffusion assay, is there an antifungal protocol that will guide us to conduct our study? thank you po.

Well Diffusion Assay is also a good method to determine the antifungal activity. I think it gives more accurate result when compared to disc diffusion assay.

Well diffusion disk is good.method to determine zone of inhibition and MIC. Plz refer my published work in IJTK, fatty acid composition and bioactivity of Sesbania sesban seed oil.

Antifungal Assay Protocol 1. Fungal Culture Preparation

• Strains: Select target fungi (e.g., Candida albicans, Aspergillus niger).
• Culturing: Grow fungi on Sabouraud Dextrose Agar or Potato Dextrose Agar plates at 25–30°C for 48–72 hours.
• Inoculum: Prepare a fungal suspension in sterile saline, adjusting to a 0.5 McFarland standard (approx. 1–5 x 10^6 CFU/mL).

2. Extract Preparation

• Stock Solution: Dissolve extract at a high concentration (e.g., 100 mg/mL in DMSO or methanol).
• Dilutions: Make serial dilutions to achieve a range of concentrations, typically 1–100 mg/mL.

3. Antifungal Testing Methods

A. Disc Diffusion Method

• Steps: Spread fungal suspension on agar plates using a sterile swab. Place filter paper discs (6 mm diameter) impregnated with 10–20 µL of each extract concentration on the agar. Include a positive control (antifungal agent like amphotericin B) and a negative control (DMSO or solvent alone). Incubate at 25–30°C for 48–72 hours. Measure inhibition zones around each disc (in mm) to assess antifungal activity.
• B. Broth Microdilution Method (for MIC determination)
• Steps: Add 100 µL broth medium to each well in a 96-well plate. Add 100 µL of each extract concentration to designated wells. Add 100 µL of fungal suspension to each well. Include positive and negative controls. Incubate at 25–30°C for 48–72 hours. Observe for fungal growth or measure OD at 600 nm. The MIC is the lowest concentration with no visible fungal growth.
• 4. Data Analysis
• Disc Diffusion: Measure inhibition zones; larger zones indicate stronger antifungal effects.
• Broth Microdilution: Determine MIC, with lower MIC values showing higher potency.

To assess the antifungal activity of plant extracts against pathogenic fungi, extracts will be prepared using solvents like ethanol, and an agar disc diffusion assay with fungal strains will be conducted on Potato Dextrose Agar. Measure the inhibition zones around the discs to evaluate effectiveness, and determine the Minimum Inhibitory Concentration (MIC) through serial dilutions.