Since your compound is entirely novel in terms of antifungal screening, you will need to perform a preliminary test to find the approximate range of activity, followed by a more precise test to determine the exact Minimum Inhibitory Concentration (MIC).

Here is a step-by-step guide on how to determine the concentrations to use.

Step 1: Preliminary Assay (Determining the Active Range)

The goal of this first test is not to get a precise MIC value, but to find out if the compound is active and in what general ballpark (e.g., µg/mL or mg/mL). This will guide your dilution scheme for the definitive test.

1. Choose a Broth Microdilution Method: This is the standard technique(based on CLSI guidelines) where you prepare a series of dilutions of your compound in a microtiter plate containing fungal culture broth.

2. Select a Broad Concentration Range: Since you have no prior data,you must start with a very wide range. A common and practical strategy is to use a logarithmic (log) dilution series.

· Recommended Starting Range: 1000 µg/mL to 0.1 µg/mL (or 1 mg/mL to 0.0001 mg/mL).

· How to prepare this series:

· Prepare a stock solution of your phytoactive compound. This is often done in DMSO (Dimethyl sulfoxide), as most phytochemicals are not water-soluble. The final concentration of DMSO in any well must not exceed 1%, as higher concentrations can themselves inhibit fungal growth and confound your results.

· Create a two-fold or ten-fold serial dilution series in the microtiter plate.

· Example of a two-fold series for a broad screen: 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9, 1.95, 0.98, 0.49 µg/mL... and so on until you cover your desired range.

3. Include Essential Controls: Your experiment is worthless without proper controls.

· Growth Control: Wells with broth + fungus + solvent (e.g., 1% DMSO). This shows the fungus can grow normally in the absence of your compound.

· Sterility Control: Wells with broth only. This confirms your broth is not contaminated.

· Negative Control: Wells with broth + your compound at the highest concentration. This checks if your compound is sterile (no microbial growth) and doesn't cause turbidity on its own.

· Positive Control: Wells with broth + fungus + a known antifungal drug (e.g., Fluconazole for yeast, Amphotericin B for a broader range). This validates that your test system is working correctly.

4. Inoculate and Incubate:

· Inoculate each well (except the sterility control) with a standardized suspension of your test fungus (e.g., Candida albicans, Aspergillus brasiliensis). The inoculum size is critical (usually 0.5-2.5 x 10³ CFU/mL for yeast).

· Incubate the plate at the appropriate temperature (e.g., 35°C) for 24-48 hours (or longer for some fungi).

5. Interpret the Preliminary Results:

· Visually or with a spectrophotometer (to measure turbidity), determine the lowest concentration that completely inhibits visible growth. This gives you a rough MIC value.

· Example Result: You see growth up to the 62.5 µg/mL well, but the 125 µg/mL well and all higher concentrations are clear. Your approximate MIC is 125 µg/mL.

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Step 2: Definitive MIC Assay (Determining the Exact Concentration)

Now that you know the compound is active around, for example, ~125 µg/mL, you can set up a second, more precise test with a narrower range of concentrations.

1. Define a Narrower and Finer Dilution Series: Center your dilution series around the approximate MIC from step 1.

· From the example above (MIC ~125 µg/mL):

· Create a two-fold dilution series covering one log above and below the rough MIC.

· Suggested concentrations: 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9 µg/mL.

· This will allow you to pinpoint the exact MIC value (e.g., is it 62.5 or 125?).

2. Repeat the Broth Microdilution: Perform the assay again with this new,finer dilution series and all the necessary controls.

3. Determine the Exact MIC: The MIC is defined as thelowest concentration of the compound that completely prevents visible growth after the incubation period.

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Summary and Practical Recommendations

Assay Phase Goal Recommended Concentrations Dilution Scheme

Preliminary (Screen) Find the range of activity 1000 µg/mL down to 0.1 µg/mL Logarithmic (e.g., 10-fold or 2-fold)

Definitive (MIC) Find the exact MIC e.g., 250 µg/mL down to ~4 µg/mL (based on preliminary results) Two-fold serial dilution

Key Considerations for Your Experiment:

1. Solubility: This is your biggest hurdle. If your compound precipitates out of solution at higher concentrations, you cannot tell if the lack of growth is due to inhibition or insolubility. Always check your wells for precipitation. DMSO is the most common solvent for this reason.

2. Test Organisms: Choose reference strains from international collections (e.g., ATCC, INCQS). Common choices for initial screening include:

· Yeast: Candida albicans (ATCC 10231)

· Filamentous Fungus: Aspergillus brasiliensis (ATCC 16404)

· You can also include clinically relevant resistant strains later.

3. Standard Guidelines: Follow established protocols like those from the Clinical and Laboratory Standards Institute (CLSI) documents M27 (for yeasts) and M38 (for filamentous fungi). These provide detailed methodologies for inoculum preparation, media, incubation time, and endpoint reading.

4. Replicates: Always perform the experiment in at least duplicate or triplicate to ensure your results are reproducible.

5. Beyond MIC - MFC: To determine if your compound is fungicidal (kills the fungus) or just fungistatic (stops its growth), you can perform a Minimum Fungicidal Concentration (MFC) test. After reading the MIC plate, take a sample from the clear wells, plate it on agar, and see if any colonies grow.

Is this an isolated chemical compound or an extract

Phil Geis it is an isolated chemical

If you have compound why not address it by weight - e.g. MIC?

To determine appropriate concentrations for testing the antifungal activity of your plant extract, a serial dilution is recommended, starting with a broad range like 200 mg/mL down to 50 mg/mL for diffusion assays or a range of µg/mL if using a microdilution method to determine minimum inhibitory concentration (MIC). These initial concentrations can then be adjusted based on the initial screening results, with lower concentrations tested if the extract shows high activity and higher concentrations if it shows little to no effect.

Common detection methods include microdilution or agar dilution, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS).